Transcriptional analysis of the bglP gene from Streptococcus mutans

PubMed Central

Cote, Christopher K; Honeyman, Allen L

2006-01-01

Background An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II. Results To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments. Conclusion The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript. PMID:16630357

Genome-Wide Identification and Expression Analysis of Homeodomain Leucine Zipper Subfamily IV (HDZ IV) Gene Family from Musa accuminata

PubMed Central

Pandey, Ashutosh; Misra, Prashant; Alok, Anshu; Kaur, Navneet; Sharma, Shivani; Lakhwani, Deepika; Asif, Mehar H.; Tiwari, Siddharth; Trivedi, Prabodh K.

2016-01-01

The homeodomain zipper family (HD-ZIP) of transcription factors is present only in plants and plays important role in the regulation of plant-specific processes. The subfamily IV of HDZ transcription factors (HD-ZIP IV) has primarily been implicated in the regulation of epidermal structure development. Though this gene family is present in all lineages of land plants, members of this gene family have not been identified in banana, which is one of the major staple fruit crops. In the present work, we identified 21 HDZIV encoding genes in banana by the computational analysis of banana genome resource. Our analysis suggested that these genes putatively encode proteins having all the characteristic domains of HDZIV transcription factors. The phylogenetic analysis of the banana HDZIV family genes further confirmed that after separation from a common ancestor, the banana, and poales lineages might have followed distinct evolutionary paths. Further, we conclude that segmental duplication played a major role in the evolution of banana HDZIV encoding genes. All the identified banana HDZIV genes expresses in different banana tissue, however at varying levels. The transcript levels of some of the banana HDZIV genes were also detected in banana fruit pulp, suggesting their putative role in fruit attributes. A large number of genes of this family showed modulated expression under drought and salinity stress. Taken together, the present work lays a foundation for elucidation of functional aspects of the banana HDZIV encoding genes and for their possible use in the banana improvement programs. PMID:26870050

Structure and transcriptional impact of divergent repetitive elements inserted within Phanerochaete chrysosporium strain RP-78 genes

Treesearch

Luis F. Larrondo; Paulo Canessa; Rafael Vicuna; Philip Stewart; Amber Vanden Wymelenberg; Dan Cullen

2007-01-01

We describe the structure, organization, and transcriptional impact of repetitive elements within the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Searches of the P. chrysosporium genome revealed five copies of pce1, a 1,750-nt non-autonomous, class II element. Alleles encoding a putative glucosyltransferase and a cytochrome P450 harbor pce insertions...

A MADS Box Protein Interacts with a Mating-Type Protein and Is Required for Fruiting Body Development in the Homothallic Ascomycete Sordaria macrospora

PubMed Central

Nolting, Nicole; Pöggeler, Stefanie

2006-01-01

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Δmcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development. PMID:16835449

Expression profiles of defence related cDNAs in oil palm (Elaeis guineensis Jacq.) inoculated with mycorrhizae and Trichoderma harzianum Rifai T32.

PubMed

Tan, Yung-Chie; Wong, Mui-Yun; Ho, Chai-Ling

2015-11-01

Basal stem rot is one of the major diseases of oil palm (Elaies guineensis Jacq.) caused by pathogenic Ganoderma species. Trichoderma and mycorrhizae were proposed to be able to reduce the disease severity. However, their roles in improving oil palm defence system by possibly inducing defence-related genes in the host are not well characterized. To better understand that, transcript profiles of eleven putative defence-related cDNAs in the roots of oil palm inoculated with Trichoderma harzianum T32 and mycorrhizae at different time points were studied. Transcripts encoding putative Bowman-Birk protease inhibitor (EgBBI2) and defensin (EgDFS) increased more than 2 fold in mycorrhizae-treated roots at 6 weeks post inoculation (wpi) compared to those in controls. Transcripts encoding putative dehydrin (EgDHN), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), type 2 ribosome inactivating protein (EgT2RIP), and EgDFS increased in the oil palm roots treated with T. harzianum at 6 and/or 12 wpi compared to those in the controls. Some of these genes were also expressed in oil palm roots treated with Ganoderma boninense. This study provides an insight of some defence-related genes induced by Trichoderma and mycorrhizae, and their roles as potential agents to boost the plant defence system. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

PubMed Central

2012-01-01

Background The expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response. Results Transcription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed. Conclusions The rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress response after the cells have overcome the stress condition. Here we propose a model of the regulation of oxidative and heat stress response including redox homeostasis by SigH, RshA and the thioredoxin system. PMID:22943411

Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum.

PubMed

Busche, Tobias; Silar, Radoslav; Pičmanová, Martina; Pátek, Miroslav; Kalinowski, Jörn

2012-09-03

The expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response. Transcription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed. The rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress response after the cells have overcome the stress condition. Here we propose a model of the regulation of oxidative and heat stress response including redox homeostasis by SigH, RshA and the thioredoxin system.

Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

PubMed Central

Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

2012-01-01

Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

Suppressor of gamma response 1 (SOG1) encodes a putative transcription factor governing multiple responses to DNA damage

PubMed Central

Yoshiyama, Kaoru; Conklin, Phillip A.; Huefner, Neil D.; Britt, Anne B.

2009-01-01

The Arabidopsis sog1-1 (suppressor of gamma response) mutant was originally isolated as a second-site suppressor of the radiosensitive phenotype of seeds defective in the repair endonuclease XPF. Here, we report that SOG1 encodes a putative transcription factor. This gene is a member of the NAC domain [petunia NAM (no apical meristem) and Arabidopsis ATAF1, 2 and CUC2] family (a family of proteins unique to land plants). Hundreds of genes are normally up-regulated in Arabidopsis within an hour of treatment with ionizing radiation; the induction of these genes requires the damage response protein kinase ATM, but not the related kinase ATR. Here, we find that SOG1 is also required for this transcriptional up-regulation. In contrast, the SOG1-dependent checkpoint response observed in xpf mutant seeds requires ATR, but does not require ATM. Thus, phenotype of the sog1-1 mutant mimics aspects of the phenotypes of both atr and atm mutants in Arabidopsis, suggesting that SOG1 participates in pathways governed by both of these sensor kinases. We propose that, in plants, signals related to genomic stress are processed through a single, central transcription factor, SOG1. PMID:19549833

[Isolation of ABA-regulated genes in Oryza sativa through fluorescent differential display PCR (FDD-PCR)].

PubMed

Xu, Shou Ling; Shen, Si Shi; Xu, Zhi Hong; Xue, Hong Wei

2002-12-01

Abscisic acid (ABA) was critical in plant seed development and response to environmental factors such as stress situations. To study the possible ABA related signaling transduction pathways, we tried to isolate the ABA-regulated genes through fluorescent differential display PCR (FDD-PCR) technology using rice seedling as materials (treated with ABA for 2, 4, 8 and 12h). In the 17 fragments isolated, 14 and 3 clones were up-and down-regulated respectively. Sequence analyses revealed that the encoded proteins were involved in photosynthesis (7 fragments), signal transduction (1 fragments), transcription (2 fragments), metabolism and resistance (6 fragments), and unknown protein (1 fragments). 3 clones, encoding putative alpha/beta hydrolase fold, putative vacuolar H+ -ATPase B subunit, putative tyrosine phosphatase, were confirmed to be regulated under ABA treatment by RT-PCR and northern blot analysis. FDD-PCR and possible functional mechanisms of ABA were discussed.

Analysis of gene expression provides insights into the mechanism of cadmium tolerance in Acidithiobacillus ferrooxidans.

PubMed

Chen, Minjie; Li, Yanjun; Zhang, Li; Wang, Jianying; Zheng, Chunli; Zhang, Xuefeng

2015-02-01

Acidithiobacillus ferrooxidans plays a critical role in metal solubilization in the biomining industry, and occupies an ecological niche characterized by high acidity and high concentrations of toxic heavy metal ions. In order to investigate the possible metal resistance mechanism, the cellular distribution of cadmium was tested. The result indicated that Cd(2+) entered the cells upon initial exposure resulting in increased intracellular concentrations, followed by its excretion from the cells during subsequent growth and adaptation. Sequence homology analyses were used to identify 10 genes predicted to participate in heavy metal homeostasis, and the expression of these genes was investigated in cells cultured in the presence of increasing concentrations of toxic divalent cadmium (Cd(2+)). The results suggested that one gene (cmtR A.f ) encoded a putative Cd(2+)/Pb(2+)-responsive transcriptional regulator; four genes (czcA1 A.f , czcA2 A.f , czcB1 A.f ; and czcC1 A.f ) encoded heavy metal efflux proteins for Cd(2+); two genes (cadA1 A.f and cadB1 A.f ) encoded putative cation channel proteins related to the transport of Cd(2+). No significant enhancement of gene expression was observed at low concentrations of Cd(2+) (5 mM) and most of the putative metal resistance genes were up-regulated except cmtR A.f , cadB3 A.f ; and czcB1 A.f at higher concentrations (15 and 30 mM) according to real-time polymerase chain reaction. A model was developed for the mechanism of resistance to cadmium ions based on homology analyses of the predicted genes, the transcription of putative Cd(2+) resistance genes, and previous work.

Global Identification and Characterization of Transcriptionally Active Regions in the Rice Genome

PubMed Central

Stolc, Viktor; Deng, Wei; He, Hang; Korbel, Jan; Chen, Xuewei; Tongprasit, Waraporn; Ronald, Pamela; Chen, Runsheng; Gerstein, Mark; Wang Deng, Xing

2007-01-01

Genome tiling microarray studies have consistently documented rich transcriptional activity beyond the annotated genes. However, systematic characterization and transcriptional profiling of the putative novel transcripts on the genome scale are still lacking. We report here the identification of 25,352 and 27,744 transcriptionally active regions (TARs) not encoded by annotated exons in the rice (Oryza. sativa) subspecies japonica and indica, respectively. The non-exonic TARs account for approximately two thirds of the total TARs detected by tiling arrays and represent transcripts likely conserved between japonica and indica. Transcription of 21,018 (83%) japonica non-exonic TARs was verified through expression profiling in 10 tissue types using a re-array in which annotated genes and TARs were each represented by five independent probes. Subsequent analyses indicate that about 80% of the japonica TARs that were not assigned to annotated exons can be assigned to various putatively functional or structural elements of the rice genome, including splice variants, uncharacterized portions of incompletely annotated genes, antisense transcripts, duplicated gene fragments, and potential non-coding RNAs. These results provide a systematic characterization of non-exonic transcripts in rice and thus expand the current view of the complexity and dynamics of the rice transcriptome. PMID:17372628

A resource for characterizing genome-wide binding and putative target genes of transcription factors expressed during secondary growth and wood formation in Populus.

PubMed

Liu, Lijun; Ramsay, Trevor; Zinkgraf, Matthew; Sundell, David; Street, Nathaniel Robert; Filkov, Vladimir; Groover, Andrew

2015-06-01

Identifying transcription factor target genes is essential for modeling the transcriptional networks underlying developmental processes. Here we report a chromatin immunoprecipitation sequencing (ChIP-seq) resource consisting of genome-wide binding regions and associated putative target genes for four Populus homeodomain transcription factors expressed during secondary growth and wood formation. Software code (programs and scripts) for processing the Populus ChIP-seq data are provided within a publically available iPlant image, including tools for ChIP-seq data quality control and evaluation adapted from the human Encyclopedia of DNA Elements (ENCODE) project. Basic information for each transcription factor (including members of Class I KNOX, Class III HD ZIP, BEL1-like families) binding are summarized, including the number and location of binding regions, distribution of binding regions relative to gene features, associated putative target genes, and enriched functional categories of putative target genes. These ChIP-seq data have been integrated within the Populus Genome Integrative Explorer (PopGenIE) where they can be analyzed using a variety of web-based tools. We present an example analysis that shows preferential binding of transcription factor ARBORKNOX1 to the nearest neighbor genes in a pre-calculated co-expression network module, and enrichment for meristem-related genes within this module including multiple orthologs of Arabidopsis KNOTTED-like Arabidopsis 2/6. © 2015 Society for Experimental Biology and John Wiley & Sons Ltd This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de

1994-04-01

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-pointmore » sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.« less

Overexpression of a cotton gene that encodes a putative transcription factor of AP2/EREBP family in Arabidopsis affects growth and development of transgenic plants.

PubMed

Zhou, Ying; Xia, Hui; Li, Xiao-Jie; Hu, Rong; Chen, Yun; Li, Xue-Bao

2013-01-01

In the study, a gene encoding a putative ethylene response factor of AP2/EREBP family was isolated from cotton (Gossypium hirsutum) and designated as GhERF12. Sequence alignment showed that GhERF12 protein contains a central AP2/ERF domain (58 amino acids) with two functional conserved amino acid residues (ala14 and asp19). Transactivation assay indicated that GhERF12 displayed strong transcription activation activity in yeast cells, suggesting that this protein may be a transcriptional activator in cotton. Quantitative RT-PCR analysis showed that GhERF12 expression in cotton was induced by ACC and IAA. Overexpression of GhERF12 in Arabidopsis affected seedling growth and development. The GhERF12 transgenic plants grew slowly, and displayed a dwarf phenotype. The mean bolting time of the transgenic plants was delayed for about 10 days, compared with that of wild type. Further study revealed that some ethylene-related and auxin-related genes were dramatically up-regulated in the transgenic plants, compared with those of wild type. Collectively, we speculated that GhERF12, as a transcription factor, may be involved in regulation of plant growth and development by activating the constitutive ethylene response likely related to auxin biosynthesis and/or signaling.

Overexpression of a Cotton Gene That Encodes a Putative Transcription Factor of AP2/EREBP Family in Arabidopsis Affects Growth and Development of Transgenic Plants

PubMed Central

Li, Xiao-Jie; Hu, Rong; Chen, Yun; Li, Xue-Bao

2013-01-01

In the study, a gene encoding a putative ethylene response factor of AP2/EREBP family was isolated from cotton (Gossypium hirsutum) and designated as GhERF12. Sequence alignment showed that GhERF12 protein contains a central AP2/ERF domain (58 amino acids) with two functional conserved amino acid residues (ala14 and asp19). Transactivation assay indicated that GhERF12 displayed strong transcription activation activity in yeast cells, suggesting that this protein may be a transcriptional activator in cotton. Quantitative RT-PCR analysis showed that GhERF12 expression in cotton was induced by ACC and IAA. Overexpression of GhERF12 in Arabidopsis affected seedling growth and development. The GhERF12 transgenic plants grew slowly, and displayed a dwarf phenotype. The mean bolting time of the transgenic plants was delayed for about 10 days, compared with that of wild type. Further study revealed that some ethylene-related and auxin-related genes were dramatically up-regulated in the transgenic plants, compared with those of wild type. Collectively, we speculated that GhERF12, as a transcription factor, may be involved in regulation of plant growth and development by activating the constitutive ethylene response likely related to auxin biosynthesis and/or signaling. PMID:24194949

An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins.

PubMed

Varmanen, P; Rantanen, T; Palva, A

1996-12-01

A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.

Burkholderia mallei tssM encodes a putative deubiquitinase that is secreted and expressed inside infected RAW 264.7 murine macrophages.

PubMed

Shanks, John; Burtnick, Mary N; Brett, Paul J; Waag, David M; Spurgers, Kevin B; Ribot, Wilson J; Schell, Mark A; Panchal, Rekha G; Gherardini, Frank C; Wilkinson, Keith D; Deshazer, David

2009-04-01

Burkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. The B. mallei VirAG two-component regulatory system activates the transcription of approximately 60 genes, including a large virulence gene cluster encoding a type VI secretion system (T6SS). The B. mallei tssM gene encodes a putative ubiquitin-specific protease that is physically linked to, and transcriptionally coregulated with, the T6SS gene cluster. Mass spectrometry and immunoblot analysis demonstrated that TssM was secreted in a virAG-dependent manner in vitro. Surprisingly, the T6SS was found to be dispensable for the secretion of TssM. The C-terminal half of TssM, which contains Cys and His box motifs conserved in eukaryotic deubiquitinases, was purified and biochemically characterized. Recombinant TssM hydrolyzed multiple ubiquitinated substrates and the cysteine at position 102 was critical for enzymatic activity. The tssM gene was expressed within 1 h after uptake of B. mallei into RAW 264.7 murine macrophages, suggesting that the TssM deubiquitinase is produced in this intracellular niche. Although the physiological substrate(s) is currently unknown, the TssM deubiquitinase may provide B. mallei a selective advantage in the intracellular environment during infection.

Negative regulation of P element excision by the somatic product and terminal sequences of P in drosophila melanogaster

USDA-ARS?s Scientific Manuscript database

A transient in vivo P element excision assay was used to test the regulatory properties of putative repressor-encoding plasmids in Drosophila melanogaster embryos. The somatic expression of an unmodified transposase transcription unit under the control of a heat shock gene promoter (phsn) effectivel...

Phylogeny of fungal hemoglobins and expression analysis of the Aspergillus oryzae flavohemoglobin gene fhbA during hyphal growth.

PubMed

te Biesebeke, Rob; Levasseur, Anthony; Boussier, Amandine; Record, Eric; van den Hondel, Cees A M J J; Punt, Peter J

2010-01-01

The fhbA genes encoding putative flavohemoglobins (FHb) from Aspergillus niger and Aspergillus oryzae were isolated. Comparison of the deduced amino acid sequence of the A. niger fhbA gene and other putative filamentous fungal FHb-encoding genes to that of Ralstonia eutropha shows an overall conserved gene structure and completely conserved catalytic amino acids. Several yeasts and filamentous fungi, including both Aspergillus species have been found to contain a small FHb gene family mostly consisting of two family members. Based on these sequences the evolutionary history of the fungal FHb family was reconstructed. The isolated fhbA genes from A. oryzae and A. niger belong to a phylogenetic group, which exclusively contains Aspergillus genes. Different experimental approaches show that fhbA transcript levels appear during active hyphal growth. Moreover, in a pclA-disrupted strain with a hyperbranching growth phenotype, the transcript levels of the fhbA gene were 2–5 times higher compared to the wild-type. These results suggest that FHb from filamentous fungi have a function that is correlated to the hyphal growth phenotype.

A cDNA from a mouse pancreatic beta cell encoding a putative transcription factor of the insulin gene.

PubMed Central

Walker, M D; Park, C W; Rosen, A; Aronheim, A

1990-01-01

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5' flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5-10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine beta cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5' flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression. Images PMID:2181401

A repertoire of the dominant transcripts from the salivary glands of the blood-sucking bug, Triatoma dimidiata, a vector of Chagas disease

PubMed Central

Kato, Hirotomo; Jochim, Ryan C.; Gomez, Eduardo A.; Sakoda, Ryo; Iwata, Hiroyuki; Valenzuela, Jesus G.; Hashiguchi, Yoshihisa

2010-01-01

Triatoma (T.) dimidiata is a hematophagous Hemiptera and a main vector of Chagas disease. The saliva of this and other blood-sucking insects contains potent pharmacologically active components that assist them in counteracting the host hemostatic and inflammatory systems during blood feeding. To describe the repertoire of potential bioactive salivary molecules from this insect, a number of randomly selected transcripts from the salivary gland cDNA library of T. dimidiata were sequenced and analyzed. This analysis showed that 77.5% of the isolated transcripts coded for putative secreted proteins, and 89.9% of these coded for variants of the lipocalin family proteins. The most abundant transcript was a homologue of procalin, the major allergen of T. protracta saliva, and contributed more than 50% of the transcripts coding for putative secreted proteins, suggesting that it may play an important role in the blood-feeding process. Other salivary transcripts encoding lipocalin family proteins had homology to triabin (a thrombin inhibitor), triafestin (an inhibitor of kallikrein–kinin system), pallidipin (an inhibitor of collagen-induced platelet aggregation) and others with unknown function. PMID:19900580

Identification of the WBSCR9 gene, encoding a novel transcriptional regulator, in the Williams-Beuren syndrome deletion at 7q11.23.

PubMed

Peoples, R J; Cisco, M J; Kaplan, P; Francke, U

1998-01-01

We have identified a novel gene (WBSCR9) within the common Williams-Beuren syndrome (WBS) deletion by interspecies sequence conservation. The WBSCR9 gene encodes a roughly 7-kb transcript with an open reading frame of 1483 amino acids and a predicted protein product size of 170.8 kDa. WBSCR9 is comprised of at least 20 exons extending over 60 kb. The transcript is expressed ubiquitously throughout development and is subject to alternative splicing. Functional motifs identified by sequence homology searches include a bromodomain; a PHD, or C4HC3, finger; several putative nuclear localization signals; four nuclear receptor binding motifs; a polyglutamate stretch and two PEST sequences. Bromodomains, PHD motifs and nuclear receptor binding motifs are cardinal features of proteins that are involved in chromatin remodeling and modulation of transcription. Haploinsufficiency for WBSCR9 gene products may contribute to the complex phenotype of WBS by interacting with tissue-specific regulatory factors during development.

Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

PubMed

Adewoye, L O; Worobec, E A

2000-08-08

The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

PubMed Central

Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

PubMed

Rice, Elena A; Khandelwal, Abha; Creelman, Robert A; Griffith, Cara; Ahrens, Jeffrey E; Taylor, J Philip; Murphy, Lesley R; Manjunath, Siva; Thompson, Rebecca L; Lingard, Matthew J; Back, Stephanie L; Larue, Huachun; Brayton, Bonnie R; Burek, Amanda J; Tiwari, Shiv; Adam, Luc; Morrell, James A; Caldo, Rico A; Huai, Qing; Kouadio, Jean-Louis K; Kuehn, Rosemarie; Sant, Anagha M; Wingbermuehle, William J; Sala, Rodrigo; Foster, Matt; Kinser, Josh D; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E; Huang, Mingya G; Kuriakose, Saritha V; Skottke, Kyle; Repetti, Peter P; Reuber, T Lynne; Ruff, Thomas G; Petracek, Marie E; Loida, Paul J

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

Transcriptional Profiles of Mating-Responsive Genes from Testes and Male Accessory Glands of the Mediterranean Fruit Fly, Ceratitis capitata

PubMed Central

Scolari, Francesca; Gomulski, Ludvik M.; Ribeiro, José M. C.; Siciliano, Paolo; Meraldi, Alice; Falchetto, Marco; Bonomi, Angelica; Manni, Mosè; Gabrieli, Paolo; Malovini, Alberto; Bellazzi, Riccardo; Aksoy, Serap; Gasperi, Giuliano; Malacrida, Anna R.

2012-01-01

Background Insect seminal fluid is a complex mixture of proteins, carbohydrates and lipids, produced in the male reproductive tract. This seminal fluid is transferred together with the spermatozoa during mating and induces post-mating changes in the female. Molecular characterization of seminal fluid proteins in the Mediterranean fruit fly, Ceratitis capitata, is limited, although studies suggest that some of these proteins are biologically active. Methodology/Principal Findings We report on the functional annotation of 5914 high quality expressed sequence tags (ESTs) from the testes and male accessory glands, to identify transcripts encoding putative secreted peptides that might elicit post-mating responses in females. The ESTs were assembled into 3344 contigs, of which over 33% produced no hits against the nr database, and thus may represent novel or rapidly evolving sequences. Extraction of the coding sequences resulted in a total of 3371 putative peptides. The annotated dataset is available as a hyperlinked spreadsheet. Four hundred peptides were identified with putative secretory activity, including odorant binding proteins, protease inhibitor domain-containing peptides, antigen 5 proteins, mucins, and immunity-related sequences. Quantitative RT-PCR-based analyses of a subset of putative secretory protein-encoding transcripts from accessory glands indicated changes in their abundance after one or more copulations when compared to virgin males of the same age. These changes in abundance, particularly evident after the third mating, may be related to the requirement to replenish proteins to be transferred to the female. Conclusions/Significance We have developed the first large-scale dataset for novel studies on functions and processes associated with the reproductive biology of Ceratitis capitata. The identified genes may help study genome evolution, in light of the high adaptive potential of the medfly. In addition, studies of male recovery dynamics in terms of accessory gland gene expression profiles and correlated remating inhibition mechanisms may permit the improvement of pest management approaches. PMID:23071645

Transcriptional regulation of dimethyl sulfoxide respiration in a haloarchaeon, Haloferax volcanii.

PubMed

Qi, Qiuzi; Ito, Yoshiyasu; Yoshimatsu, Katsuhiko; Fujiwara, Taketomo

2016-01-01

The halophilic euryarchaeon Haloferax volcanii can grow anaerobically by DMSO respiration. DMSO reductase was induced by DMSO respiration not only under anaerobic growth conditions but also in denitrifying cells of H. volcanii. Deletion of the dmsR gene, encoding a putative regulator for the DMSO reductase, resulted in the loss of anaerobic growth by DMSO respiration. Reporter experiments revealed that only the anaerobic condition was essential for transcription of the dmsEABCD genes encoding DMSO reductase and that transcription was enhanced threefold by supplementation of DMSO. In the ∆dmsR mutant, transcription of the dmsEABCD genes induced by the anaerobic condition was not enhanced by DMSO, suggesting that DmsR is a DMSO-responsive regulator. Transcriptions of the dmsR and mgd genes for Mo-bisMGD biosynthesis were regulated in the same manner as the dmsEABCD genes. These results suggest that the genetic regulation of DMSO respiration in H. volcanii is controlled by at least two systems: one is the DMSO-responsive DmsR, and the other is an unknown anaerobic regulator.

Burkholderia mallei tssM Encodes a Putative Deubiquitinase That Is Secreted and Expressed inside Infected RAW 264.7 Murine Macrophages▿ †

PubMed Central

Shanks, John; Burtnick, Mary N.; Brett, Paul J.; Waag, David M.; Spurgers, Kevin B.; Ribot, Wilson J.; Schell, Mark A.; Panchal, Rekha G.; Gherardini, Frank C.; Wilkinson, Keith D.; DeShazer, David

2009-01-01

Burkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. The B. mallei VirAG two-component regulatory system activates the transcription of ∼60 genes, including a large virulence gene cluster encoding a type VI secretion system (T6SS). The B. mallei tssM gene encodes a putative ubiquitin-specific protease that is physically linked to, and transcriptionally coregulated with, the T6SS gene cluster. Mass spectrometry and immunoblot analysis demonstrated that TssM was secreted in a virAG-dependent manner in vitro. Surprisingly, the T6SS was found to be dispensable for the secretion of TssM. The C-terminal half of TssM, which contains Cys and His box motifs conserved in eukaryotic deubiquitinases, was purified and biochemically characterized. Recombinant TssM hydrolyzed multiple ubiquitinated substrates and the cysteine at position 102 was critical for enzymatic activity. The tssM gene was expressed within 1 h after uptake of B. mallei into RAW 264.7 murine macrophages, suggesting that the TssM deubiquitinase is produced in this intracellular niche. Although the physiological substrate(s) is currently unknown, the TssM deubiquitinase may provide B. mallei a selective advantage in the intracellular environment during infection. PMID:19168747

Changes in regulation of a transcription factor lead to autogamy in cultivated tomatoes.

PubMed

Chen, Kai-Yi; Cong, Bin; Wing, Rod; Vrebalov, Julia; Tanksley, Steven D

2007-10-26

We report the cloning of Style2.1, the major quantitative trait locus responsible for a key floral attribute (style length) associated with the evolution of self-pollination in cultivated tomatoes. The gene encodes a putative transcription factor that regulates cell elongation in developing styles. The transition from cross-pollination to self-pollination was accompanied, not by a change in the STYLE2.1 protein, but rather by a mutation in the Style2.1 promoter that results in a down-regulation of Style2.1 expression during flower development.

Regulation of melanin biosynthesis via the dihydroxynaphthalene pathway is dependent on sexual development in the ascomycete Sordaria macrospora.

PubMed

Engh, Ines; Nowrousian, Minou; Kück, Ulrich

2007-10-01

The filamentous ascomycete Sordaria macrospora accumulates melanin during sexual development. The four melanin biosynthesis genes pks, teh, sdh and tih were isolated and their homology to genes involved in 1,8 dihydroxynaphthalene (DHN) melanin biosynthesis was shown. The presence of DHN melanin in S. macrospora was further confirmed by disrupting the pks gene encoding a putative polyketide synthase and by RNA interference-mediated silencing of the sdh gene encoding a putative scytalone dehydratase. Because melanin occurs in fruiting bodies that develop through several intermediate stages within 7 days of growth, a Northern analysis of a developmental time-course was conducted. These data revealed a time-dependent regulation of teh and sdh transcript levels. Comparing the transcriptional expression by real-time PCR of melanin biosynthesis genes in the wild type under conditions allowing or repressing sexual development, a significant downregulation during vegetative growth was detected. Quantitative real-time PCR and Northern blot analysis of melanin biosynthesis gene expression in different developmental mutants confirmed that melanin biosynthesis is linked to fruiting body development and is under the control of specific regulatory genes that participate in sexual differentiation.

Putative Monofunctional Type I Polyketide Synthase Units: A Dinoflagellate-Specific Feature?

PubMed Central

Eichholz, Karsten; Beszteri, Bánk; John, Uwe

2012-01-01

Marine dinoflagellates (alveolata) are microalgae of which some cause harmful algal blooms and produce a broad variety of most likely polyketide synthesis derived phycotoxins. Recently, novel polyketide synthesase (PKS) transcripts have been described from the Florida red tide dinoflagellate Karenia brevis (gymnodiniales) which are evolutionarily related to Type I PKS but were apparently expressed as monofunctional proteins, a feature typical of Type II PKS. Here, we investigated expression units of PKS I-like sequences in Alexandrium ostenfeldii (gonyaulacales) and Heterocapsa triquetra (peridiniales) at the transcript and protein level. The five full length transcripts we obtained were all characterized by polyadenylation, a 3′ UTR and the dinoflagellate specific spliced leader sequence at the 5′end. Each of the five transcripts encoded a single ketoacylsynthase (KS) domain showing high similarity to K. brevis KS sequences. The monofunctional structure was also confirmed using dinoflagellate specific KS antibodies in Western Blots. In a maximum likelihood phylogenetic analysis of KS domains from diverse PKSs, dinoflagellate KSs formed a clade placed well within the protist Type I PKS clade between apicomplexa, haptophytes and chlorophytes. These findings indicate that the atypical PKS I structure, i.e., expression as putative monofunctional units, might be a dinoflagellate specific feature. In addition, the sequenced transcripts harbored a previously unknown, apparently dinoflagellate specific conserved N-terminal domain. We discuss the implications of this novel region with regard to the putative monofunctional organization of Type I PKS in dinoflagellates. PMID:23139807

Induction of multixenobiotic defense mechanisms in resistant Daphnia magna clones as a general cellular response to stress.

PubMed

Jordão, Rita; Campos, Bruno; Lemos, Marco F L; Soares, Amadeu M V M; Tauler, Romà; Barata, Carlos

2016-06-01

Multixenobiotic resistance mechanisms (MXR) were recently identified in Daphnia magna. Previous results characterized gene transcripts of genes encoding and efflux activities of four putative ABCB1 and ABCC transporters that were chemically induced but showed low specificity against model transporter substrates and inhibitors, thus preventing us from distinguishing between activities of different efflux transporter types. In this study we report on the specificity of induction of ABC transporters and of the stress protein hsp70 in clones selected to be genetically resistant to ABCB1 chemical substrates. Clones resistant to mitoxantrone, ivermectin and pentachlorophenol showed distinctive transcriptional responses of transporter protein coding genes and of putative transporter dye activities. Expression of hsp70 proteins also varied across resistant clones. Clones resistant to mitoxantrone and pentachlorophenol showed high constitutive levels of hsp70. Transcriptional levels of the abcb1 gene transporter and of putative dye transporter activity were also induced to a greater extent in the pentachlorophenol resistant clone. Observed higher dye transporter activities in individuals from clones resistant to mitoxantrone and ivermectin were unrelated with transcriptional levels of the studied four abcc and abcb1 transporter genes. These findings suggest that Abcb1 induction in D. magna may be a part of a general cellular stress response. Copyright © 2016 Elsevier B.V. All rights reserved.

Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses.

PubMed

Komatsu, Ken; Hirata, Hisae; Fukagawa, Takako; Yamaji, Yasuyuki; Okano, Yukari; Ishikawa, Kazuya; Adachi, Tatsushi; Maejima, Kensaku; Hashimoto, Masayoshi; Namba, Shigetou

2012-07-01

The first open-reading frame (ORF) of apple stem grooving virus (ASGV), of the genus Capillovirus, encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP). However, our previous study revealed that ASGV mutants with distinct and discontinuous Rep- and CP-coding regions successfully infect plants, indicating that CP expressed via a subgenomic RNA (sgRNA) is sufficient for viability of the virus. Here we identified a transcription start site of the CP sgRNA and revealed that CP translated from the sgRNA is essential for ASGV infection. We mapped the transcription start sites of both the CP and the movement protein (MP) sgRNAs of ASGV and found a hexanucleotide motif, UUAGGU, conserved upstream from both sgRNA transcription start sites. Mutational analysis of the putative CP initiation codon and of the UUAGGU sequence upstream from the transcription start site of CP sgRNA demonstrated their importance for ASGV accumulation. Our results also demonstrated that potato virus T (PVT), an unassigned species closely related to ASGV, produces two sgRNAs putatively deployed for the CP and MP expression and that the same hexanucleotide motif as found in ASGV is located upstream from the transcription start sites of both sgRNAs. This motif, which constituted putative core elements of the sgRNA promoter, is broadly conserved among viruses in the families Alphaflexiviridae and Betaflexiviridae, suggesting that the gene expression strategy of the viruses in both families has been conserved throughout evolution. Copyright © 2012 Elsevier B.V. All rights reserved.

RegA, an AraC-like protein, is a global transcriptional regulator that controls virulence gene expression in Citrobacter rodentium.

PubMed

Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J; Frankel, Gad; Hartland, Elizabeth L; Robins-Browne, Roy M

2008-11-01

Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5alpha, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.

RegA, an AraC-Like Protein, Is a Global Transcriptional Regulator That Controls Virulence Gene Expression in Citrobacter rodentium▿

PubMed Central

Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J.; Frankel, Gad; Hartland, Elizabeth L.; Robins-Browne, Roy M.

2008-01-01

Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5α, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization. PMID:18765720

Differential gene expression in ripening banana fruit.

PubMed Central

Clendennen, S K; May, G D

1997-01-01

During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants. PMID:9342866

Chitinase Expression in Listeria monocytogenes Is Influenced by lmo0327, Which Encodes an Internalin-Like Protein.

PubMed

Paspaliari, Dafni Katerina; Kastbjerg, Vicky Gaedt; Ingmer, Hanne; Popowska, Magdalena; Larsen, Marianne Halberg

2017-11-15

The chitinolytic system of Listeria monocytogenes thus far comprises two chitinases, ChiA and ChiB, and a lytic polysaccharide monooxygenase, Lmo2467. The role of the system in the bacterium appears to be pleiotropic, as besides mediating the hydrolysis of chitin, the second most ubiquitous carbohydrate in nature, the chitinases have been deemed important for the colonization of unicellular molds, as well as mammalian hosts. To identify additional components of the chitinolytic system, we screened a transposon mutant library for mutants exhibiting impaired chitin hydrolysis. The screening yielded a mutant with a transposon insertion in a locus corresponding to lmo0327 of the EGD-e strain. lmo0327 encodes a large (1,349 amino acids [aa]) cell wall-associated protein that has been proposed to possess murein hydrolase activity. The single inactivation of lmo0327 , as well as of lmo0325 that codes for a putative transcriptional regulator functionally related to lmo0327 , led to an almost complete abolishment of chitinolytic activity. The effect could be traced at the transcriptional level, as both chiA and chiB transcripts were dramatically decreased in the lmo0327 mutant. In accordance with that, we could barely detect ChiA and ChiB in the culture supernatants of the mutant strain. Our results provide new information regarding the function of the lmo0325-lmo0327 locus in L. monocytogenes and link it to the expression of chitinolytic activity. IMPORTANCE Many bacteria from terrestrial and marine environments express chitinase activities enabling them to utilize chitin as the sole source of carbon and nitrogen. Interestingly, several bacterial chitinases may also be involved in host pathogenesis. For example, in the important foodborne pathogen Listeria monocytogenes , the chitinases ChiA and ChiB and the lytic polysaccharide monooxygenase Lmo2467 are implicated in chitin assimilation but also act as virulence factors during the infection of mammalian hosts. Therefore, it is important to identify their regulators and induction cues to understand how the different roles of the chitinolytic system are controlled and mediated. Here, we provide evidence for the importance of lmo0327 and lmo0325 , encoding a putative internalin/autolysin and a putative transcriptional activator, respectively, in the efficient expression of chitinase activity in L. monocytogenes and thereby provide new information regarding the function of the lmo0325-lmo0327 locus. Copyright © 2017 Paspaliari et al.

Transcriptome-wide profiling and expression analysis of transcription factor families in a liverwort, Marchantia polymorpha

PubMed Central

2013-01-01

Background Transcription factors (TFs) are vital elements that regulate transcription and the spatio-temporal expression of genes, thereby ensuring the accurate development and functioning of an organism. The identification of TF-encoding genes in a liverwort, Marchantia polymorpha, offers insights into TF organization in the members of the most basal lineages of land plants (embryophytes). Therefore, a comparison of Marchantia TF genes with other land plants (monocots, dicots, bryophytes) and algae (chlorophytes, rhodophytes) provides the most comprehensive view of the rates of expansion or contraction of TF genes in plant evolution. Results In this study, we report the identification of TF-encoding transcripts in M. polymorpha for the first time, as evidenced by deep RNA sequencing data. In total, 3,471 putative TF encoding transcripts, distributed in 80 families, were identified, representing 7.4% of the generated Marchantia gametophytic transcriptome dataset. Overall, TF basic functions and distribution across families appear to be conserved when compared to other plant species. However, it is of interest to observe the genesis of novel sequences in 24 TF families and the apparent termination of 2 TF families with the emergence of Marchantia. Out of 24 TF families, 6 are known to be associated with plant reproductive development processes. We also examined the expression pattern of these TF-encoding transcripts in six male and female developmental stages in vegetative and reproductive gametophytic tissues of Marchantia. Conclusions The analysis highlighted the importance of Marchantia, a model plant system, in an evolutionary context. The dataset generated here provides a scientific resource for TF gene discovery and other comparative evolutionary studies of land plants. PMID:24365221

Differential Expression Patterns in Chemosensory and Non-Chemosensory Tissues of Putative Chemosensory Genes Identified by Transcriptome Analysis of Insect Pest the Purple Stem Borer Sesamia inferens (Walker)

PubMed Central

Zhang, Ya-Nan; Jin, Jun-Yan; Jin, Rong; Xia, Yi-Han; Zhou, Jing-Jiang; Deng, Jian-Yu; Dong, Shuang-Lin

2013-01-01

Background A large number of insect chemosensory genes from different gene subfamilies have been identified and annotated, but their functional diversity and complexity are largely unknown. A systemic examination of expression patterns in chemosensory organs could provide important information. Methodology/Principal Findings We identified 92 putative chemosensory genes by analysing the transcriptome of the antennae and female sex pheromone gland of the purple stem borer Sesamia inferens, among them 87 are novel in this species, including 24 transcripts encoding for odorant binding proteins (OBPs), 24 for chemosensory proteins (CSPs), 2 for sensory neuron membrane proteins (SNMPs), 39 for odorant receptors (ORs) and 3 for ionotropic receptors (IRs). The transcriptome analyses were validated and quantified with a detailed global expression profiling by Reverse Transcription-PCR for all 92 transcripts and by Quantitative Real Time RT-PCR for selected 16 ones. Among the chemosensory gene subfamilies, CSP transcripts are most widely and evenly expressed in different tissues and stages, OBP transcripts showed a clear antenna bias and most of OR transcripts are only detected in adult antennae. Our results also revealed that some OR transcripts, such as the transcripts of SNMP2 and 2 IRs were expressed in non-chemosensory tissues, and some CSP transcripts were antenna-biased expression. Furthermore, no chemosensory transcript is specific to female sex pheromone gland and very few are found in the heads. Conclusion Our study revealed that there are a large number of chemosensory genes expressed in S. inferens, and some of them displayed unusual expression profile in non-chemosensory tissues. The identification of a large set of putative chemosensory genes of each subfamily from a single insect species, together with their different expression profiles provide further information in understanding the functions of these chemosensory genes in S. inferens as well as other insects. PMID:23894529

Differential expression patterns in chemosensory and non-chemosensory tissues of putative chemosensory genes identified by transcriptome analysis of insect pest the purple stem borer Sesamia inferens (Walker).

PubMed

Zhang, Ya-Nan; Jin, Jun-Yan; Jin, Rong; Xia, Yi-Han; Zhou, Jing-Jiang; Deng, Jian-Yu; Dong, Shuang-Lin

2013-01-01

A large number of insect chemosensory genes from different gene subfamilies have been identified and annotated, but their functional diversity and complexity are largely unknown. A systemic examination of expression patterns in chemosensory organs could provide important information. We identified 92 putative chemosensory genes by analysing the transcriptome of the antennae and female sex pheromone gland of the purple stem borer Sesamia inferens, among them 87 are novel in this species, including 24 transcripts encoding for odorant binding proteins (OBPs), 24 for chemosensory proteins (CSPs), 2 for sensory neuron membrane proteins (SNMPs), 39 for odorant receptors (ORs) and 3 for ionotropic receptors (IRs). The transcriptome analyses were validated and quantified with a detailed global expression profiling by Reverse Transcription-PCR for all 92 transcripts and by Quantitative Real Time RT-PCR for selected 16 ones. Among the chemosensory gene subfamilies, CSP transcripts are most widely and evenly expressed in different tissues and stages, OBP transcripts showed a clear antenna bias and most of OR transcripts are only detected in adult antennae. Our results also revealed that some OR transcripts, such as the transcripts of SNMP2 and 2 IRs were expressed in non-chemosensory tissues, and some CSP transcripts were antenna-biased expression. Furthermore, no chemosensory transcript is specific to female sex pheromone gland and very few are found in the heads. Our study revealed that there are a large number of chemosensory genes expressed in S. inferens, and some of them displayed unusual expression profile in non-chemosensory tissues. The identification of a large set of putative chemosensory genes of each subfamily from a single insect species, together with their different expression profiles provide further information in understanding the functions of these chemosensory genes in S. inferens as well as other insects.

Characterization of the SPI-1 and Rsp type three secretion systems in Pseudomonas fluorescens F113.

PubMed

Barret, Matthieu; Egan, Frank; Moynihan, Jennifer; Morrissey, John P; Lesouhaitier, Olivier; O'Gara, Fergal

2013-06-01

Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) isolated from the sugar beet rhizosphere. The recent annotation of the F113 genome sequence has revealed that this strain encodes a wide array of secretion systems, including two complete type three secretion systems (T3SSs) belonging to the Hrp1 and SPI-1 families. While Hrp1 T3SSs are frequently encoded in other P. fluorescens strains, the presence of a SPI-1 T3SS in a plant-beneficial bacterial strain was unexpected. In this work, the genetic organization and expression of these two T3SS loci have been analysed by a combination of transcriptional reporter fusions and transcriptome analyses. Overexpression of two transcriptional activators has shown a number of genes encoding putative T3 effectors. In addition, the influence of these two T3SSs during the interaction of P. fluorescens F113 with some bacterial predators was also assessed. Our data revealed that the transcriptional activator hilA is induced by amoeba and that the SPI-1 T3SS could potentially be involved in resistance to amoeboid grazing. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans.

PubMed

Eshghi, Azad; Becam, Jérôme; Lambert, Ambroise; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Wunder, Elsio A; Ko, Albert I; Coppee, Jean-Yves; Goarant, Cyrille; Picardeau, Mathieu

2014-06-01

Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139(-) mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1.

PubMed

Zhu, Hongwei; Li, Huixin; Han, Zongxi; Shao, Yuhao; Wang, Yu; Kong, Xiangang

2011-04-06

In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV), the causative agent of duck viral enteritis (DVE), the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. DEV UL15 consists of two exons with a 3.5 kilobases (kb) inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa), whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp) ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s) in the cytoplasm at 6 h post infection (h p. i.) and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s) in the cytoplasm in the absence of any other viral protein. DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15 are very similar to those of alphaherpesviruses and most similar to the genus Mardivirus. The UL15 and/or UL15.5 accumulate(s) in the cytoplasm during early times post-infection and then are translocated to the nucleus at late times.

A putative APSES transcription factor is necessary for normal growth and development of Aspergillus nidulans.

PubMed

Lee, Ji-Yeon; Kim, Lee-Han; Kim, Ha-Eun; Park, Jae-Sin; Han, Kap-Hoon; Han, Dong-Min

2013-12-01

The nsdD gene encoding a GATA type transcription factor positively controls sexual development in Aspergillus nidulans. According to microarray data, 20 genes that were upregulated by deleting nsdD during various life cycle stages were randomly selected and deleted for functional analysis. None of the mutants showed apparent changes in growth or development compared with those of the wild-type except the AN3154 gene that encodes a putative APSES transcription factor and is an ortholog of Saccharomyces cerevisiae swi4. Deleting AN3154 resulted in retarded growth and development, and the gene was named rgdA (retared growth and development). The rgdA deletion mutant developed a reduced number of conidia even under favorable conditions for asexual development. The retarded growth and development was partially suppressed by the veA1 mutation. The conidial heads of the mutant aborted, showing reduced and irregular shaped phialides. Fruiting body development was delayed compared with that in the wild-type. The mutant did not respond to various nutritional or environmental factors that affected the development patterns. The rgdA gene was expressed at low levels throughout the life cycle and was not significantly affected by several regulators of sexual and asexual development such as nsdD, veA, stuA, or brlA. However, the rgdA gene affected brlA and abaA expression, which function as key regulators of asexual sporulation, suggesting that rgdA functions upstream of those genes.

Molecular cloning, sequence characterization and expression analysis of a CD63 homologue from the coleopteran beetle, Tenebrio molitor.

PubMed

Patnaik, Bharat Bhusan; Kang, Seong Min; Seo, Gi Won; Lee, Hyo Jeong; Patnaik, Hongray Howrelia; Jo, Yong Hun; Tindwa, Hamisi; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Bang, In Seok; Han, Yeon Soo

2013-10-15

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic "Cys-Cys-Gly" motif and "Cys188" residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Molecular Cloning, Sequence Characterization and Expression Analysis of a CD63 Homologue from the Coleopteran Beetle, Tenebrio molitor

PubMed Central

Patnaik, Bharat Bhusan; Kang, Seong Min; Seo, Gi Won; Lee, Hyo Jeong; Patnaik, Hongray Howrelia; Jo, Yong Hun; Tindwa, Hamisi; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Bang, In Seok; Han, Yeon Soo

2013-01-01

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens. PMID:24132157

A Deeper Examination of Thorellius atrox Scorpion Venom Components with Omic Techonologies.

PubMed

Romero-Gutierrez, Teresa; Peguero-Sanchez, Esteban; Cevallos, Miguel A; Batista, Cesar V F; Ortiz, Ernesto; Possani, Lourival D

2017-12-12

This communication reports a further examination of venom gland transcripts and venom composition of the Mexican scorpion Thorellius atrox using RNA-seq and tandem mass spectrometry. The RNA-seq, which was performed with the Illumina protocol, yielded more than 20,000 assembled transcripts. Following a database search and annotation strategy, 160 transcripts were identified, potentially coding for venom components. A novel sequence was identified that potentially codes for a peptide with similarity to spider ω-agatoxins, which act on voltage-gated calcium channels, not known before to exist in scorpion venoms. Analogous transcripts were found in other scorpion species. They could represent members of a new scorpion toxin family, here named omegascorpins. The mass fingerprint by LC-MS identified 135 individual venom components, five of which matched with the theoretical masses of putative peptides translated from the transcriptome. The LC-MS/MS de novo sequencing allowed to reconstruct and identify 42 proteins encoded by assembled transcripts, thus validating the transcriptome analysis. Earlier studies conducted with this scorpion venom permitted the identification of only twenty putative venom components. The present work performed with more powerful and modern omic technologies demonstrates the capacity of accomplishing a deeper characterization of scorpion venom components and the identification of novel molecules with potential applications in biomedicine and the study of ion channel physiology.

Transcriptome Profiling of Selectively Bred Pacific Oyster Crassostrea gigas Families that Differ in Tolerance of Heat Shock

PubMed Central

Bayne, Christopher J.; Camara, Mark D.; Cunningham, Charles; Jenny, Matthew J.; Langdon, Christopher J.

2010-01-01

Sessile inhabitants of marine intertidal environments commonly face heat stress, an important component of summer mortality syndrome in the Pacific oyster Crassostrea gigas. Marker-aided selection programs would be useful for developing oyster strains that resist summer mortality; however, there is currently a need to identify candidate genes associated with stress tolerance and to develop molecular markers associated with those genes. To identify candidate genes for further study, we used cDNA microarrays to test the hypothesis that oyster families that had high (>64%) or low (<29%) survival of heat shock (43°C, 1 h) differ in their transcriptional responses to stress. Based upon data generated by the microarray and by real-time quantitative PCR, we found that transcription after heat shock increased for genes putatively encoding heat shock proteins and genes for proteins that synthesize lipids, protect against bacterial infection, and regulate spawning, whereas transcription decreased for genes for proteins that mobilize lipids and detoxify reactive oxygen species. RNAs putatively identified as heat shock protein 27, collagen, peroxinectin, S-crystallin, and two genes with no match in Genbank had higher transcript concentrations in low-surviving families than in high-surviving families, whereas concentration of putative cystatin B mRNA was greater in high-surviving families. These ESTs should be studied further for use in marker-aided selection programs. Low survival of heat shock could result from a complex interaction of cell damage, opportunistic infection, and metabolic exhaustion. PMID:19205802

Regulation of the grapevine polygalacturonase-inhibiting protein encoding gene: expression pattern, induction profile and promoter analysis.

PubMed

Joubert, D Albert; de Lorenzo, Giulia; Vivier, Melané A

2013-03-01

Regulation of defense in plants is a complex process mediated by various signaling pathways. Promoter analysis of defense-related genes is useful to understand these signaling pathways involved in regulation. To this end, the regulation of the polygalacturonase-inhibiting protein encoding gene from Vitis vinifera L. (Vvpgip1) was analyzed with regard to expression pattern and induction profile as well as the promoter in terms of putative regulatory elements present, core promoter size and the start of transcription. Expression of Vvpgip1 is tissue-specific and developmentally regulated. Vvpgip1 expression was induced in response to auxin, salicylic acid and sugar treatment, wounding and pathogen infection. The start of transcription was mapped to 17 bp upstream of the ATG and the core promoter was mapped to the 137 bp upstream of the ATG. Fructose- and Botrytis responsiveness were identified in the region between positions -3.1 and -1.5 kb. The analyses showed induction in water when the leaves were submersed and this response and the response to wounding mapped to the region between positions -1.1 and -0.1 kb. In silico analyses revealed putative cis-acting elements in these areas that correspond well to the induction stimuli tested.

The OSU1/QUA2/TSD2-Encoded Putative Methyltransferase Is a Critical Modulator of Carbon and Nitrogen Nutrient Balance Response in Arabidopsis

PubMed Central

Zheng, Zhi-Liang

2008-01-01

The balance between carbon (C) and nitrogen (N) nutrients must be tightly coordinated so that cells can optimize their opportunity for metabolism, growth and development. However, the C and N nutrient balance perception and signaling mechanism remains poorly understood. Here, we report the isolation and characterization of two allelic oversensitive to sugar1 mutants (osu1-1, osu1-2) in Arabidopsis thaliana. Using the cotyledon anthocyanin accumulation and root growth inhibition assays, we show that the osu1 mutants are more sensitive than wild-type to both of the imbalanced C/N conditions, high C/low N and low C/high N. However, under the balanced C/N conditions (low C/low N or high C/high N), the osu1 mutants have similar anthocyanin levels and root lengths as wild-type. Consistently, the genes encoding two MYB transcription factors (MYB75 and MYB90) and an Asn synthetase isoform (ASN1) are strongly up-regulated by the OSU1 mutation in response to high C/low N and low C/high N, respectively. Furthermore, the enhanced sensitivity of osu1-1 to high C/low N with respect to anthocyanin accumulation but not root growth inhibition can be suppressed by co-suppression of MYB75, indicating that MYB75 acts downstream of OSU1 in the high C/low N imbalance response. Map-based cloning reveals that OSU1 encodes a member of a large family of putative methyltransferases and is allelic to the recently reported QUA2/TSD2 locus identified in genetic screens for cell-adhesion-defective mutants. Accumulation of OSU1/QUA2/TSD2 transcript was not regulated by C and N balance, but the OSU1 promoter was slightly more active in the vascular system. Taken together, our results show that the OSU1/QUA2/TSD2-encoded putative methyltransferase is required for normal C/N nutrient balance response in plants. PMID:18167546

Overexpression of a partial fragment of the salt-responsive gene OsNUC1 enhances salt adaptation in transgenic Arabidopsis thaliana and rice (Oryza sativa L.) during salt stress.

PubMed

Sripinyowanich, Siriporn; Chamnanmanoontham, Nontalee; Udomchalothorn, Thanikarn; Maneeprasopsuk, Somporn; Santawee, Panudda; Buaboocha, Teerapong; Qu, Li-Jia; Gu, Hongya; Chadchawan, Supachitra

2013-12-01

The rice (Oryza sativa L.) nucleolin gene, OsNUC1, transcripts were expressed in rice leaves, flowers, seeds and roots but differentially expressed within and between two pairs of salt-sensitive and salt-resistant rice lines when subjected to salt stress. Salt-resistant lines exhibited higher OsNUC1 transcript expression levels than salt-sensitive lines during 0.5% (w/v) NaCl salt stress for 6d. Two sizes of OsNUC1 full-length cDNA were found in the rice genome database and northern blot analysis confirmed their existence in rice tissues. The longer transcript (OsNUC1-L) putatively encodes for a protein with a serine rich N-terminal, RNA recognition motifs in the central domain and a glycine- and arginine-rich repeat in the C-terminal domain, while the shorter one (OsNUC1-S) putatively encodes for the similar protein without the N-terminus. Without salt stress, OsNUC1-L expressing Arabidopsis thaliana Atnuc1-L1 plants displayed a substantial but incomplete revertant phenotype, whereas OsNUC1-S expression only induced a weak effect. However, under 0.5% (w/v) NaCl salt stress they displayed a higher relative growth rate, longer root length and a lower H2O2 level than the wild type plants, suggesting a higher salt resistance. Moreover, they displayed elevated AtSOS1 and AtP5CS1 transcript levels. We propose that OsNUC1-S plays an important role in salt resistance during salt stress, a new role for nucleolin in plants. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

PubMed

Qin, Jin-Hong; Zhang, Qing; Zhang, Zhi-Ming; Zhong, Yi; Yang, Yang; Hu, Bao-Yu; Zhao, Guo-Ping; Guo, Xiao-Kui

2008-06-01

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

Differential gene expression profiles in the venom gland/sac of Eumenes pomiformis (Hymenoptera: Eumenidae).

PubMed

Baek, Ji Hyeong; Lee, Si Hyeock

2010-06-01

To search for novel transcripts encoding biologically active venom components, a subtractive cDNA library specific to the venom gland and sac (gland/sac) of a solitary hunting wasp species, Eumenes pomiformis Fabricius (1781), was constructed by suppression subtractive hybridization. A total of 541 expressed sequence tags (ESTs) were clustered and assembled into 102 contigs (31 multiple sequences and 71 singletons). In total, 37 cDNAs were found in the library via BLASTx searching and manual annotation. Eight contigs (337 ESTs) encoding short venom peptides (10 to 16 amino acids) occupied 62% of the library. The deduced amino acid sequence (78 amino acids) of a novel venom peptide transcript shared sequence similarity with trypsin inhibitors and dendrotoxin-like venom peptides known to be K(+) channel blockers, implying that this novel peptide may play a role in the paralysis of prey. In addition to phospholipase A2 and hyaluronidase, which are known to be the main components of wasp venoms, several transcripts encoding enzymes, including three metallopeptidases and a decarboxylase likely involved in the processing and activation of venomous proteins, peptides, amines, and neurotransmitters, were also isolated from the library. The presence of a transcript encoding a putative insulin/insulin-like peptide binding protein suggests that solitary hunting wasps use their venom to control their prey, leading to larval growth cessation. The abundance of these venom components in the venom gland/sac and in the alimentary canal was confirmed by quantitative real-time PCR. Discovery of venom gland/sac-specific transcripts should promote further studies on biologically active components in the venom of solitary hunting wasps. Copyright 2010 Elsevier Ltd. All rights reserved.

Cloning and characterization of a novel zinc finger gene in Xp11.2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Derry, J.M.J.; Jess, U.; Francke, U.

1995-11-20

During a systematic search for open reading frames in chromosome band Xp11.2, a novel gene (ZNF157) that encodes a putative 506-amino-acid protein with the sequence characteristics of a zinc-finger-containing transcription factor was isolated. ZNF157 is encoded by four exons distributed over >20 kb of genomic DNA. The second and third exons contain sequences similar to those of the previously described KRAB-A and KRAB-B domains, motifs that have been shown to mediate transcriptional repression in other members of the protein family. A fourth exon contains 12 zinc finger DNA binding motifs and finger linking regions characteristic of ZNF proteins of themore » Krueppel family. ZNF157 maps to the telomeric end of a cluster of ZNF genes that includes ZNF21, ZNF41, and ZNF81. 19 refs., 2 figs.« less

MHY1 Encodes a C2H2-Type Zinc Finger Protein That Promotes Dimorphic Transition in the Yeast Yarrowia lipolytica

PubMed Central

Hurtado, Cleofe A. R.; Rachubinski, Richard A.

1999-01-01

The yeast-to-hypha morphological transition (dimorphism) is typical of many pathogenic fungi. Dimorphism has been attributed to changes in temperature and nutritional status and is believed to constitute a mechanism of response to adverse conditions. We have isolated and characterized a gene, MHY1, whose transcription is dramatically increased during the yeast-to-hypha transition in Yarrowia lipolytica. Deletion of MHY1 is viable and has no effect on mating, but it does result in a complete inability of cells to undergo mycelial growth. MHY1 encodes a C2H2-type zinc finger protein, Mhy1p, which can bind putative cis-acting DNA stress response elements, suggesting that Mhy1p may act as a transcription factor. Interestingly, Mhy1p tagged with a hemagglutinin epitope was concentrated in the nuclei of actively growing cells found at the hyphal tip. PMID:10322005

PigZ, a TetR/AcrR family repressor, modulates secondary metabolism via the expression of a putative four-component resistance-nodulation-cell-division efflux pump, ZrpADBC, in Serratia sp. ATCC 39006.

PubMed

Gristwood, Tamzin; Fineran, Peter C; Everson, Lee; Salmond, George P C

2008-07-01

The Gram-negative enterobacterium, Serratia sp. ATCC 39006 synthesizes several secondary metabolites, including prodigiosin (Pig) and a carbapenem antibiotic (Car). A complex hierarchical network of regulatory proteins control Pig and Car production. In this study we characterize a TetR family regulator, PigZ, which represses transcription of a divergently transcribed putative resistance-nodulation-cell-division (RND) efflux pump, encoded by zrp (PigZ repressed pump) ADBC, via direct binding to the zrpA-pigZ intergenic region. Unusually, this putative RND pump contains two predicted membrane fusion proteins (MFPs), ZrpA and ZrpD. A mutation in pigZ resulted in multiple phenotypic changes, including exoenzyme production, motility and differential regulation of Pig and Car production. A polar suppressor mutation, within zrpA, restored all tested phenotypes to parental strain levels, indicating that the changes observed are due to the increase in expression of ZrpADBC in the absence of the repressor, PigZ. Genomic deletions of zrpA and zrpD indicate that the MFP ZrpD, but not ZrpA, is essential for activity of the putative pump. Bioinformatic analysis revealed that putative RND efflux pumps encoding two MFP components are not uncommon, particularly among plant-associated, Gram-negative bacteria. In addition, based on phylogenetic analysis, we propose that these pairs of MFPs consist of two distinct subtypes.

Putative Porin of Bradyrhizobium sp. (Lupinus) Bacteroids Induced by Glyphosate▿

PubMed Central

de María, Nuria; Guevara, Ángeles; Serra, M. Teresa; García-Luque, Isabel; González-Sama, Alfonso; de Lacoba, Mario García; de Felipe, M. Rosario; Fernández-Pascual, Mercedes

2007-01-01

Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed. PMID:17557843

Analyses of advanced rice anther transcriptomes reveal global tapetum secretory functions and potential proteins for lipid exine formation.

PubMed

Huang, Ming-Der; Wei, Fu-Jin; Wu, Cheng-Cheih; Hsing, Yue-Ie Caroline; Huang, Anthony H C

2009-02-01

The anthers in flowers perform important functions in sexual reproduction. Several recent studies used microarrays to study anther transcriptomes to explore genes controlling anther development. To analyze the secretion and other functions of the tapetum, we produced transcriptomes of anthers of rice (Oryza sativa subsp. japonica) at six progressive developmental stages and pollen with sequencing-by-synthesis technology. The transcriptomes included at least 18,000 unique transcripts, about 25% of which had antisense transcripts. In silico anther-minus-pollen subtraction produced transcripts largely unique to the tapetum; these transcripts include all the reported tapetum-specific transcripts of orthologs in other species. The differential developmental profiles of the transcripts and their antisense transcripts signify extensive regulation of gene expression in the anther, especially the tapetum, during development. The transcriptomes were used to dissect two major cell/biochemical functions of the tapetum. First, we categorized and charted the developmental profiles of all transcripts encoding secretory proteins present in the cellular exterior; these transcripts represent about 12% and 30% of the those transcripts having more than 100 and 1,000 transcripts per million, respectively. Second, we successfully selected from hundreds of transcripts several transcripts encoding potential proteins for lipid exine synthesis during early anther development. These proteins include cytochrome P450, acyltransferases, and lipid transfer proteins in our hypothesized mechanism of exine synthesis in and export from the tapetum. Putative functioning of these proteins in exine formation is consistent with proteins and metabolites detected in the anther locule fluid obtained by micropipetting.

Nucleotide sequence and transcriptional start site of the Methylobacterium organophilum XX methanol dehydrogenase structural gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Machlin, S.M.; Hanson, R.S.

The nucleotide sequence of a cloned 2.5-kilobase-pair SmaI fragment containing the methanol dehydrogenase (MDH) structural gene from Methylobacterium organophilum XX was determined. A single open reading frame with a coding capacity of 626 amino acids (molecular weight, 66,000) was identified on one stand, and N-terminal sequencing of purified MDH revealed that 27 of these residues constituted a putative signal peptide. Primer extension mapping of in vivo transcripts indicated that the start of mRNA synthesis was 160 to 170 base pairs upstream of the ATG codon. Northern (RNA) blot analysis further demonstrated that the transcript was 2.1 kilobase pairs in lengthmore » and therefore appeared to encode only MDH.« less

A deep learning method for lincRNA detection using auto-encoder algorithm.

PubMed

Yu, Ning; Yu, Zeng; Pan, Yi

2017-12-06

RNA sequencing technique (RNA-seq) enables scientists to develop novel data-driven methods for discovering more unidentified lincRNAs. Meantime, knowledge-based technologies are experiencing a potential revolution ignited by the new deep learning methods. By scanning the newly found data set from RNA-seq, scientists have found that: (1) the expression of lincRNAs appears to be regulated, that is, the relevance exists along the DNA sequences; (2) lincRNAs contain some conversed patterns/motifs tethered together by non-conserved regions. The two evidences give the reasoning for adopting knowledge-based deep learning methods in lincRNA detection. Similar to coding region transcription, non-coding regions are split at transcriptional sites. However, regulatory RNAs rather than message RNAs are generated. That is, the transcribed RNAs participate the biological process as regulatory units instead of generating proteins. Identifying these transcriptional regions from non-coding regions is the first step towards lincRNA recognition. The auto-encoder method achieves 100% and 92.4% prediction accuracy on transcription sites over the putative data sets. The experimental results also show the excellent performance of predictive deep neural network on the lincRNA data sets compared with support vector machine and traditional neural network. In addition, it is validated through the newly discovered lincRNA data set and one unreported transcription site is found by feeding the whole annotated sequences through the deep learning machine, which indicates that deep learning method has the extensive ability for lincRNA prediction. The transcriptional sequences of lincRNAs are collected from the annotated human DNA genome data. Subsequently, a two-layer deep neural network is developed for the lincRNA detection, which adopts the auto-encoder algorithm and utilizes different encoding schemes to obtain the best performance over intergenic DNA sequence data. Driven by those newly annotated lincRNA data, deep learning methods based on auto-encoder algorithm can exert their capability in knowledge learning in order to capture the useful features and the information correlation along DNA genome sequences for lincRNA detection. As our knowledge, this is the first application to adopt the deep learning techniques for identifying lincRNA transcription sequences.

Olive phenolic compounds: metabolic and transcriptional profiling during fruit development

PubMed Central

2012-01-01

Background Olive (Olea europaea L.) fruits contain numerous secondary metabolites, primarily phenolics, terpenes and sterols, some of which are particularly interesting for their nutraceutical properties. This study will attempt to provide further insight into the profile of olive phenolic compounds during fruit development and to identify the major genetic determinants of phenolic metabolism. Results The concentration of the major phenolic compounds, such as oleuropein, demethyloleuropein, 3–4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans, were measured in the developing fruits of 12 olive cultivars. The content of these compounds varied significantly among the cultivars and decreased during fruit development and maturation, with some compounds showing specificity for certain cultivars. Thirty-five olive transcripts homologous to genes involved in the pathways of the main secondary metabolites were identified from the massive sequencing data of the olive fruit transcriptome or from cDNA-AFLP analysis. Their mRNA levels were determined using RT-qPCR analysis on fruits of high- and low-phenolic varieties (Coratina and Dolce d’Andria, respectively) during three different fruit developmental stages. A strong correlation was observed between phenolic compound concentrations and transcripts putatively involved in their biosynthesis, suggesting a transcriptional regulation of the corresponding pathways. OeDXS, OeGES, OeGE10H and OeADH, encoding putative 1-deoxy-D-xylulose-5-P synthase, geraniol synthase, geraniol 10-hydroxylase and arogenate dehydrogenase, respectively, were almost exclusively present at 45 days after flowering (DAF), suggesting that these compounds might play a key role in regulating secoiridoid accumulation during fruit development. Conclusions Metabolic and transcriptional profiling led to the identification of some major players putatively involved in biosynthesis of secondary compounds in the olive tree. Our data represent the first step towards the functional characterisation of important genes for the determination of olive fruit quality. PMID:22963618

Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project.

PubMed

Gerstein, Mark B; Lu, Zhi John; Van Nostrand, Eric L; Cheng, Chao; Arshinoff, Bradley I; Liu, Tao; Yip, Kevin Y; Robilotto, Rebecca; Rechtsteiner, Andreas; Ikegami, Kohta; Alves, Pedro; Chateigner, Aurelien; Perry, Marc; Morris, Mitzi; Auerbach, Raymond K; Feng, Xin; Leng, Jing; Vielle, Anne; Niu, Wei; Rhrissorrakrai, Kahn; Agarwal, Ashish; Alexander, Roger P; Barber, Galt; Brdlik, Cathleen M; Brennan, Jennifer; Brouillet, Jeremy Jean; Carr, Adrian; Cheung, Ming-Sin; Clawson, Hiram; Contrino, Sergio; Dannenberg, Luke O; Dernburg, Abby F; Desai, Arshad; Dick, Lindsay; Dosé, Andréa C; Du, Jiang; Egelhofer, Thea; Ercan, Sevinc; Euskirchen, Ghia; Ewing, Brent; Feingold, Elise A; Gassmann, Reto; Good, Peter J; Green, Phil; Gullier, Francois; Gutwein, Michelle; Guyer, Mark S; Habegger, Lukas; Han, Ting; Henikoff, Jorja G; Henz, Stefan R; Hinrichs, Angie; Holster, Heather; Hyman, Tony; Iniguez, A Leo; Janette, Judith; Jensen, Morten; Kato, Masaomi; Kent, W James; Kephart, Ellen; Khivansara, Vishal; Khurana, Ekta; Kim, John K; Kolasinska-Zwierz, Paulina; Lai, Eric C; Latorre, Isabel; Leahey, Amber; Lewis, Suzanna; Lloyd, Paul; Lochovsky, Lucas; Lowdon, Rebecca F; Lubling, Yaniv; Lyne, Rachel; MacCoss, Michael; Mackowiak, Sebastian D; Mangone, Marco; McKay, Sheldon; Mecenas, Desirea; Merrihew, Gennifer; Miller, David M; Muroyama, Andrew; Murray, John I; Ooi, Siew-Loon; Pham, Hoang; Phippen, Taryn; Preston, Elicia A; Rajewsky, Nikolaus; Rätsch, Gunnar; Rosenbaum, Heidi; Rozowsky, Joel; Rutherford, Kim; Ruzanov, Peter; Sarov, Mihail; Sasidharan, Rajkumar; Sboner, Andrea; Scheid, Paul; Segal, Eran; Shin, Hyunjin; Shou, Chong; Slack, Frank J; Slightam, Cindie; Smith, Richard; Spencer, William C; Stinson, E O; Taing, Scott; Takasaki, Teruaki; Vafeados, Dionne; Voronina, Ksenia; Wang, Guilin; Washington, Nicole L; Whittle, Christina M; Wu, Beijing; Yan, Koon-Kiu; Zeller, Georg; Zha, Zheng; Zhong, Mei; Zhou, Xingliang; Ahringer, Julie; Strome, Susan; Gunsalus, Kristin C; Micklem, Gos; Liu, X Shirley; Reinke, Valerie; Kim, Stuart K; Hillier, LaDeana W; Henikoff, Steven; Piano, Fabio; Snyder, Michael; Stein, Lincoln; Lieb, Jason D; Waterston, Robert H

2010-12-24

We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor-binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor-binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

Ovarian-specific expression of a new gene regulated by the goat PIS region and transcribed by a FOXL2 bidirectional promoter.

PubMed

Pannetier, Maëlle; Renault, Lauriane; Jolivet, Geneviève; Cotinot, Corinne; Pailhoux, Eric

2005-06-01

Studies on XX sex reversal in polled goats (PIS mutation: polled intersex syndrome) have led to the discovery of a female-specific locus crucial for ovarian differentiation. This genomic region is composed of at least two genes, FOXL2 and PISRT1, sharing a common transcriptional regulatory region, PIS. In this paper, we describe a third gene, PFOXic (promoter FOXL2 inverse complementary), located near FOXL2 in the opposite orientation. This gene composed of five exons encodes a 1723-bp cDNA, enclosing two repetitive elements in its 3' end. PFOXic mRNA encodes a putative protein of 163 amino acids with no homologies in any of the databases tested. The transcriptional expression of PFOXic is driven by a bidirectional promoter also enhancing FOXL2 transcription. In goats, PFOXic is expressed in developing ovaries, from 36 days postcoitum until adulthood. Ovarian-specific expression of PFOXic is regulated by the PIS region. PFOXic is found conserved only in Bovidae. But, a human gene located in the opposite orientation relative to FOXL2 can be considered a human PFOXic. Finally, we discuss evidence arguing for regulation of the level of FOXL2 transcription via the bidirectional promoter and the level of transcription of PFOXic.

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

PubMed

Reid, S D; Green, N M; Buss, J K; Lei, B; Musser, J M

2001-06-19

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

Transcriptional Reprogramming of the Mycoparasitic Fungus Ampelomyces quisqualis During the Powdery Mildew Host-Induced Germination.

PubMed

Siozios, Stefanos; Tosi, Lorenzo; Ferrarini, Alberto; Ferrari, Alessandro; Tononi, Paola; Bellin, Diana; Maurhofer, Monika; Gessler, Cesare; Delledonne, Massimo; Pertot, Ilaria

2015-02-01

Ampelomyces quisqualis is a mycoparasite of a diverse range of phytopathogenic fungi associated with the powdery mildew disease. Among them are several Erysiphaceae species with great economic impact on high-value crops such as grape. Due to its ability to parasitize and prevent the spread of powdery mildews, A. quisqualis has received considerable attention for its biocontrol potential. However, and in sharp contrast to the extensively studied biocontrol species belonging to the genus Trichoderma, little is known about the biology of A. quisqualis at the molecular and genetic levels. We present the first genome-wide transcription profiling in A. quisqualis during host-induced germination. A total of 1,536 putative genes showed significant changes in transcription during the germination of A. quisqualis. This finding denotes an extensive transcriptional reprogramming of A. quisqualis induced by the presence of the host. Several upregulated genes were predicted to encode for putative mycoparasitism-related proteins such as secreted proteases, virulence factors, and proteins related to toxin biosynthesis. Our data provide the most comprehensive sequence resource currently available for A. quisqualis in addition to offering valuable insights into the biology of A. quisqualis and its mycoparasitic lifestyle. Eventually, this may improve the biocontrol capacity of this mycoparasite.

A transferrin gene associated with development and 2-tridecanone tolerance in Helicoverpa armigera

PubMed Central

Zhang, L; Shang, Q; Lu, Y; Zhao, Q; Gao, X

2015-01-01

The full-length cDNA (2320 bp) encoding a putative iron-binding transferrin protein from Helicoverpa armigera was cloned and named HaTrf. The putative HaTrf sequence included 670 amino acids with a molecular mass of approximately 76 kDa. Quantitative PCR results demonstrated that the transcriptional level of HaTrf was significantly higher in the sixth instar and pupa stages as compared with other developmental stages. HaTrf transcripts were more abundant in fat bodies and in the epidermis than in malpighian tubules. Compared with the control, the expression of HaTrf increased dramatically 24 h after treatment with 2-tridecanone. Apparent growth inhibition with a dramatic body weight decrease was observed in larvae fed with HaTrf double-stranded RNA (dsRNA), as compared with those fed with green fluorescent protein dsRNA. RNA interference of HaTrf also significantly increased the susceptibility of larvae to 2-tridecanone. These results indicate the possible involvement of HaTrf in tolerance to plant secondary chemicals. PMID:25430818

Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

PubMed Central

Khairy, Heba; Wübbeler, Jan Hendrik

2015-01-01

Four Rhodococcus spp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. Because Rhodococcus erythropolis strain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis of R. erythropolis MI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity of noxR and nox. The interruption mutant generated, R. erythropolis MI2 noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently, nox was overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB). PMID:26407888

Structural and transcriptional characterization of a novel member of the soybean urease gene family.

PubMed

Wiebke-Strohm, Beatriz; Ligabue-Braun, Rodrigo; Rechenmacher, Ciliana; De Oliveira-Busatto, Luisa Abruzzi; Carlini, Célia Regina; Bodanese-Zanettini, Maria Helena

2016-04-01

In plants, ureases have been related to urea degradation, to defense against pathogenic fungi and phytophagous insects, and to the soybean-Bradyrhizobium japonicum symbiosis. Two urease isoforms have been described for soybean: the embryo-specific, encoded by Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding locus exists in the completed soybean genome. The gene was designated Eu5 and the putative product of its ORF as SBU-III. Phylogenetic analysis shows that 41 plant, moss and algal ureases have diverged from a common ancestor protein, but ureases from monocots, eudicots and ancient species have evolved independently. Genomes of ancient organisms present a single urease-encoding gene and urease-encoding gene duplication has occurred independently along the evolution of some eudicot species. SBU-III has a shorter amino acid sequence, since many gaps are found when compared to other sequences. A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity, but the overall protein architecture remains very similar to the other ureases. The expression profile of urease-encoding genes in different organs and developmental stages was determined by RT-qPCR. Eu5 transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Eu1 and Eu4 transcripts were found in all analyzed organs, but Eu4 expression was more prominent in seeds one day after dormancy break whereas Eu1 predominated in developing seeds. The evidence suggests that SBU-III may not be involved in nitrogen availability to plants, but it could be involved in other biological role(s). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A Deeper Examination of Thorellius atrox Scorpion Venom Components with Omic Techonologies

PubMed Central

Romero-Gutierrez, Teresa; Batista, Cesar V. F.

2017-01-01

This communication reports a further examination of venom gland transcripts and venom composition of the Mexican scorpion Thorellius atrox using RNA-seq and tandem mass spectrometry. The RNA-seq, which was performed with the Illumina protocol, yielded more than 20,000 assembled transcripts. Following a database search and annotation strategy, 160 transcripts were identified, potentially coding for venom components. A novel sequence was identified that potentially codes for a peptide with similarity to spider ω-agatoxins, which act on voltage-gated calcium channels, not known before to exist in scorpion venoms. Analogous transcripts were found in other scorpion species. They could represent members of a new scorpion toxin family, here named omegascorpins. The mass fingerprint by LC-MS identified 135 individual venom components, five of which matched with the theoretical masses of putative peptides translated from the transcriptome. The LC-MS/MS de novo sequencing allowed to reconstruct and identify 42 proteins encoded by assembled transcripts, thus validating the transcriptome analysis. Earlier studies conducted with this scorpion venom permitted the identification of only twenty putative venom components. The present work performed with more powerful and modern omic technologies demonstrates the capacity of accomplishing a deeper characterization of scorpion venom components and the identification of novel molecules with potential applications in biomedicine and the study of ion channel physiology. PMID:29231872

Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta.

PubMed

Zhu, Yu-Cheng; Specht, Charles A; Dittmer, Neal T; Muthukrishnan, Subbaratnam; Kanost, Michael R; Kramer, Karl J

2002-11-01

Glycosyltransferases are enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. One type of glycosyltransferase is chitin synthase, a very important enzyme in biology, which is utilized by insects, fungi, and other invertebrates to produce chitin, a polysaccharide of beta-1,4-linked N-acetylglucosamine. Chitin is an important component of the insect's exoskeletal cuticle and gut lining. To identify and characterize a chitin synthase gene of the tobacco hornworm, Manduca sexta, degenerate primers were designed from two highly conserved regions in fungal and nematode chitin synthase protein sequences and then used to amplify a similar region from Manduca cDNA. A full-length cDNA of 5152 nucleotides was assembled for the putative Manduca chitin synthase gene, MsCHS1, and sequencing of genomic DNA verified the contiguity of the sequence. The MsCHS1 cDNA has an ORF of 4692 nucleotides that encodes a transmembrane protein of 1564 amino acid residues with a mass of approximately 179 kDa (GenBank no. AY062175). It is most similar, over its entire length of protein sequence, to putative chitin synthases from other insects and nematodes, with 68% identity to enzymes from both the blow fly, Lucilia cuprina, and the fruit fly, Drosophila melanogaster. The similarity with fungal chitin synthases is restricted to the putative catalytic domain, and the MsCHS1 protein has, at equivalent positions, several amino acids that are essential for activity as revealed by mutagenesis of the fungal enzymes. A 5.3-kb transcript of MsCHS1 was identified by northern blot hybridization of RNA from larval epidermis, suggesting that the enzyme functions to make chitin deposited in the cuticle. Further examination by RT-PCR showed that MsCHS1 expression is regulated in the epidermis, with the amount of transcript increasing during phases of cuticle deposition.

Identification of Putative Olfactory Genes from the Oriental Fruit Moth Grapholita molesta via an Antennal Transcriptome Analysis

PubMed Central

Li, Yiping; Wu, Junxiang

2015-01-01

Background The oriental fruit moth, Grapholita molesta, is an extremely important oligophagous pest species of stone and pome fruits throughout the world. As a host-switching species, adult moths, especially females, depend on olfactory cues to a large extent in locating host plants, finding mates, and selecting oviposition sites. The identification of olfactory genes can facilitate investigation on mechanisms for chemical communications. Methodology/Principal Finding We generated transcriptome of female antennae of G.molesta using the next-generation sequencing technique, and assembled transcripts from RNA-seq reads using Trinity, SOAPdenovo-trans and Abyss-trans assemblers. We identified 124 putative olfactory genes. Among the identified olfactory genes, 118 were novel to this species, including 28 transcripts encoding for odorant binding proteins, 17 chemosensory proteins, 48 odorant receptors, four gustatory receptors, 24 ionotropic receptors, two sensory neuron membrane proteins, and one odor degrading enzyme. The identified genes were further confirmed through semi-quantitative reverse transcription PCR for transcripts coding for 26 OBPs and 17 CSPs. OBP transcripts showed an obvious antenna bias, whereas CSP transcripts were detected in different tissues. Conclusion Antennal transcriptome data derived from the oriental fruit moth constituted an abundant molecular resource for the identification of genes potentially involved in the olfaction process of the species. This study provides a foundation for future research on the molecules involved in olfactory recognition of this insect pest, and in particular, the feasibility of using semiochemicals to control this pest. PMID:26540284

Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

PubMed

van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

2016-01-01

RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

Transcriptome and gene expression analysis during flower blooming in Rosa chinensis 'Pallida'.

PubMed

Yan, Huijun; Zhang, Hao; Chen, Min; Jian, Hongying; Baudino, Sylvie; Caissard, Jean-Claude; Bendahmane, Mohammed; Li, Shubin; Zhang, Ting; Zhou, Ningning; Qiu, Xianqin; Wang, Qigang; Tang, Kaixue

2014-04-25

Rosa chinensis 'Pallida' (Rosa L.) is one of the most important ancient rose cultivars originating from China. It contributed the 'tea scent' trait to modern roses. However, little information is available on the gene regulatory networks involved in scent biosynthesis and metabolism in Rosa. In this study, the transcriptome of R. chinensis 'Pallida' petals at different developmental stages, from flower buds to senescent flowers, was investigated using Illumina sequencing technology. De novo assembly generated 89,614 clusters with an average length of 428bp. Based on sequence similarity search with known proteins, 62.9% of total clusters were annotated. Out of these annotated transcripts, 25,705 and 37,159 sequences were assigned to gene ontology and clusters of orthologous groups, respectively. The dataset provides information on transcripts putatively associated with known scent metabolic pathways. Digital gene expression (DGE) was obtained using RNA samples from flower bud, open flower and senescent flower stages. Comparative DGE and quantitative real time PCR permitted the identification of five transcripts encoding proteins putatively associated with scent biosynthesis in roses. The study provides a foundation for scent-related gene discovery in roses. Copyright © 2014. Published by Elsevier B.V.

White collar 2, a partner in blue-light signal transduction, controlling expression of light-regulated genes in Neurospora crassa.

PubMed Central

Linden, H; Macino, G

1997-01-01

A saturating genetic dissection of 'blind' mutants in Neurospora crassa has identified a total of two non-redundant loci (wc-1 and wc-2) each of which is required for blue-light perception/signal transduction. Previously, we demonstrated that WC1 is a putative zinc finger transcription factor able to bind specifically to a light-regulated promoter. Here, we present the cloning and characterization of the wc-2 gene. We demonstrate using mutation analysis and in vitro DNA-binding assays that WC2, the second partner of this light signal transduction system, encodes a functional zinc finger DNA-binding protein with putative PAS dimerization and transcription activation domains. This molecular genetic dissection of the second of two components of this light signal transduction system has enabled us to devise a model whereby WC1 and WC2 are proposed to interact via homologous PAS domains, bind to promoters of light-regulated genes and activate transcription. As such, this study provides the first insight into two co-operating partners in blue-light signal transduction in any organism and describes the molecular tools with which to dissect this enigmatic process. PMID:9009271

Transcriptional Regulation and Characterization of a Novel β-Fructofuranosidase-Encoding Gene from Bifidobacterium breve UCC2003

PubMed Central

Ryan, Sinéad M.; Fitzgerald, Gerald F.; van Sinderen, Douwe

2005-01-01

An operon involved in fructooligosaccharide breakdown was identified in the genome of Bifidobacterium breve UCC2003. This 2.6-kb transcriptional unit was comprised of three genes that encoded a putative permease, a conserved hypothetical protein, and a β-fructofuranosidase. Active transcription of the operon was observed when B. breve UCC2003 was grown on sucrose or Actilight, while transcription appeared to be repressed when the organism was grown on glucose, fructose, a combination of glucose and sucrose, or a combination of fructose and sucrose. The β-fructofuranosidase encoded by this operon was purified and biochemically characterized. The optimum pH and temperature for catalytic activity were determined to be pH 6.0 and 37°C, respectively, and there was a dependence on bivalent cations, particularly manganese. The Km and Vmax values for sucrose hydrolysis were determined to be 25 ± 2 mM and 24 ± 3 μmol min−1 mg−1, respectively. Interestingly, the enzyme was shown to specifically catalyze cleavage of the β(2-1) glycosidic bond between glucose and its neighboring fructose moiety in sucrose and other fructooligosaccharides with a relatively low degree of polymerization, and there was no detectable activity towards the β(2-1) glycosidic bond between two fructose moieties within the same substrate. To our knowledge, such an enzymatic activity has not previously been described in bifidobacteria or other gram-positive bacteria. PMID:16000751

Regulation of Lactobacillus casei Sorbitol Utilization Genes Requires DNA-Binding Transcriptional Activator GutR and the Conserved Protein GutM▿

PubMed Central

Alcántara, Cristina; Sarmiento-Rubiano, Luz Adriana; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar; Yebra, María J.

2008-01-01

Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encoding Lactobacillus casei gutF gene revealed that they constitute a sorbitol (glucitol) utilization operon. The gutRM genes encode putative regulators, while the gutCBA genes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTSGut). The gut operon is transcribed as a polycistronic gutFRMCBA messenger, the expression of which is induced by sorbitol and repressed by glucose. gutR encodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIBGat domain) and a mannitol/fructose-specific EIIA-like domain (EIIAMtl domain). Its inactivation abolished gut operon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying a gutB mutation expressed the gut operon constitutively, but they failed to transport sorbitol, indicating that EIIBCGut negatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from the gut promoter. A sequence comparison with the presumed promoter region of gut operons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of the L. casei gut operon is therefore likely to be operative in other firmicutes. Finally, gutM codes for a conserved protein of unknown function present in all sequenced gut operons. A gutM mutant, the first constructed in a firmicute, showed drastically reduced gut operon expression and sorbitol uptake, indicating a regulatory role also for GutM. PMID:18676710

Characterization and mapping of cDNA encoding aspartate aminotransferase in rice, Oryza sativa L.

PubMed

Song, J; Yamamoto, K; Shomura, A; Yano, M; Minobe, Y; Sasaki, T

1996-10-31

Fifteen cDNA clones, putatively identified as encoding aspartate aminotransferase (AST, EC 2.6.1.1.), were isolated and partially sequenced. Together with six previously isolated clones putatively identified to encode ASTs (Sasaki, et al. 1994, Plant Journal 6, 615-624), their sequences were characterized and classified into 4 cDNA species. Two of the isolated clones, C60213 and C2079, were full-length cDNAs, and their complete nucleotide sequences were determined. C60213 was 1612 bp long and its deduced amino acid sequence showed 88% homology with that of Panicum miliaceum L. mitochondrial AST. The C60213-encoded protein had an N-terminal amino acid sequence that was characteristic of a mitochondrial transit peptide. On the other hand, C2079 was 1546 bp long and had 91% amino acid sequence homology with P. miliaceum L. cytosolic AST but lacked in the transit peptide sequence. The homologies of nucleotide sequences and deduced amino acid sequences of C2079 and C60213 were 54% and 52%, respectively. C2079 and C60213 were mapped on chromosomes 1 and 6, respectively, by restriction fragment length polymorphism linkage analysis. Northern blot analysis using C2079 as a probe revealed much higher transcript levels in callus and root than in green and etiolated shoots, suggesting tissue-specific variations of AST gene expression.

Expression profiles of putative defence-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense.

PubMed

Tan, Yung-Chie; Yeoh, Keat-Ai; Wong, Mui-Yun; Ho, Chai-Ling

2013-11-01

Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection. Copyright © 2013 Elsevier GmbH. All rights reserved.

Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

PubMed Central

Pickl, Andreas; Johnsen, Ulrike

2012-01-01

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea. PMID:22493022

Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.

PubMed

Jin, Zhehao; Kim, Jin-Hee; Park, Sang Un; Kim, Soo-Un

2016-12-01

Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein.

PubMed Central

Ballario, P; Vittorioso, P; Magrelli, A; Talora, C; Cabibbo, A; Macino, G

1996-01-01

The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation. Images PMID:8612589

Type 3 fimbriae and biofilm formation are regulated by the transcriptional regulators MrkHI in Klebsiella pneumoniae.

PubMed

Johnson, Jeremiah G; Murphy, Caitlin N; Sippy, Jean; Johnson, Tylor J; Clegg, Steven

2011-07-01

Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production. One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate type 3 fimbrial expression.

Type 3 Fimbriae and Biofilm Formation Are Regulated by the Transcriptional Regulators MrkHI in Klebsiella pneumoniae▿

PubMed Central

Johnson, Jeremiah G.; Murphy, Caitlin N.; Sippy, Jean; Johnson, Tylor J.; Clegg, Steven

2011-01-01

Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production. One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate type 3 fimbrial expression. PMID:21571997

Identification of differentially expressed small non-coding RNAs in the legume endosymbiont Sinorhizobium meliloti by comparative genomics

PubMed Central

del Val, Coral; Rivas, Elena; Torres-Quesada, Omar; Toro, Nicolás; Jiménez-Zurdo, José I

2007-01-01

Bacterial small non-coding RNAs (sRNAs) are being recognized as novel widespread regulators of gene expression in response to environmental signals. Here, we present the first search for sRNA-encoding genes in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, performed by a genome-wide computational analysis of its intergenic regions. Comparative sequence data from eight related α-proteobacteria were obtained, and the interspecies pairwise alignments were scored with the programs eQRNA and RNAz as complementary predictive tools to identify conserved and stable secondary structures corresponding to putative non-coding RNAs. Northern experiments confirmed that eight of the predicted loci, selected among the original 32 candidates as most probable sRNA genes, expressed small transcripts. This result supports the combined use of eQRNA and RNAz as a robust strategy to identify novel sRNAs in bacteria. Furthermore, seven of the transcripts accumulated differentially in free-living and symbiotic conditions. Experimental mapping of the 5′-ends of the detected transcripts revealed that their encoding genes are organized in autonomous transcription units with recognizable promoter and, in most cases, termination signatures. These findings suggest novel regulatory functions for sRNAs related to the interactions of α-proteobacteria with their eukaryotic hosts. PMID:17971083

The Zur regulon of Corynebacterium glutamicum ATCC 13032

PubMed Central

2010-01-01

Background Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators. Results The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays. Conclusion Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum. PMID:20055984

Expression-based clustering of CAZyme-encoding genes of Aspergillus niger.

PubMed

Gruben, Birgit S; Mäkelä, Miia R; Kowalczyk, Joanna E; Zhou, Miaomiao; Benoit-Gelber, Isabelle; De Vries, Ronald P

2017-11-23

The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX that were grown on their specific inducing compounds. The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.

Identification of the Pr1 Gene Product Completes the Anthocyanin Biosynthesis Pathway of Maize

PubMed Central

Sharma, Mandeep; Cortes-Cruz, Moises; Ahern, Kevin R.; McMullen, Michael; Brutnell, Thomas P.; Chopra, Surinder

2011-01-01

In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3′H encoding gene (Zmf3′h1) and showed by segregation analysis that the red kernel phenotype is linked to this gene. Genetic mapping using SNP markers confirms its position on chromosome 5L. Furthermore, genetic complementation experiments using a CaMV 35S::ZmF3′H1 promoter–gene construct established that the encoded protein product was sufficient to perform a 3′-hydroxylation reaction. The Zmf3′h1-specific transcripts were detected in floral and vegetative tissues of Pr1 plants and were absent in pr1. Four pr1 alleles were characterized: two carry a 24 TA dinucleotide repeat insertion in the 5′-upstream promoter region, a third has a 17-bp deletion near the TATA box, and a fourth contains a Ds insertion in exon1. Genetic and transcription assays demonstrated that the pr1 gene is under the regulatory control of anthocyanin transcription factors red1 and colorless1. The cloning and characterization of pr1 completes the molecular identification of all genes encoding structural enzymes of the anthocyanin pathway of maize. PMID:21385724

The Anaerobe-Specific Orange Protein Complex of Desulfovibrio vulgaris Hildenborough Is Encoded by Two Divergent Operons Coregulated by σ54 and a Cognate Transcriptional Regulator▿†

PubMed Central

Fiévet, Anouchka; My, Laetitia; Cascales, Eric; Ansaldi, Mireille; Pauleta, Sofia R.; Moura, Isabel; Dermoun, Zorah; Bernard, Christophe S.; Dolla, Alain; Aubert, Corinne

2011-01-01

Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the σ54-RNA polymerase. We further demonstrate that the σ54-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with σ54-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the σ70-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed. PMID:21531797

Differentially expressed microRNAs associated with changes of transcript levels in detoxification pathways and DDT-resistance in the Drosophila melanogaster strain 91-R.

PubMed

Seong, Keon Mook; Coates, Brad S; Kim, Do-Hyup; Hansen, Allison K; Pittendrigh, Barry R

2018-01-01

Dichloro-diphenyl-trichloroethane (DDT) resistance among arthropod species is a model for understanding the molecular adaptations in response to insecticide exposures. Previous studies reported that DDT resistance may involve one or multiple detoxification genes, such as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), esterases, and ATP binding cassette (ABC) transporters, or changes in the voltage-sensitive sodium channel. However, the possible involvement of microRNAs (miRNAs) in the post-transcriptional regulation of genes associated with DDT resistance in the Drosophila melanogaster strain 91-R remains poorly understood. In this study, the majority of the resulting miRNAs discovered in small RNA libraries from 91-R and the susceptible control strain, 91-C, ranged from 16-25 nt, and contained 163 precursors and 256 mature forms of previously-known miRNAs along with 17 putative novel miRNAs. Quantitative analyses predicted the differential expression of ten miRNAs between 91-R and 91-C, and, based on Gene Ontology and pathway analysis, these ten miRNAs putatively target transcripts encoding proteins involved in detoxification mechanisms. RT-qPCR validated an inverse correlation between levels of differentially-expressed miRNAs and their putatively targeted transcripts, which implies a role of these miRNAs in the differential regulation of detoxification pathways in 91-R compared to 91-C. This study provides evidence associating the differential expression of miRNAs in response to multigenerational DDT selection in Drosophila melanogaster and provides important clues for understanding the possible roles of miRNAs in mediating insecticide resistance traits.

De novo sequencing analysis of the Rosa roxburghii fruit transcriptome reveals putative ascorbate biosynthetic genes and EST-SSR markers.

PubMed

Yan, Xiuqin; Zhang, Xue; Lu, Min; He, Yong; An, Huaming

2015-04-25

Rosa roxburghii Tratt. is a well-known ornamental rose species native to China. In addition, the fruits of this species are valued for their nutritional and medicinal characteristics, especially their high ascorbic acid (AsA) levels. Nevertheless, AsA biosynthesis in R. roxburghii fruit has not been explored in detail because of a lack of genomic resources for this species. High-throughput transcriptomic sequencing generating large volumes of transcript sequence data can aid in gene discovery and molecular marker development. In this study, we generated more than 53 million clean reads using Illumina paired-end sequencing technology. De novo assembly yielded 106,590 unigenes, with an average length of 343 bp. On the basis of sequence similarity to known proteins, 9301 and 2393 unigenes were classified into Gene Ontology and Clusters of Orthologous Group categories, respectively. There were 7480 unigenes assigned to 124 pathways in the Kyoto Encyclopedia of Gene and Genome pathway database. BLASTx searches identified 498 unique putative transcripts encoding various transcription factors, some known to regulate fruit development. qRT-PCR validated the expressions of most of the genes encoding the main enzymes involved in ascorbate biosynthesis. In addition, 9131 potential simple sequence repeat (SSR) loci were identified among the unigenes. One hundred and two primer pairs were synthesized and 71 pairs produced an amplification product during initial screening. Among the amplified products, 30 were polymorphic in the 16 R. roxburghii germplasms tested. Our study was the first to produce a large volume of transcriptome data from R. roxburghii. The resulting sequence collection is a valuable resource for gene discovery and marker-assisted selective breeding in this rose species. Copyright © 2015 Elsevier B.V. All rights reserved.

Transcriptional analysis of the Escherichia coli ColV-Ia plasmid pS88 during growth in human serum and urine.

PubMed

Lemaître, Chloé; Bidet, Philippe; Bingen, Edouard; Bonacorsi, Stéphane

2012-06-21

The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulence plasmid. To identify possible genetic determinants of pS88 virulence, we examined the transcriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes, and 35 ORFs of unknown function. Quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted RNA, normalized on three housekeeping genes. The transcriptome of E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtained during growth in Luria Bertani broth, with and without iron depletion. We also analyzed the transcriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection. The transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted LB broth. Genes encoding iron acquisition systems were strongly upregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. Four ORFs were induced ex vivo, independently of the iron concentration. Other putative virulence genes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. We identify new pS88 genes potentially involved in the growth of E. coli meningitis strain S88 in human serum and urine.

Characterization of cDNA encoding molt-inhibiting hormone of the crab, Cancer pagurus; expression of MIH in non-X-organ tissues.

PubMed

Lu, W; Wainwright, G; Olohan, L A; Webster, S G; Rees, H H; Turner, P C

2001-10-31

Synthesis of ecdysteroids (molting hormones) by crustacean Y-organs is regulated by a neuropeptide, molt-inhibiting hormone (MIH), produced in eyestalk neural ganglia. We report here the molecular cloning of a cDNA encoding MIH of the edible crab, Cancer pagurus. Full-length MIH cDNA was obtained by using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides based upon the amino acid sequence of MIH, in conjunction with 5'- and 3'-RACE. Full-length clones of MIH cDNA were obtained that encoded a 35 amino acid putative signal peptide and the mature 78 amino acid peptide. Of various tissues examined by Northern blot analysis, the X-organ was the sole major site of expression of the MIH gene. However, a nested-PCR approach using non-degenerate MIH-specific primers indicated the presence of MIH transcripts in other tissues. Southern blot analysis indicated a simple gene arrangement with at least two copies of the MIH gene in the genome of C. pagurus. Additional Southern blotting experiments detected MIH-hybridizing bands in another Cancer species, Cancer antennarius and another crab species, Carcinus maenas.

[Divergence of paralogous growth-hormone-encoding genes and their promoters in Salmonidae].

PubMed

Kamenskaya, D N; Pankova, M V; Atopkin, D M; Brykov, V A

2017-01-01

In many fish species, including salmonids, the growth-hormone is encoded by two duplicated paralogous genes, gh1 and gh2. Both genes were already in place at the time of divergence of species in this group. A comparison of the entire sequence of these genes of salmonids has shown that their conserved regions are associated with exons, while their most variable regions correspond to introns. Introns C and D include putative regulatory elements (sites Pit-1, CRE, and ERE), that are also conserved. In chars, the degree of polymorphism of gh2 gene is 2-3 times as large as that in gh1 gene. However, a comparison across all Salmonidae species would not extent this observation to other species. In both these chars' genes, the promoters are conserved mainly because they correspond to putative regulatory sequences (TATA box, binding sites for the pituitary transcription factor Pit-1 (F1-F4), CRE, GRE and RAR/RXR elements). The promoter of gh2 gene has a greater degree of polymorphism compared with gh1 gene promoter in all investigated species of salmonids. The observed differences in the rates of accumulation of changes in growth hormone encoding paralogs could be explained by differences in the intensity of selection.

Characterization of Hydrogen Metabolism in the Multicellular Green Alga Volvox carteri.

PubMed

Cornish, Adam J; Green, Robin; Gärtner, Katrin; Mason, Saundra; Hegg, Eric L

2015-01-01

Hydrogen gas functions as a key component in the metabolism of a wide variety of microorganisms, often acting as either a fermentative end-product or an energy source. The number of organisms reported to utilize hydrogen continues to grow, contributing to and expanding our knowledge of biological hydrogen processes. Here we demonstrate that Volvox carteri f. nagariensis, a multicellular green alga with differentiated cells, evolves H2 both when supplied with an abiotic electron donor and under physiological conditions. The genome of Volvox carteri contains two genes encoding putative [FeFe]-hydrogenases (HYDA1 and HYDA2), and the transcripts for these genes accumulate under anaerobic conditions. The HYDA1 and HYDA2 gene products were cloned, expressed, and purified, and both are functional [FeFe]-hydrogenases. Additionally, within the genome the HYDA1 and HYDA2 genes cluster with two putative genes which encode hydrogenase maturation proteins. This gene cluster resembles operon-like structures found within bacterial genomes and may provide further insight into evolutionary relationships between bacterial and algal [FeFe]-hydrogenase genes.

Characterization of Hydrogen Metabolism in the Multicellular Green Alga Volvox carteri

PubMed Central

Cornish, Adam J.; Green, Robin; Gärtner, Katrin; Mason, Saundra; Hegg, Eric L.

2015-01-01

Hydrogen gas functions as a key component in the metabolism of a wide variety of microorganisms, often acting as either a fermentative end-product or an energy source. The number of organisms reported to utilize hydrogen continues to grow, contributing to and expanding our knowledge of biological hydrogen processes. Here we demonstrate that Volvox carteri f. nagariensis, a multicellular green alga with differentiated cells, evolves H2 both when supplied with an abiotic electron donor and under physiological conditions. The genome of Volvox carteri contains two genes encoding putative [FeFe]-hydrogenases (HYDA1 and HYDA2), and the transcripts for these genes accumulate under anaerobic conditions. The HYDA1 and HYDA2 gene products were cloned, expressed, and purified, and both are functional [FeFe]-hydrogenases. Additionally, within the genome the HYDA1 and HYDA2 genes cluster with two putative genes which encode hydrogenase maturation proteins. This gene cluster resembles operon-like structures found within bacterial genomes and may provide further insight into evolutionary relationships between bacterial and algal [FeFe]-hydrogenase genes. PMID:25927230

Characterization of Hydrogen Metabolism in the Multicellular Green Alga Volvox carteri

DOE PAGES

Cornish, Adam J.; Green, Robin; Gärtner, Katrin; ...

2015-04-30

Hydrogen gas functions as a key component in the metabolism of a wide variety of microorganisms, often acting as either a fermentative end-product or an energy source. The number of organisms reported to utilize hydrogen continues to grow, contributing to and expanding our knowledge of biological hydrogen processes. Here we demonstrate that Volvox carteri f. nagariensis, a multicellular green alga with differentiated cells, evolves H 2 both when supplied with an abiotic electron donor and under physiological conditions. The genome of Volvox carteri contains two genes encoding putative [FeFe]-hydrogenases (HYDA1 and HYDA2), and the transcripts for these genes accumulate undermore » anaerobic conditions. The HYDA1 and HYDA2 gene products were cloned, expressed, and purified, and both are functional [FeFe]-hydrogenases. Additionally, within the genome the HYDA1 and HYDA2 genes cluster with two putative genes which encode hydrogenase maturation proteins. This gene cluster resembles operon-like structures found within bacterial genomes and may provide further insight into evolutionary relationships between bacterial and algal [FeFe]-hydrogenase genes.« less

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children.

PubMed

Vignali, Marissa; Armour, Christopher D; Chen, Jingyang; Morrison, Robert; Castle, John C; Biery, Matthew C; Bouzek, Heather; Moon, Wonjong; Babak, Tomas; Fried, Michal; Raymond, Christopher K; Duffy, Patrick E

2011-03-01

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.

NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children

PubMed Central

Vignali, Marissa; Armour, Christopher D.; Chen, Jingyang; Morrison, Robert; Castle, John C.; Biery, Matthew C.; Bouzek, Heather; Moon, Wonjong; Babak, Tomas; Fried, Michal; Raymond, Christopher K.; Duffy, Patrick E.

2011-01-01

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases. PMID:21317536

The First Step of Gibberellin Biosynthesis in Pumpkin Is Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by Differentially Regulated Genes

PubMed Central

Smith, Maria W.; Yamaguchi, Shinjiro; Ait-Ali, Tahar; Kamiya, Yuji

1998-01-01

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression. PMID:9847116

Molecular cloning and identification of the transcriptional regulatory domain of the goat neurokinin B gene TAC3.

PubMed

Suetomi, Yuta; Matsuda, Fuko; Uenoyama, Yoshihisa; Maeda, Kei-ichiro; Tsukamura, Hiroko; Ohkura, Satoshi

2013-10-01

Neurokinin B (NKB), encoded by TAC3, is thought to be an important accelerator of pulsatile gonadotropin-releasing hormone release. This study aimed to clarify the transcriptional regulatory mechanism of goat TAC3. First, we determined the full-length mRNA sequence of goat TAC3 from the hypothalamus to be 820 b, including a 381 b coding region, with the putative transcription start site located 143-b upstream of the start codon. The deduced amino acid sequence of NKB, which is produced from preproNKB, was completely conserved among goat, cattle, and human. Next, we cloned 5'-upstream region of goat TAC3 up to 3400 b from the translation initiation site, and this region was highly homologous with cattle TAC3 (89%). We used this goat TAC3 5'-upstream region to perform luciferase assays. We created a luciferase reporter vector containing DNA constructs from -2706, -1837, -834, -335, or -197 to +166 bp (the putative transcription start site was designated as +1) of goat TAC3 and these were transiently transfected into mouse hypothalamus-derived N7 cells and human neuroblastoma-derived SK-N-AS cells. The luciferase activity gradually increased with the deletion of the 5'-upstream region, suggesting that the transcriptional suppressive region is located between -2706 and -336 bp and that the core promoter exists downstream of -197 bp. Estradiol treatment did not lead to significant suppression of luciferase activity of any constructs, suggesting the existence of other factor(s) that regulate goat TAC3 transcription.

Prediction of the protein components of a putative Calanus finmarchicus (Crustacea, Copepoda) circadian signaling system using a de novo assembled transcriptome

PubMed Central

Christie, Andrew E.; Fontanilla, Tiana M.; Nesbit, Katherine T.; Lenz, Petra H.

2013-01-01

Diel vertical migration and seasonal diapause are critical life history events for the copepod Calanus finmarchicus. While much is known about these behaviors phenomenologically, little is known about their molecular underpinnings. Recent studies in insects suggest that some circadian genes/proteins also contribute to the establishment of seasonal diapause. Thus, it is possible that in Calanus these distinct timing regimes share some genetic components. To begin to address this possibility, we used the well-established Drosophila melanogaster circadian system as a reference for mining clock transcripts from a 200,000+ sequence Calanus transcriptome; the proteins encoded by the identified transcripts were also deduced and characterized. Sequences encoding homologs of the Drosophila core clock proteins CLOCK, CYCLE, PERIOD and TIMELESS were identified, as was one encoding CRYPTOCHROME 2, a core clock protein in ancestral insect systems, but absent in Drosophila. Calanus transcripts encoding proteins known to modulate the Drosophila core clock were also identified and characterized, e.g. CLOCKWORK ORANGE, DOUBLETIME, SHAGGY and VRILLE. Alignment and structural analyses of the deduced Calanus proteins with their Drosophila counterparts revealed extensive sequence conservation, particularly in functional domains. Interestingly, reverse BLAST analyses of these sequences against all arthropod proteins typically revealed non-Drosophila isoforms to be most similar to the Calanus queries. This, in combination with the presence of both CRYPTOCHROME 1 (a clock input pathway protein) and CRYPTOCHROME 2 in Calanus, suggests that the organization of the copepod circadian system is an ancestral one, more similar to that of insects like Danaus plexippus than to that of Drosophila. PMID:23727418

Identification of regulatory targets for the bacterial Nus factor complex.

PubMed

Baniulyte, Gabriele; Singh, Navjot; Benoit, Courtney; Johnson, Richard; Ferguson, Robert; Paramo, Mauricio; Stringer, Anne M; Scott, Ashley; Lapierre, Pascal; Wade, Joseph T

2017-12-11

Nus factors are broadly conserved across bacterial species, and are often essential for viability. A complex of five Nus factors (NusB, NusE, NusA, NusG and SuhB) is considered to be a dedicated regulator of ribosomal RNA folding, and has been shown to prevent Rho-dependent transcription termination. Here, we identify an additional cellular function for the Nus factor complex in Escherichia coli: repression of the Nus factor-encoding gene, suhB. This repression occurs primarily by translation inhibition, followed by Rho-dependent transcription termination. Thus, the Nus factor complex can prevent or promote Rho activity depending on the gene context. Conservation of putative NusB/E binding sites upstream of Nus factor genes suggests that Nus factor autoregulation occurs in many bacterial species. Additionally, many putative NusB/E binding sites are also found upstream of other genes in diverse species, and we demonstrate Nus factor regulation of one such gene in Citrobacter koseri. We conclude that Nus factors have an evolutionarily widespread regulatory function beyond ribosomal RNA, and that they are often autoregulatory.

Eucalypt NADP-Dependent Isocitrate Dehydrogenase1

PubMed Central

Boiffin, Vincent; Hodges, Michael; Gálvez, Susana; Balestrini, Raffaella; Bonfante, Paola; Gadal, Pierre; Martin, Francis

1998-01-01

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activity is increased in roots of Eucalyptus globulus subsp. bicostata ex Maiden Kirkp. during colonization by the ectomycorrhizal fungus Pisolithus tinctorius Coker and Couch. To investigate the regulation of the enzyme expression, a cDNA (EgIcdh) encoding the NADP-ICDH was isolated from a cDNA library of E. globulus-P. tinctorius ectomycorrhizae. The putative polypeptide sequence of EgIcdh showed a high amino acid similarity with plant NADP-ICDHs. Because the deduced EgICDH protein lacks an amino-terminal targeting sequence and shows highest similarity to plant cytosolic ICDHs, it probably represents a cytoplasmic isoform. RNA analysis showed that the steady-state level of EgIcdh transcripts was enhanced nearly 2-fold in ectomycorrhizal roots compared with nonmycorrhizal roots. Increased accumulation of NADP-ICDH transcripts occurred as early as 2 d after contact and likely led to the observed increased enzyme activity. Indirect immunofluorescence microscopy indicated that NADP-ICDH was preferentially accumulated in the epidermis and stele parenchyma of nonmycorrhizal and ectomycorrhizal lateral roots. The putative role of cytosolic NADP-ICDH in ectomycorrhizae is discussed. PMID:9662536

A mutation in a new gene bglJ, activates the bgl operon in Escherichia coli K-12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giel, M.; Desnoyer, M.; Lopilato, J.

1996-06-01

A new mutation , bglJ4, has been characterized that results in the expression of the silent bgl operon. The bgl operon encodes proteins necessary for the transport and utilization of the aromatic {beta}-glucosides arbutin and salicin. A variety of mutations activate the operon and result in a Bgl{sup +} phenotype. Activating mutations are located upstream of the bgl promoter and in genes located elsewhere on the chromosome. Mutations outside of the bgl operon occur in the genes encoding DNA gyrase and in the gene encoding the nucleoid associated protein H-NS. The mutation described here, bglJ4, has been mapped to amore » new locus at min 99 on the Escherichia coli K-12 genetic map. The putative protein encoded by the bglJ gene has homology to a family of transcriptional activators. Evidence is presented that increased expression of the bglJ product is needed for activation of the bgl operon. 56 refs., 3 figs., 3 tabs.« less

Polymorphism in exon 2 encoding the putative ligand binding pocket of the bovine insulin-like growth factor 1 receptor affects milk traits in four different cattle breeds.

PubMed

Szewczuk, M

2017-02-01

As a member of the somatotropic axis, insulin-like growth factor I receptor (IGF1R) seems to be a promising candidate gene. Two silent polymorphisms, identified by MspI and TaqI restriction enzymes, were selected within exon 2, encoding the majority of the putative ligand binding pocket. A total of 1169 cows of four pure breeds (Polish Holstein Friesian, Montbeliarde, Jersey and Holstein Friesian) were genotyped. The T (IGF1R/e2/MspI) and G (IGF1R/e2/TaqI) alleles were found to be prevalent. Three combinations of genotypes (TT/GG, TT/AG and CT/GG) were associated with the highest productivity (milk, protein and fat yields) among all breeds under study, as opposed to individuals carrying the worst CC/AA combination. In view of the specific structure of the ligand binding pocket and the significance of insulin-like growth factor I signalling promoting the development and differentiation in a variety of tissues (not only limited to mammary gland), the existence of missense mutation is unlikely. Potential mutations are likely limited to mRNA transcription and further post-transcriptional modifications. Further investigations should follow searching for the most useful IGF1R haplotypes, associated with higher milk production traits, exerting at the same time positive or neutral impact on health and welfare of individuals. © 2016 Blackwell Verlag GmbH.

Gravity-regulated gene expression in Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Sederoff, Heike; Brown, Christopher S.; Heber, Steffen; Kajla, Jyoti D.; Kumar, Sandeep; Lomax, Terri L.; Wheeler, Benjamin; Yalamanchili, Roopa

Plant growth and development is regulated by changes in environmental signals. Plants sense environmental changes and respond to them by modifying gene expression programs to ad-just cell growth, differentiation, and metabolism. Functional expression of genes comprises many different processes including transcription, translation, post-transcriptional and post-translational modifications, as well as the degradation of RNA and proteins. Recently, it was discovered that small RNAs (sRNA, 18-24 nucleotides long), which are heritable and systemic, are key elements in regulating gene expression in response to biotic and abiotic changes. Sev-eral different classes of sRNAs have been identified that are part of a non-cell autonomous and phloem-mobile network of regulators affecting transcript stability, translational kinetics, and DNA methylation patterns responsible for heritable transcriptional silencing (epigenetics). Our research has focused on gene expression changes in response to gravistimulation of Arabidopsis roots. Using high-throughput technologies including microarrays and 454 sequencing, we iden-tified rapid changes in transcript abundance of genes as well as differential expression of small RNA in Arabidopsis root apices after minutes of reorientation. Some of the differentially regu-lated transcripts are encoded by genes that are important for the bending response. Functional mutants of those genes respond faster to reorientation than the respective wild type plants, indicating that these proteins are repressors of differential cell elongation. We compared the gravity responsive sRNAs to the changes in transcript abundances of their putative targets and identified several potential miRNA: target pairs. Currently, we are using mutant and transgenic Arabidopsis plants to characterize the function of those miRNAs and their putative targets in gravitropic and phototropic responses in Arabidopsis.

Identification and expression analysis of a putative fatty acidbinding protein gene in the Asian honeybee, Apis cerana cerana.

PubMed

Yu, Xiaoli; Kang, Mingjiang; Liu, Li; Guo, Xingqi; Xu, Baohua

2013-01-01

Fatty acid-binding proteins (FABPs) play pivotal roles in cellular signaling, gene transcription, and lipid metabolism in vertebrates and invertebrates. In this study, a putative FABP gene, referred to as AccFABP, was isolated from the Asian honeybee, Apis cerana cerana Fabricius (Hymenoptera: Apidae). The full-length cDNA consisted of 725 bp, and encoded a protein of 204 amino acids. Homology and phylogenetic analysis indicated that AccFABP was a member of the FABP multifamily. The genomic structure of this gene, which was common among FABP multifamily members, spanned 1,900 bp, and included four exons and three introns. Gene expression analysis revealed that AccFABP was highly expressed in the dark-pigmented phase of pupal development, with peak expression observed in the fat bodies of the dark-pigmented phase pupae. The AccFABP transcripts in the fat body were upregulated by exposure to dietary fatty acids such as conjugated linoleic acid, docosahexaenoic acid, and arachidonic acid. Transcription factor binding sites for Caudal-Related Homeobox and functional CCAAT/enhancer binding site, which were respectively associated with tissue expression and lipid metabolism, were detected in the 5' promoter sequence. The evidence provided in the present study suggests that AccFABP may regulate insect growth and development, and lipid metabolism.

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

PubMed

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional enrichments were related to the cellular functions. The normalized number of functional enrichments of human putative transcriptional target genes changed according to the criteria of enhancer-promoter assignments and correlated with the median expression level of the target genes. These analyses and characters of human putative transcriptional target genes would be useful to examine the criteria of enhancer-promoter assignments and to predict the novel mechanisms and factors such as DNA binding proteins and DNA sequences of enhancer-promoter interactions.

A Fruit-Specific Putative Dihydroflavonol 4-Reductase Gene Is Differentially Expressed in Strawberry during the Ripening Process1

PubMed Central

Moyano, Enriqueta; Portero-Robles, Ignacio; Medina-Escobar, Nieves; Valpuesta, Victoriano; Muñoz-Blanco, Juan; Luis Caballero, José

1998-01-01

A cDNA clone encoding a putative dihydroflavonol 4-reductase gene has been isolated from a strawberry (Fragaria × ananassa cv Chandler) DNA subtractive library. Northern analysis showed that the corresponding gene is predominantly expressed in fruit, where it is first detected during elongation (green stages) and then declines and sharply increases when the initial fruit ripening events occur, at the time of initiation of anthocyanin accumulation. The transcript can be induced in unripe green fruit by removing the achenes, and this induction can be partially inhibited by treatment of de-achened fruit with naphthylacetic acid, indicating that the expression of this gene is under hormonal control. We propose that the putative dihydroflavonol 4-reductase gene in strawberry plays a main role in the biosynthesis of anthocyanin during color development at the late stages of fruit ripening; during the first stages the expression of this gene could be related to the accumulation of condensed tannins. PMID:9625725

Structure, Regulation, and Putative Function of the Arginine Deiminase System of Streptococcus suis

PubMed Central

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative β-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression. Furthermore, comparing an arcA knockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that the arcABC operon of S. suis contributes to survival under acidic conditions. PMID:16385025

Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis.

PubMed

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative beta-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression. Furthermore, comparing an arcA knockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that the arcABC operon of S. suis contributes to survival under acidic conditions.

Biodegradation of the organic disulfide 4,4'-dithiodibutyric acid by Rhodococcus spp.

PubMed

Khairy, Heba; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

2015-12-01

Four Rhodococcus spp. exhibited the ability to use 4,4'-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. Because Rhodococcus erythropolis strain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis of R. erythropolis MI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity of noxR and nox. The interruption mutant generated, R. erythropolis MI2 noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently, nox was overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Sugar regulation of SUGAR TRANSPORTER PROTEIN 1 (STP1) expression in Arabidopsis thaliana

PubMed Central

Cordoba, Elizabeth; Aceves-Zamudio, Denise Lizeth; Hernández-Bernal, Alma Fabiola; Ramos-Vega, Maricela; León, Patricia

2015-01-01

Sugars regulate the expression of many genes at the transcriptional level. In Arabidopsis thaliana, sugars induce or repress the expression of >1800 genes, including the STP1 (SUGAR TRANSPORTER PROTEIN 1) gene, which encodes an H+/monosaccharide cotransporter. STP1 transcript levels decrease more rapidly after the addition of low concentrations of sugars than the levels of other repressed genes, such as DIN6 (DARK-INDUCED 6). We found that this regulation is exerted at the transcriptional level and is initiated by phosphorylatable sugars. Interestingly, the sugar signal that modulates STP1 expression is transmitted through a HEXOKINASE 1-independent signalling pathway. Finally, analysis of the STP1 5′ regulatory region allowed us to delimit a region of 309bp that contains the cis elements implicated in the glucose regulation of STP1 expression. Putative cis-acting elements involved in this response were identified. PMID:25281700

The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and β-Lactams in Staphylococcus aureus▿

PubMed Central

Truong-Bolduc, Que Chi; Hooper, David C.

2007-01-01

MgrA is a known regulator of the expression of several multidrug transporters in Staphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression of norG produces a threefold increase in norB transcripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption of norG produces no change in the level of transcripts of norA, norB, and norC but causes an increase of at least threefold in the transcript level of abcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of cloned abcA caused an 8- to 128-fold increase in the level of resistance to all four β-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA acts as an indirect repressor for norB and a direct activator for abcA, whereas NorG acts as a direct activator for norB and a direct repressor for abcA. PMID:17277059

Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis.

PubMed

Sato, Takeo; Maekawa, Shugo; Konishi, Mineko; Yoshioka, Nozomi; Sasaki, Yuki; Maeda, Haruna; Ishida, Tetsuya; Kato, Yuki; Yamaguchi, Junji; Yanagisawa, Shuichi

2017-01-29

Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis. Copyright © 2016 Elsevier Inc. All rights reserved.

Nitrogen metabolism and nitrogen control in corynebacteria: variations of a common theme.

PubMed

Walter, Britta; Hänssler, Eva; Kalinowski, Jörn; Burkovski, Andreas

2007-01-01

The published genome sequences of Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum and Corynebacterium jeikeium were screened for genes encoding central components of nitrogen source uptake, nitrogen assimilation and nitrogen control systems. Interestingly, the soil-living species C. efficiens and C. glutamicum exhibit a broader spectrum of genes for nitrogen transport and metabolism than the pathogenic species C. diphtheriae and C. jeikeium. The latter are characterized by gene decay and loss of functions like urea metabolism and nitrogen-dependent transcription control. The global regulator of nitrogen regulation AmtR and its DNA-binding motif are conserved in C. diphtheriae, C. efficiens and C. glutamicum, while in C. jeikeium, an AmtR-encoding gene as well as putative AmtR-binding motifs are missing. Copyright (c) 2007 S. Karger AG, Basel.

Contribution of AmyA, an extracellular α-glucan degrading enzyme, to group A streptococcal host-pathogen interaction

PubMed Central

Shelburne, Samuel A.; Keith, David B.; Davenport, Michael T.; Beres, Stephen B.; Carroll, Ronan K.; Musser, James M.

2010-01-01

α-glucans such as starch and glycogen are abundant in the human oropharynx, the main site of group A Streptococcus (GAS) infection. However, the role in pathogenesis of GAS extracellular α-glucan binding and degrading enzymes is unknown. The serotype M1 GAS genome encodes two extracellular proteins putatively involved in α-glucan binding and degradation; pulA encodes a cell-wall anchored pullulanase and amyA encodes a freely secreted putative cyclomaltodextrin α-glucanotransferase. Genetic inactivation of amyA, but not pulA, abolished GAS α-glucan degradation. The ΔamyA strain had a slower rate of translocation across human pharyngeal epithelial cells. Consistent with this finding, the ΔamyA strain was less virulent following mouse mucosal challenge. Recombinant AmyA degraded α-glucans into β-cyclomaltodextrins that reduced pharyngeal cell transepithelial resistance, providing a physiologic explanation for the observed transepithelial migration phenotype. Higher amyA transcript levels were present in serotype M1 GAS strains causing invasive infection compared to strains causing pharyngitis. GAS proliferation in a defined α-glucan-containing medium was dependent on the presence of human salivary α-amylase. These data delineate the molecular mechanisms by which α-glucan degradation contributes to GAS host-pathogen interaction including how GAS employs human salivary α-amylase for its own metabolic benefit. PMID:19735442

Transcript Profiling Reveals New Insights into the Acclimation of the Mesophilic Fresh-Water Cyanobacterium Synechococcus elongatus PCC 7942 to Iron Starvation1[W

PubMed Central

Nodop, Anke; Pietsch, Daniel; Höcker, Ralf; Becker, Anke; Pistorius, Elfriede K.; Forchhammer, Karl; Michel, Klaus-Peter

2008-01-01

The regulatory network for acclimation of the obligate photoautotrophic fresh water cyanobacterium Synechococcus elongatus PCC 7942 to iron (Fe) limitation was studied by transcript profiling with an oligonucleotide whole genome DNA microarray. Six regions on the chromosome with several Fe-regulated genes each were identified. The irpAB and fut region encode putative Fe uptake systems, the suf region participates in [Fe-sulfur] cluster assembly under oxidative stress and Fe limitation, the isiAB region encodes CP43′ and flavodoxin, the idiCB region encodes the NuoE-like electron transport associated protein IdiC and the transcriptional activator IdiB, and the ackA/pgam region encodes an acetate kinase and a phosphoglycerate mutase. We also investigated the response of two S. elongatus PCC 7942 mutants to Fe starvation. These were mutant K10, lacking IdiB but containing IdiC, and mutant MuD, representing a idiC-merodiploid mutant with a strongly reduced amount of IdiC as well as IdiB. The absence of IdiB in mutant K10 or the strongly reduced amount of IdiB in mutant MuD allowed for the identification of additional members of the Fe-responsive IdiB regulon. Besides idiA and the irpAB operon somB(1), somA(2), ftr1, ackA, pgam, and nat also seem to be regulated by IdiB. In addition to the reduced amount of IdiB in MuD, the low concentration of IdiC may be responsible for a number of additional changes in the abundance of mainly photosynthesis-related transcripts as compared to the wild type and mutant K10. This fact may explain why it has been impossible to obtain a fully segregated IdiC-free mutant, whereas it was possible to obtain a fully segregated IdiB-free mutant. PMID:18424627

The C. elegans ceh-36 gene encodes a putative homemodomain transcription factor involved in chemosensory functions of ASE and AWC neurons.

PubMed

Koga, Makoto; Ohshima, Yasumi

2004-02-20

Chemotaxis to water-soluble chemicals such as sodium ion is an important behavior of Caenorhabditis elegans for seeking food, and ASE chemosensory neurons have a major role in this behavior. We isolated mutants defective in chemotaxis to sodium acetate. We show here that among them ks86 had a mutation in the ceh-36 gene. ceh-36 :: gfp reporter constructs were expressed in ASE and AWC neurons. In a mutant of the che-1 gene, which encodes another transcription factor and is required for specification of ASE neurons, expression of the ceh-36 :: gfp reporter in ASE is lost. This indicates that the ceh-36 gene functions downstream of the che-1 gene in ASE. In the ceh-36(ks86) mutant, expression of the tax-2 gene encoding a cyclic nucleotide-gated channel was reduced in ASE and AWC. This affords an explanation for defects of the ceh-36 mutant in the chemotaxis mediated by ASE and AWC. When a ceh-36 cDNA was expressed in an adult ceh-36 mutant by a heat shock promoter, chemotaxis to sodium acetate was recovered. These results suggest that ceh-36 is required for functions, and not for development, of ASE.

Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR.

PubMed

Chen, Jin-Zhong; Wang, Shu; Tang, Rong; Yang, Quan-Sheng; Zhao, Enpeng; Chao, Yaoqiong; Ying, Kang; Xie, Yi; Mao, Yu-Min

2002-09-01

A cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The 2390 bp cDNA with an open reading fragment (ORF) of 816 bp encodes a 272 amino acids putative protein with a thrombospondin type I repeat (TSR) domain and a cysteine-rich region at the N-terminus, so it is named hPWTSR. We used Northern blot detected two bands with length of about 3 kb and 4 kb respectively, which expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were expressed ubiquitously in fetal tissues and human tumor tissues too. However, the transcript was detected neither in ovarian carcinoma GI-102 nor in lung carcinoma LX-1. Blast analysis against NCBI database revealed that the new gene contained at least 5 exons and located in human chromosome 6q22.33. Our results demonstrate that the gene is a novel member of TSR supergene family.

A Polyketide Synthase Encoded by the Gene An15g07920 Is Involved in the Biosynthesis of Ochratoxin A in Aspergillus niger.

PubMed

Zhang, Jian; Zhu, Liuyang; Chen, Haoyu; Li, Min; Zhu, Xiaojuan; Gao, Qiang; Wang, Depei; Zhang, Ying

2016-12-28

The polyketide synthase gene An15g07920 was known in Aspergillus niger CBS 513.88 as putatively involved in the production of ochratoxin A (OTA). Genome resequencing analysis revealed that the gene An15g07920 is also present in the ochratoxin-producing A. niger strain 1062. Disruption of An15g07920 in A. niger 1062 removed its capacity to biosynthesize ochratoxin β (OTβ), ochratoxin α (OTα), and OTA. These results indicate that the polyketide synthase encoded by An15g07920 is a crucial player in the biosynthesis of OTA, in the pathway prior to the phenylalanine ligation step. The gene An15g07920 reached its maximum transcription level before OTA accumulation reached its highest level, confirming that gene transcription precedes OTA production. These findings will not only help explain the mechanism of OTA production in A. niger but also provide necessary information for the development of effective diagnostic, preventive, and control strategies to reduce the risk of OTA contamination in foods.

Transcriptome analysis of trichothecene-induced gene expression in barley.

PubMed

Boddu, Jayanand; Cho, Seungho; Muehlbauer, Gary J

2007-11-01

Fusarium head blight, caused primarily by Fusarium graminearum, is a major disease problem on barley (Hordeum vulgare L.). Trichothecene mycotoxins produced by the fungus during infection increase the aggressiveness of the fungus and promote infection in wheat (Triticum aestivum L.). Loss-of-function mutations in the TRI5 gene in F. graminearum result in the inability to synthesize trichothecenes and in reduced virulence on wheat. We examined the impact of pathogen-derived trichothecenes on virulence and the transcriptional differences in barley spikes infected with a trichothecene-producing wild-type strain and a loss-of-function tri5 trichothecene nonproducing mutant. Disease severity, fungal biomass, and floret necrosis and bleaching were reduced in spikes inoculated with the tri5 mutant strain compared with the wild-type strain, indicating that the inability to synthesize trichothecenes results in reduced virulence in barley. We detected 63 transcripts that were induced during trichothecene accumulation, including genes encoding putative trichothecene detoxification and transport proteins, ubiquitination-related proteins, programmed cell death-related proteins, transcription factors, and cytochrome P450s. We also detected 414 gene transcripts that were designated as basal defense response genes largely independent of trichothecene accumulation. Our results show that barley exhibits a specific response to trichothecene accumulation that can be separated from the basal defense response. We propose that barley responds to trichothecene accumulation by inducing at least two general responses. One response is the induction of genes encoding trichothecene detoxification and transport activities that may reduce the impact of trichothecenes. The other response is to induce genes encoding proteins associated with ubiquitination and cell death which may promote successful establishment of the disease.

Molecular cloning and characterization of a membrane associated NAC family gene, SiNAC from foxtail millet [Setaria italica (L.) P. Beauv].

PubMed

Puranik, Swati; Bahadur, Ranjit Prasad; Srivastava, Prem S; Prasad, Manoj

2011-10-01

The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A transcript encoding NAC protein, termed SiNAC was identified from a salt stress subtractive cDNA library of S. italica seedling (Puranik et al., J Plant Physiol 168:280-287, 2011). This single/low copy gene containing four exons and four introns within the genomic-sequence encoded a protein of 462 amino acids. Structural analysis revealed that highly divergent C terminus contains a transmembrane domain. The NAC domain consisted of a twisted antiparallel beta-sheet packing against N terminal alpha helix on one side and a shorter helix on the other side. The domain was predicted to homodimerize and control DNA-binding specificity. The physicochemical features of the SiNAC homodimer interface justified the dimeric form of the predicted model. A 1539 bp fragment upstream to the start codon of SiNAC gene was cloned and in silico analysis revealed several putative cis-acting regulatory elements within the promoter sequence. Transactivation analysis indicated that SiNAC activated expression of reporter gene and the activation domain lied at the C terminal. The SiNAC:GFP was detected in the nucleus and cytoplasm while SiNAC ΔC(1-158):GFP was nuclear localized in onion epidermal cells. SiNAC transcripts mostly accumulated in young spikes and were strongly induced by dehydration, salinity, ethephon, and methyl jasmonate. These results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants.

Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.

PubMed

Lüneberg, E; Mayer, B; Daryab, N; Kooistra, O; Zähringer, U; Rohde, M; Swanson, J; Frosch, M

2001-03-01

We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

PubMed

Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

1992-06-01

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.

PubMed

Graham, Morag R; Smoot, Laura M; Migliaccio, Cristi A Lux; Virtaneva, Kimmo; Sturdevant, Daniel E; Porcella, Stephen F; Federle, Michael J; Adams, Gerald J; Scott, June R; Musser, James M

2002-10-15

Two-component gene regulatory systems composed of a membrane-bound sensor and cytoplasmic response regulator are important mechanisms used by bacteria to sense and respond to environmental stimuli. Group A Streptococcus, the causative agent of mild infections and life-threatening invasive diseases, produces many virulence factors that promote survival in humans. A two-component regulatory system, designated covRS (cov, control of virulence; csrRS), negatively controls expression of five proven or putative virulence factors (capsule, cysteine protease, streptokinase, streptolysin S, and streptodornase). Inactivation of covRS results in enhanced virulence in mouse models of invasive disease. Using DNA microarrays and quantitative RT-PCR, we found that CovR influences transcription of 15% (n = 271) of all chromosomal genes, including many that encode surface and secreted proteins mediating host-pathogen interactions. CovR also plays a central role in gene regulatory networks by influencing expression of genes encoding transcriptional regulators, including other two-component systems. Differential transcription of genes influenced by covR also was identified in mouse soft-tissue infection. This analysis provides a genome-scale overview of a virulence gene network in an important human pathogen and adds insight into the molecular mechanisms used by group A Streptococcus to interact with the host, promote survival, and cause disease.

Palmate-like pentafoliata1 encodes a novel Cys(2)His(2) zinc finger transcription factor essential for compound leaf morphogenesis in Medicago truncatula

PubMed Central

2010-01-01

As the primary site for photosynthetic carbon fixation and the interface between plants and the environment, plant leaves play a key role in plant growth, biomass production and survival, and global carbon and oxygen cycles. Leaves can be simple with a single blade or compound with multiple units of blades known as leaflets. In a palmate-type compound leaf, leaflets are clustered at the tip of the leaf. In a pinnate-type compound leaf, on the other hand, leaflets are placed on a rachis in distance from each other. Higher orders of complexities such as bipinnate compound leaves of the “sensitive” plant, Mimosa pudica, also occur in nature. However, how different leaf morphologies are determined is still poorly understood. Medicago truncatula is a model legume closely related to alfalfa and soybean with trifoliate compound leaves. Recently, we have shown that Palmate-like Pentafoliata1 (PALM1) encodes a putative Cys(2) His(2) zinc finger transcription factor essential for compound leaf morphogenesis in M. truncatula. Here, we present our phylogenetic relationship analysis of PALM1 homologs from different species and demonstrate that PALM1 has transcriptional activity in the transactivation assay in yeast. PMID:20724826

Heterologous expression of a Tpo1 homolog from Arabidopsis thaliana confers resistance to the herbicide 2,4-D and other chemical stresses in yeast.

PubMed

Cabrito, Tânia R; Teixeira, Miguel C; Duarte, Alexandra A; Duque, Paula; Sá-Correia, Isabel

2009-10-01

The understanding of the molecular mechanisms underlying acquired herbicide resistance is crucial in dealing with the emergence of resistant weeds. Saccharomyces cerevisiae has been used as a model system to gain insights into the mechanisms underlying resistance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The TPO1 gene, encoding a multidrug resistance (MDR) plasma membrane transporter of the major facilitator superfamily (MFS), was previously found to confer resistance to 2,4-D in yeast and to be transcriptionally activated in response to the herbicide. In this work, we demonstrate that Tpo1p is required to reduce the intracellular concentration of 2,4-D. ScTpo1p homologs encoding putative plasma membrane MFS transporters from the plant model Arabidopsis thaliana were analyzed for a possible role in 2,4-D resistance. At5g13750 was chosen for further analysis, as its transcript levels were found to increase in 2,4-D stressed plants. The functional heterologous expression of this plant open reading frame in yeast was found to confer increased resistance to the herbicide in Deltatpo1 and wild-type cells, through the reduction of the intracellular concentration of 2,4-D. Heterologous expression of At5g13750 in yeast also leads to increased resistance to indole-3-acetic acid (IAA), Al(3+) and Tl(3+). At5g13750 is the first plant putative MFS transporter to be suggested as possibly involved in MDR.

Characterization of a Thioredoxin-1 Gene from Taenia solium and Its Encoding Product

PubMed Central

Jiménez, Lucía; Rodríguez-Lima, Oscar; Ochoa-Sánchez, Alicia; Landa, Abraham

2015-01-01

Taenia solium thioredoxin-1 gene (TsTrx-1) has a length of 771 bp with three exons and two introns. The core promoter gene presents two putative stress transcription factor binding sites, one putative TATA box, and a transcription start site (TSS). TsTrx-1 mRNA is expressed higher in larvae than in adult. This gene encodes a protein of 107 amino acids that presents the Trx active site (CGPC), the classical secondary structure of the thioredoxin fold, and the highest degree of identity with the Echinococcus granulosus Trx. A recombinant TsTrx-1 (rTsTrx-1) was produced in Escherichia coli with redox activity. Optimal activity for rTsTrx-1 was at pH 6.5 in the range of 15 to 25°C. The enzyme conserved activity for 3 h and lost it in 24 h at 37°C. rTsTrx-1 lost 50% activity after 1 h and lost activity completely in 24 h at temperatures higher than 55°C. Best storage temperature for rTsTrx-1 was at −70°C. It was inhibited by high concentrations of H2O2 and methylglyoxal (MG), but it was inhibited neither by NaCl nor by anti-rTsTrx-1 rabbit antibodies that strongly recognized a ~12 kDa band in extracts from several parasites. These TsTrx-1 properties open the opportunity to study its role in relationship T. solium-hosts. PMID:26090410

Transcription of the Streptococcus pyogenes Hyaluronic Acid Capsule Biosynthesis Operon Is Regulated by Previously Unknown Upstream Elements

PubMed Central

Falaleeva, Marina; Zurek, Oliwia W.; Watkins, Robert L.; Reed, Robert W.; Ali, Hadeel; Sumby, Paul; Voyich, Jovanka M.

2014-01-01

The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence. PMID:25287924

Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.

PubMed

Zhang, Fei; Bai, Fengwu; Zhao, Xinqing

2016-10-01

Trichoderma reesei Rut-C30 is a well-known cellulase producer, and improvement of its cellulase production is of great interest. An artificial zinc finger protein (AZFP) library is constructed for expression in T. reesei Rut-C30, and a mutant strain T. reesei U3 is selected based on its enhanced cellulase production. The U3 mutant shows a 55% rise in filter paper activity and 8.1-fold increased β-glucosidase activity, when compared to the native strain T. reesei Rut-C30. It is demonstrated that enhanced β-glucosidase activity was due to elevated transcription level of β-glucosidase gene in the U3 mutant. Moreover, significant elevation in transcription levels of several putative Azfp-U3 target genes is detected in the U3 mutant, including genes encoding hypothetical transcription factors and a putative glycoside hydrolase. Furthermore, U3 cellulase shows 115% higher glucose yield from pretreated corn stover, when compared to the cellulase of T. reesei Rut-C30. These results demonstrate that AZFP can be used to improve cellulase production in T. reesei Rut-C30. Our current work offers the establishment of an alternative strategy to develop fungal cell factories for improved production of high value industrial products. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes.

PubMed

McDowell, T L; Gibbons, R J; Sutherland, H; O'Rourke, D M; Bickmore, W A; Pombo, A; Turley, H; Gatter, K; Picketts, D J; Buckle, V J; Chapman, L; Rhodes, D; Higgs, D R

1999-11-23

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein.

PubMed

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-06-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

Characterization of the Tupaia Rhabdovirus Genome Reveals a Long Open Reading Frame Overlapping with P and a Novel Gene Encoding a Small Hydrophobic Protein

PubMed Central

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-01-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses. PMID:15890917

Regulatory role of XynR (YagI) in catabolism of xylonate in Escherichia coli K-12.

PubMed

Shimada, Tomohiro; Momiyama, Eri; Yamanaka, Yuki; Watanabe, Hiroki; Yamamoto, Kaneyoshi; Ishihama, Akira

2017-12-01

The genome of Escherichia coli K-12 contains ten cryptic phages, altogether constituting about 3.6% of the genome in sequence. Among more than 200 predicted genes in these cryptic phages, 14 putative transcription factor (TF) genes exist, but their regulatory functions remain unidentified. As an initial attempt to make a breakthrough for understanding the regulatory roles of cryptic phage-encoded TFs, we tried to identify the regulatory function of CP4-6 cryptic prophage-encoded YagI with unknown function. After SELEX screening, YagI was found to bind mainly at a single site within the spacer of bidirectional transcription units, yagA (encoding another uncharacterized TF) and yagEF (encoding 2-keto-3-deoxy gluconate aldolase, and dehydratase, respectively) within this prophage region. YagEF enzymes are involved in the catabolism of xylose downstream from xylonate. We then designated YagI as XynR (regulator of xylonate catabolism), one of the rare single-target TFs. In agreement with this predicted regulatory function, the activity of XynR was suggested to be controlled by xylonate. Even though low-affinity binding sites of XynR were identified in the E. coli K-12 genome, they all were inside open reading frames, implying that the regulation network of XynR is still fixed within the CR4-6 prophage without significant influence over the host E. coli K-12. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

PubMed

Sarmiento, José M; Añazco, Carolina C; Campos, Danae M; Prado, Gregory N; Navarro, Javier; González, Carlos B

2004-11-05

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

Comprehensive annotation of Glossina pallidipes salivary gland hypertrophy virus from Ethiopian tsetse flies: a proteogenomics approach

PubMed Central

Kariithi, Henry M.; Cousserans, François; Parker, Nicolas J.; İnce, İkbal Agah; Scully, Erin D.; Boeren, Sjef; Geib, Scott M.; Mekonnen, Solomon; Vlak, Just M.; Parker, Andrew G.; Vreysen, Marc J. B.; Bergoin, Max

2016-01-01

Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) can establish asymptomatic and symptomatic infection in its tsetse fly host. Here, we present a comprehensive annotation of the genome of an Ethiopian GpSGHV isolate (GpSGHV-Eth) compared with the reference Ugandan GpSGHV isolate (GpSGHV-Uga; GenBank accession number EF568108). GpSGHV-Eth has higher salivary gland hypertrophy syndrome prevalence than GpSGHV-Uga. We show that the GpSGHV-Eth genome has 190 291 nt, a low G+C content (27.9 %) and encodes 174 putative ORFs. Using proteogenomic and transcriptome mapping, 141 and 86 ORFs were mapped by transcripts and peptides, respectively. Furthermore, of the 174 ORFs, 132 had putative transcriptional signals [TATA-like box and poly(A) signals]. Sixty ORFs had both TATA-like box promoter and poly(A) signals, and mapped by both transcripts and peptides, implying that these ORFs encode functional proteins. Of the 60 ORFs, 10 ORFs are homologues to baculovirus and nudivirus core genes, including three per os infectivity factors and four RNA polymerase subunits (LEF4, 5, 8 and 9). Whereas GpSGHV-Eth and GpSGHV-Uga are 98.1 % similar at the nucleotide level, 37 ORFs in the GpSGHV-Eth genome had nucleotide insertions (n = 17) and deletions (n = 20) compared with their homologues in GpSGHV-Uga. Furthermore, compared with the GpSGHV-Uga genome, 11 and 24 GpSGHV ORFs were deleted and novel, respectively. Further, 13 GpSGHV-Eth ORFs were non-canonical; they had either CTG or TTG start codons instead of ATG. Taken together, these data suggest that GpSGHV-Eth and GpSGHV-Uga represent two different lineages of the same virus. Genetic differences combined with host and environmental factors possibly explain the differential GpSGHV pathogenesis observed in different G. pallidipes colonies. PMID:26801744

Molecular characterization of two serine proteases expressed in gut tissue of the African trypanosome vector, Glossina morsitans morsitans.

PubMed

Yan, J; Cheng, Q; Li, C B; Aksoy, S

2001-02-01

Serine proteases are major insect gut enzymes involved in digestion of dietary proteins, and in addition they have been implicated in the process of pathogen establishment in several vector insects. The medically important vector, tsetse fly (Diptera:Glossinidiae), is involved in the transmission of African trypanosomes, which cause devastating diseases in animals and humans. Both the male and female tsetse can transmit trypanosomes and both are strict bloodfeeders throughout all stages of their development. Here, we describe the characterization of two putative serine protease-encoding genes, Glossina serine protease-1 (Gsp1) and Glossina serine protease-2 (Gsp2) from gut tissue. Both putative cDNA products represent prepro peptides with hydrophobic signal peptide sequences associated with their 5'-end terminus. The Gsp1 cDNA encodes a putative mature protein of 245 amino acids with a molecular mass of 26 428 Da, while the predicted size of the 228 amino acid mature peptide encoded by Gsp2 cDNA is 24 573 Da. Both deduced peptides contain the Asp/His/Ser catalytic triad and the conserved residues surrounding it which are characteristic of serine proteases. In addition, both proteins have the six-conserved cysteine residues to form the three-cysteine bonds typically present in invertebrate serine proteases. Based on the presence of substrate specific residues, the Gsp1 gene encodes a chymotrypsin-like protease while Gsp2 gene encodes for a protein with trypsin-like activity. Both proteins are encoded by few loci in tsetse genome, being present in one or two copies only. The mRNA expression levels for the genes do not vary extensively throughout the digestive cycle, and high levels of mRNAs can be readily detected in the gut tissue of newly emerged flies. The levels of trypsin and chymotrypsin activities in the gut lumen increase following blood feeding and change significantly in the gut cells throughout the digestion cycle. Hence, the regulation of expression for trypsin and chymotrypsin occurs at the post-transcriptional level in tsetse. Both the coding sequences and patterns of expression of Gsp1 and Gsp2 genes are similar to the serine proteases that have been reported from the bloodfeeding insect Stomoxys calcitrans.

Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network.

PubMed

Fish, Jason E; Cantu Gutierrez, Manuel; Dang, Lan T; Khyzha, Nadiya; Chen, Zhiqi; Veitch, Shawn; Cheng, Henry S; Khor, Melvin; Antounians, Lina; Njock, Makon-Sébastien; Boudreau, Emilie; Herman, Alexander M; Rhyner, Alexander M; Ruiz, Oscar E; Eisenhoffer, George T; Medina-Rivera, Alejandra; Wilson, Michael D; Wythe, Joshua D

2017-07-01

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis. © 2017. Published by The Company of Biologists Ltd.

Identification and Characterization of microRNA319a and Its Putative Target Gene, PvPCF5, in the Bioenergy Grass Switchgrass (Panicum virgatum).

PubMed

Xie, Qi; Liu, Xue; Zhang, Yinbing; Tang, Jinfu; Yin, Dedong; Fan, Bo; Zhu, Lihuang; Han, Liebao; Song, Guilong; Li, Dayong

2017-01-01

Due to its high biomass yield, low environmental impact, and widespread adaptability to poor soils and harsh conditions, switchgrass ( Panicum virgatum L.), a warm-region perennial herbaceous plant, has attracted much attention in recent years. However, little is known about microRNAs (miRNAs) and their functions in this bioenergy grass. Here, we identified and characterized a miRNA gene, Pvi-MIR319a , encoding microRNA319a in switchgrass. Transgenic rice lines generated by overexpressing the Pvi-MIR319a precursor gene exhibited broader leaves and delayed flowering compared with the control. Gene expression analysis indicated at least four putative target genes were downregulated. Additionally, we cloned a putative target gene ( PvPCF5 ) of Pvi-MIR319a from switchgrass. PvPCF5, a TCP transcription factor, is a nuclear-localized protein with transactivation activity and control the development of leaf. Our results suggest that Pvi-MIR319a and its target genes may be used as potential genetic regulators for future switchgrass genetic improvement.

Neuropeptide discovery in Eucyclops serrulatus (Crustacea, Copepoda): in silico prediction of the first peptidome for a member of the Cyclopoida.

PubMed

Christie, Andrew E

2015-01-15

Crustaceans of the subclass Copepoda are key components of essentially all aquatic ecosystems as they serve both as the primary consumers of phytoplankton and/or as major food sources for a wide variety of higher-level consumers. The dominant group of copepods in most freshwater ecosystems is the Cyclopoida; members of this order are routinely used as environmental indicators, and some predatory species are used for the biological control of disease-causing mosquitoes. Given their ecological and disease control importance, it is surprising that little is known about endocrine control in cyclopoids. Here, as part of an ongoing effort to identify and characterize the neurochemical signaling systems of members of the Copepoda, the extant transcriptome shotgun assembly for Eucyclops serrulatus, a member of the Cyclopoida, was mined for transcripts encoding putative peptide hormone-encoding transcripts. Via queries using known arthropod pre/preprohormone sequences, primarily ones from other copepod species, 36 E. serrulatus peptide-encoding transcripts were identified. The proteins deduced from these sequences allowed for the prediction of 160 unique mature neuropeptides, including the first copepod isoform of pigment dispersing hormone, as well as isoforms of adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, allatotropin, crustacean hyperglycemic hormone, diuretic hormone 31, DXXRLamide, FLRFamide, FXGGXamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F and tachykinin-related peptide. These peptides are currently the only ones known from any member of the Cyclopoida, and as such, provide a new resource for investigating peptidergic signaling in this important copepod order. Copyright © 2014 Elsevier Inc. All rights reserved.

Transcriptome changes associated with delayed flower senescence on transgenic petunia by inducing expression of etr1-1, a mutant ethylene receptor.

PubMed

Wang, Hong; Stier, Genevieve; Lin, Jing; Liu, Gang; Zhang, Zhen; Chang, Youhong; Reid, Michael S; Jiang, Cai-Zhong

2013-01-01

Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr1-1), a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr1-1 gene was over-expressed under the control of a chemically-inducible promoter, which would allow expression of etr1-1 to be initiated at the desired time and stage of development. Here, we showed that transgenic plants grew and developed normally without a chemical inducer. Semi-quantitative RT-PCR demonstrated that the abundance of transcripts of Arabidopsis etr1-1 gene was substantially induced in flowers with 30 μM dexamethasone (DEX). Consequently, t he life of the flowers was almost doubled and the peak of ethylene production was delayed. We compared gene expression changes of petals with DEX to those without DEX at 24 h and 48 h by microarray. Our results indicated that transcripts of many putative genes encoding transcription factors were down-regulated by etr1-1 induced expression at the early stage. In addition, putative genes involved in gibberellin biosynthesis, response to jasmonic acid/gibberellins stimulus, cell wall modification, ethylene biosynthesis, and cell death were down-regulated associating with etr1-1 induced expression. We investigated time-course gene expression profiles and found two profiles which displayed totally opposite expression patterns under these two treatments. In these profiles, 'the regulation of transcription' was predominant in GO categories. Taking all results together, we concluded those transcription factors down-regulated at early stage might exert a major role in regulating the senescence process which were consequently characterized by cell wall modification and cell death.

Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana.

PubMed

Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

1996-10-03

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Arabidopsis thaliana (At). The transcription unit of the At gene spans approximately 1900 bp and specifies a protein of 503 amino acids with a calculated relative molecular mass (M(r)) of 54,190. The gene is comprised of a minimum of four introns and five exons with all donor and acceptor splice sequences conforming to previously proposed consensus sequences. The deduced IMPDH amino-acid sequence from At shows a remarkable similarity to other eukaryotic IMPDH sequences, with a 48% identity to human Type II enzyme. Allowing for conservative substitutions, the enzyme is 69% similar to human Type II IMPDH. The putative active-site sequence of At IMPDH conforms to the IMP dehydrogenase/guanosine monophosphate reductase motif and contains an essential active-site cysteine residue.

Global gene expression during stringent response in Corynebacterium glutamicum in presence and absence of the rel gene encoding (p)ppGpp synthase

PubMed Central

Brockmann-Gretza, Olaf; Kalinowski, Jörn

2006-01-01

Background The stringent response is the initial reaction of microorganisms to nutritional stress. During stringent response the small nucleotides (p)ppGpp act as global regulators and reprogram bacterial transcription. In this work, the genetic network controlled by the stringent response was characterized in the amino acid-producing Corynebacterium glutamicum. Results The transcriptome of a C. glutamicum rel gene deletion mutant, unable to synthesize (p)ppGpp and to induce the stringent response, was compared with that of its rel-proficient parent strain by microarray analysis. A total of 357 genes were found to be transcribed differentially in the rel-deficient mutant strain. In a second experiment, the stringent response was induced by addition of DL-serine hydroxamate (SHX) in early exponential growth phase. The time point of the maximal effect on transcription was determined by real-time RT-PCR using the histidine and serine biosynthetic genes. Transcription of all of these genes reached a maximum at 10 minutes after SHX addition. Microarray experiments were performed comparing the transcriptomes of SHX-induced cultures of the rel-proficient strain and the rel mutant. The differentially expressed genes were grouped into three classes. Class A comprises genes which are differentially regulated only in the presence of an intact rel gene. This class includes the non-essential sigma factor gene sigB which was upregulated and a large number of genes involved in nitrogen metabolism which were downregulated. Class B comprises genes which were differentially regulated in response to SHX in both strains, independent of the rel gene. A large number of genes encoding ribosomal proteins fall into this class, all being downregulated. Class C comprises genes which were differentially regulated in response to SHX only in the rel mutant. This class includes genes encoding putative stress proteins and global transcriptional regulators that might be responsible for the complex transcriptional patterns detected in the rel mutant when compared directly with its rel-proficient parent strain. Conclusion In C. glutamicum the stringent response enfolds a fast answer to an induced amino acid starvation on the transcriptome level. It also showed some significant differences to the transcriptional reactions occuring in Escherichia coli and Bacillus subtilis. Notable are the rel-dependent regulation of the nitrogen metabolism genes and the rel-independent regulation of the genes encoding ribosomal proteins. PMID:16961923

Transcriptional response of Medicago truncatula sulphate transporters to arbuscular mycorrhizal symbiosis with and without sulphur stress.

PubMed

Casieri, Leonardo; Gallardo, Karine; Wipf, Daniel

2012-06-01

Sulphur is an essential macronutrient for plant growth, development and response to various abiotic and biotic stresses due to its key role in the biosynthesis of many S-containing compounds. Sulphate represents a very small portion of soil S pull and it is the only form that plant roots can uptake and mobilize through H(+)-dependent co-transport processes implying sulphate transporters. Unlike the other organically bound forms of S, sulphate is normally leached from soils due to its solubility in water, thus reducing its availability to plants. Although our knowledge of plant sulphate transporters has been growing significantly in the past decades, little is still known about the effect of the arbuscular mycorrhiza interaction on sulphur uptake. Carbon, nitrogen and sulphur measurements in plant parts and expression analysis of genes encoding putative Medicago sulphate transporters (MtSULTRs) were performed to better understand the beneficial effects of mycorrhizal interaction on Medicago truncatula plants colonized by Glomus intraradices at different sulphate concentrations. Mycorrhization significantly promoted plant growth and sulphur content, suggesting increased sulphate absorption. In silico analyses allowed identifying eight putative MtSULTRs phylogenetically distributed over the four sulphate transporter groups. Some putative MtSULTRs were transcribed differentially in roots and leaves and affected by sulphate concentration, while others were more constitutively transcribed. Mycorrhizal-inducible and -repressed MtSULTRs transcripts were identified allowing to shed light on the role of mycorrhizal interaction in sulphate uptake.

CalA, a Cyanobacterial AbrB Protein, Interacts with the Upstream Region of hypC and Acts as a Repressor of Its Transcription in the Cyanobacterium Nostoc sp. Strain PCC 7120▿ †

PubMed Central

Agervald, Åsa; Zhang, Xiaohui; Stensjö, Karin; Devine, Ellenor; Lindblad, Peter

2010-01-01

The filamentous, heterocystous, nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain, depending on growth conditions, up to two hydrogenases directly involved in hydrogen metabolism. HypC is one out of at least seven auxiliary gene products required for synthesis of a functional hydrogenase, specifically involved in the maturation of the large subunit. In this study we present a protein, CalA (Alr0946 in the genome), belonging to the transcription regulator family AbrB, which in protein-DNA assays was found to interact with the upstream region of hypC. Transcriptional investigations showed that calA is cotranscribed with the downstream gene alr0947, which encodes a putative protease from the abortive infection superfamily, Abi. CalA was shown to interact specifically not only with the upstream region of hypC but also with its own upstream region, acting as a repressor on hypC. The bidirectional hydrogenase activity was significantly downregulated when CalA was overexpressed, demonstrating a correlation with the transcription factor, either direct or indirect. In silico studies showed that homologues to both CalA and Alr0947 are highly conserved proteins within cyanobacteria with very similar physical organizations of the corresponding structural genes. Possible functions of the cotranscribed downstream protein Alr0947 are presented. In addition, we present a three-dimensional (3D) model of the DNA binding domain of CalA and putative DNA binding mechanisms are discussed. PMID:20023111

Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit.

PubMed

Grassi, Stefania; Piro, Gabriella; Lee, Je Min; Zheng, Yi; Fei, Zhangjun; Dalessandro, Giuseppe; Giovannoni, James J; Lenucci, Marcello S

2013-11-12

Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement. Total carotenoids progressively increased during fruit ripening up to ~55 μg g(-1) fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening. Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening.

Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

PubMed Central

2013-01-01

Background Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement. Results Total carotenoids progressively increased during fruit ripening up to ~55 μg g-1 fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening. Conclusions Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening. PMID:24219562

Trees, fungi and bacteria: tripartite metatranscriptomics of a root microbiome responding to soil contamination.

PubMed

Gonzalez, E; Pitre, F E; Pagé, A P; Marleau, J; Guidi Nissim, W; St-Arnaud, M; Labrecque, M; Joly, S; Yergeau, E; Brereton, N J B

2018-03-21

One method for rejuvenating land polluted with anthropogenic contaminants is through phytoremediation, the reclamation of land through the cultivation of specific crops. The capacity for phytoremediation crops, such as Salix spp., to tolerate and even flourish in contaminated soils relies on a highly complex and predominantly cryptic interacting community of microbial life. Here, Illumina HiSeq 2500 sequencing and de novo transcriptome assembly were used to observe gene expression in washed Salix purpurea cv. 'Fish Creek' roots from trees pot grown in petroleum hydrocarbon-contaminated or non-contaminated soil. All 189,849 assembled contigs were annotated without a priori assumption as to sequence origin and differential expression was assessed. The 839 contigs differentially expressed (DE) and annotated from S. purpurea revealed substantial increases in transcripts encoding abiotic stress response equipment, such as glutathione S-transferases, in roots of contaminated trees as well as the hallmarks of fungal interaction, such as SWEET2 (Sugars Will Eventually Be Exported Transporter). A total of 8252 DE transcripts were fungal in origin, with contamination conditions resulting in a community shift from Ascomycota to Basidiomycota genera. In response to contamination, 1745 Basidiomycota transcripts increased in abundance (the majority uniquely expressed in contaminated soil) including major monosaccharide transporter MST1, primary cell wall and lamella CAZy enzymes, and an ectomycorrhiza-upregulated exo-β-1,3-glucanase (GH5). Additionally, 639 DE polycistronic transcripts from an uncharacterised Enterobacteriaceae species were uniformly in higher abundance in contamination conditions and comprised a wide spectrum of genes cryptic under laboratory conditions but considered putatively involved in eukaryotic interaction, biofilm formation and dioxygenase hydrocarbon degradation. Fungal gene expression, representing the majority of contigs assembled, suggests out-competition of white rot Ascomycota genera (dominated by Pyronema), a sometimes ectomycorrhizal (ECM) Ascomycota (Tuber) and ECM Basidiomycota (Hebeloma) by a poorly characterised putative ECM Basidiomycota due to contamination. Root and fungal expression involved transcripts encoding carbohydrate/amino acid (C/N) dialogue whereas bacterial gene expression included the apparatus necessary for biofilm interaction and direct reduction of contamination stress, a potential bacterial currency for a role in tripartite mutualism. Unmistakable within the metatranscriptome is the degree to which the landscape of rhizospheric biology, particularly the important but predominantly uncharacterised fungal genetics, is yet to be discovered.

Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa

PubMed Central

Gonçalves, A. P.; Monteiro, João; Lucchi, Chiara; Kowbel, David J.; Cordeiro, J. M.; Correia-de-Sá, Paulo; Rigden, Daniel J.; Glass, N. L.; Videira, Arnaldo

2014-01-01

Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca2+ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS). Conversely, an approximately 30-fold excess of external Ca2+ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca2+ signaling profiles were observed in the absence or presence of excessive external Ca2+. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca2+ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca2+-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca2+ in fungal biology. PMID:28357255

Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster.

PubMed

Tolhuis, Bas; de Wit, Elzo; Muijrers, Inhua; Teunissen, Hans; Talhout, Wendy; van Steensel, Bas; van Lohuizen, Maarten

2006-06-01

Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila melanogaster Kc cells. We found that Pc associates with large genomic regions of up to approximately 150 kb in size, hereafter referred to as 'Pc domains'. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 Lys27 and is generally transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains.

The mRNA-bound proteome of the early fly embryo

PubMed Central

Wessels, Hans-Hermann; Imami, Koshi; Baltz, Alexander G.; Kolinski, Marcin; Beldovskaya, Anastasia; Selbach, Matthias; Small, Stephen; Ohler, Uwe; Landthaler, Markus

2016-01-01

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation. PMID:27197210

The Landscape of Somatic Chromosomal Copy Number Aberrations in GEM Models of Prostate Carcinoma

PubMed Central

Bianchi-Frias, Daniella; Hernandez, Susana A.; Coleman, Roger; Wu, Hong; Nelson, Peter S.

2015-01-01

Human prostate cancer (PCa) is known to harbor recurrent genomic aberrations consisting of chromosomal losses, gains, rearrangements and mutations that involve oncogenes and tumor suppressors. Genetically engineered mouse (GEM) models have been constructed to assess the causal role of these putative oncogenic events and provide molecular insight into disease pathogenesis. While GEM models generally initiate neoplasia by manipulating a single gene, expression profiles of GEM tumors typically comprise hundreds of transcript alterations. It is unclear whether these transcriptional changes represent the pleiotropic effects of single oncogenes, and/or cooperating genomic or epigenomic events. Therefore, it was determined if structural chromosomal alterations occur in GEM models of PCa and whether the changes are concordant with human carcinomas. Whole genome array-based comparative genomic hybridization (CGH) was used to identify somatic chromosomal copy number aberrations (SCNAs) in the widely used TRAMP, Hi-Myc, Pten-null and LADY GEM models. Interestingly, very few SCNAs were identified and the genomic architecture of Hi-Myc, Pten-null and LADY tumors were essentially identical to the germline. TRAMP neuroendocrine carcinomas contained SCNAs, which comprised three recurrent aberrations including a single copy loss of chromosome 19 (encoding Pten). In contrast, cell lines derived from the TRAMP, Hi-Myc, and Pten-null tumors were notable for numerous SCNAs that included copy gains of chromosome 15 (encoding Myc) and losses of chromosome 11 (encoding p53). PMID:25298407

Specific DNA binding of a potential transcriptional regulator, inosine 5'-monophosphate dehydrogenase-related protein VII, to the promoter region of a methyl coenzyme m reductase I-encoding operon retrieved from Methanothermobacter thermautotrophicus strain DeltaH.

PubMed

Shinzato, Naoya; Enoki, Miho; Sato, Hiroaki; Nakamura, Kohei; Matsui, Toru; Kamagata, Yoichi

2008-10-01

Two methyl coenzyme M reductases (MCRs) encoded by the mcr and mrt operons of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus DeltaH are expressed in response to H(2) availability. In the present study, cis elements and trans-acting factors responsible for the gene expression of MCRs were investigated by using electrophoretic mobility shift assay (EMSA) and affinity particle purification. A survey of their operator regions by EMSA with protein extracts from mrt-expressing cultures restricted them to 46- and 41-bp-long mcr and mrt upstream regions, respectively. Affinity particle purification of DNA-binding proteins conjugated with putative operator regions resulted in the retrieval of a protein attributed to IMP dehydrogenase-related protein VII (IMPDH VII). IMPDH VII is predicted to have a winged helix-turn-helix DNA-binding motif and two cystathionine beta-synthase domains, and it has been suspected to be an energy-sensing module. EMSA with oligonucleotide probes with unusual sequences showed that the binding site of IMPDH VII mostly overlaps the factor B-responsible element-TATA box of the mcr operon. The results presented here suggest that IMPDH VII encoded by MTH126 is a plausible candidate for the transcriptional regulator of the mcr operon in this methanogen.

Open reading frames in a 4556 nucleotide sequence within MDV-1 BamHI-D DNA fragment: evidence for splicing of mRNA from a new viral glycoprotein gene.

PubMed

Becker, Y; Asher, Y; Tabor, E; Davidson, I; Malkinson, M

1994-01-01

A DNA segment of the MDV-1 BamHI-D fragment was sequenced, and the open reading frames (ORFs) present in the 4556 nucleotide fragment were analyzed by computer programs. Computer analysis identified 19 putative ORFs in the sequence ranging from a coding capacity of 37 amino acids (aa) (ORF-1a) to 684aa (ORF-1). The special properties of four ORFs (1a, 1, 2, and 3) were investigated. Two adjacent ORFs, ORF-1a and ORF-1, were found by computer analysis to have the properties of two introns encoding a glycoprotein: ORF-1a encodes an aa sequence with the properties of a signal peptide, and ORF-1 encodes a polypeptide with a membrane anchor domain and putative N-glycosylation sites in the aa sequence. ORF-1a and ORF-1 were found to be transcribed in MDV-1-infected cells. Two RNA transcripts were detected: a precursor RNA and its spliced form. Both are transcribed from a promoter located 5' to ORF-1a, and splice donor and acceptor sites are used to splice the mRNA after cleavage of a 71-nucleotide sequence. This finding suggest that ORF-1a and ORF-1 are two introns of a new MDV-1 glycoprotein gene. The DNA sequence containing ORF-1 was transiently expressed in COS-1 cells, and the viral protein produced in these cells was found to react with anti-MDV serotype-1 Antigen B-specific monoclonal antibodies. These studies indicate that the protein encoded by ORF-1 has antigenic properties resembling Antigen B of MDV-1. A gene homologous to ORF-1 was detected in the genome of both MDV-2(SB1) and MDV-3(HVT), which serve as commercial vaccine strains. Two additional ORFs were noted in the 4556 nucleotide sequence: ORF-2, which encodes a 333 aa polypeptide initiating in the UL and terminating in the TRL prior to the putative origin of replication, and ORF-3, which encodes a 155 aa polypeptide that is partly homologous to the phosphoprotein pp38 encoded by the BamHI-H sequence. The 65 N-terminal aa of the two gene products are identical, both being derived from the nucleotide sequences in the TRL and IRL, respectively. Additional homologous aa sequences are the hydrophobic aa domain in the middle of both proteins. The functions of ORF-2, ORF-3, and additional ORFs are under study.

Inference of Expanded Lrp-Like Feast/Famine Transcription Factor Targets in a Non-Model Organism Using Protein Structure-Based Prediction

PubMed Central

Ashworth, Justin; Plaisier, Christopher L.; Lo, Fang Yin; Reiss, David J.; Baliga, Nitin S.

2014-01-01

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer. PMID:25255272

Inference of expanded Lrp-like feast/famine transcription factor targets in a non-model organism using protein structure-based prediction.

PubMed

Ashworth, Justin; Plaisier, Christopher L; Lo, Fang Yin; Reiss, David J; Baliga, Nitin S

2014-01-01

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer.

Open chromatin encoded in DNA sequence is the signature of ‘master’ replication origins in human cells

PubMed Central

Audit, Benjamin; Zaghloul, Lamia; Vaillant, Cédric; Chevereau, Guillaume; d'Aubenton-Carafa, Yves; Thermes, Claude; Arneodo, Alain

2009-01-01

For years, progress in elucidating the mechanisms underlying replication initiation and its coupling to transcriptional activities and to local chromatin structure has been hampered by the small number (approximately 30) of well-established origins in the human genome and more generally in mammalian genomes. Recent in silico studies of compositional strand asymmetries revealed a high level of organization of human genes around 1000 putative replication origins. Here, by comparing with recently experimentally identified replication origins, we provide further support that these putative origins are active in vivo. We show that regions ∼300-kb wide surrounding most of these putative replication origins that replicate early in the S phase are hypersensitive to DNase I cleavage, hypomethylated and present a significant enrichment in genomic energy barriers that impair nucleosome formation (nucleosome-free regions). This suggests that these putative replication origins are specified by an open chromatin structure favored by the DNA sequence. We discuss how this distinctive attribute makes these origins, further qualified as ‘master’ replication origins, priviledged loci for future research to decipher the human spatio-temporal replication program. Finally, we argue that these ‘master’ origins are likely to play a key role in genome dynamics during evolution and in pathological situations. PMID:19671527

Genome-wide transcriptome analysis of Clavibacter michiganensis subsp. michiganensis grown in xylem mimicking medium.

PubMed

Hiery, Eva; Adam, Susanne; Reid, Stephen; Hofmann, Jörg; Sonnewald, Sophia; Burkovski, Andreas

2013-12-01

The interaction between Clavibacter michiganensis subsp. michiganensis with its host, the tomato plant (Solanum lycopersicum), is poorly understood and only few virulence factors are known. While studying of the bacteria in planta is time-consuming and difficult, the analysis in vitro would facilitate research. Therefore, a xylem mimicking medium (XMM) for C. michiganensis subsp. michiganensis was established in this study based on an apoplast medium for Xanthomonas campestris pv. vesicatoria. In contrast to the apoplast medium, XMM contains no sugars, but amino acids which serve as nitrogen and carbon source. As a result, growth in XMM induced transcriptional changes of genes encoding putative sugar, amino acid and iron uptake systems. In summary, mRNA levels of about 8% of all C. michiganensis subsp. michiganensis genes were changed when XMM-grown bacteria were compared to M9 minimal medium-grown cells. Almost no transcriptional changes of genes encoding hydrolytic enzymes were detected, leading to the idea that XMM reflects the situation in the beginning of infection and therefore allows the characterization of virulence factors in this early stage of infection. The addition of the plant wound substance acetosyringone to the XMM medium led to a change in transcript amount, including genes coding for proteins involved in protein transport, iron uptake and regulation processes. Copyright © 2013 Elsevier B.V. All rights reserved.

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

Pseudomonas aeruginosa PumA acts on an endogenous phenazine to promote self-resistance.

PubMed

Sporer, Abigail J; Beierschmitt, Christopher; Bendebury, Anastasia; Zink, Katherine E; Price-Whelan, Alexa; Buzzeo, Marisa C; Sanchez, Laura M; Dietrich, Lars E P

2018-05-01

The activities of critical metabolic and regulatory proteins can be altered by exposure to natural or synthetic redox-cycling compounds. Many bacteria, therefore, possess mechanisms to transport or transform these small molecules. The opportunistic pathogen Pseudomonas aeruginosa PA14 synthesizes phenazines, redox-active antibiotics that are toxic to other organisms but have beneficial effects for their producer. Phenazines activate the redox-sensing transcription factor SoxR and thereby induce the transcription of a small regulon, including the operon mexGHI-opmD, which encodes an efflux pump that transports phenazines, and PA14_35160 (pumA), which encodes a putative monooxygenase. Here, we provide evidence that PumA contributes to phenazine resistance and normal biofilm development, particularly during exposure to or production of strongly oxidizing N-methylated phenazines. We show that phenazine resistance depends on the presence of residues that are conserved in the active sites of other putative and characterized monooxygenases found in the antibiotic producer Streptomyces coelicolor. We also show that during biofilm growth, PumA is required for the conversion of phenazine methosulfate to unique phenazine metabolites. Finally, we compare ∆mexGHI-opmD and ∆pumA strains in assays for colony biofilm morphogenesis and SoxR activation, and find that these deletions have opposing phenotypic effects. Our results suggest that, while MexGHI-OpmD-mediated efflux has the effect of making the cellular phenazine pool more reducing, PumA acts on cellular phenazines to make the pool more oxidizing. We present a model in which these two SoxR targets function simultaneously to control the biological activity of the P. aeruginosa phenazine pool.

Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans

PubMed Central

2004-01-01

Numerous invertebrate species belonging to several phyla cannot synthesize sterols de novo and rely on a dietary source of the compound. SCPx (sterol carrier protein 2/3-oxoacyl-CoA thiolase) is a protein involved in the trafficking of sterols and oxidation of branched-chain fatty acids. We have isolated SCPx protein from Spodoptera littoralis (cotton leafworm) and have subjected it to limited amino acid sequencing. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.9 kb), which encodes a 57 kDa protein. Northern blotting detected two mRNA transcripts, one of 1.9 kb, encoding SCPx, and one of 0.95 kb, presumably encoding SCP2 (sterol carrier protein 2). The former mRNA was highly expressed in midgut and Malpighian tubules during the last larval instar. Furthermore, constitutive expression of the gene was detected in the prothoracic glands, which are the main tissue producing the insect moulting hormone. There was no significant change in the 1.9 kb mRNA in midgut throughout development, but slightly higher expression in the early stages. Conceptual translation of the cDNA and a database search revealed that the gene includes the SCP2 sequence and a putative peroxisomal targeting signal in the C-terminal region. Also a cysteine residue at the putative active site for the 3-oxoacyl-CoA thiolase is conserved. Southern blotting showed that SCPx is likely to be encoded by a single-copy gene. The mRNA expression pattern and the gene structure suggest that SCPx from S. littoralis (a lepidopteran) is evolutionarily closer to that of mammals than to that of dipterans. PMID:15149283

Functional Analysis of the Lactobacillus casei BL23 Sortases

PubMed Central

Muñoz-Provencio, Diego; Rodríguez-Díaz, Jesús; Collado, María Carmen; Langella, Philippe; Bermúdez-Humarán, Luis G.

2012-01-01

Sortases are a class of enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria. Lactobacillus casei BL23 harbors four sortase genes, two belonging to class A (srtA1 and srtA2) and two belonging to class C (srtC1 and srtC2). Class C sortases were clustered with genes encoding their putative substrates that were homologous to the SpaEFG and SpaCBA proteins that encode mucus adhesive pili in Lactobacillus rhamnosus GG. Twenty-three genes encoding putative sortase substrates were identified in the L. casei BL23 genome with unknown (35%), enzymatic (30%), or adhesion-related (35%) functions. Strains disrupted in srtA1, srtA2, srtC1, and srtC2 and an srtA1 srtA2 double mutant were constructed. The transcription of all four sortase encoding genes was detected, but only the mutation of srtA1 resulted in a decrease in bacterial surface hydrophobicity. The β-N-acetyl-glucosaminidase and cell wall proteinase activities of whole cells diminished in the srtA1 mutant and, to a greater extent, in the srtA1 srtA2 double mutant. Cell wall anchoring of the staphylococcal NucA reporter protein fused to a cell wall sorting sequence was also affected in the srtA mutants, and the percentages of adhesion to Caco-2 and HT-29 intestinal epithelial cells were reduced for the srtA1 srtA2 strain. Mutations in srtC1 or srtC2 result in an undetectable phenotype. Together, these results suggest that SrtA1 is the housekeeping sortase in L. casei BL23 and SrtA2 would carry out redundant or complementary functions that become evident when SrtA1 activity is absent. PMID:23042174

Genome-wide Identification of TCP Family Transcription Factors from Populus euphratica and Their Involvement in Leaf Shape Regulation

PubMed Central

Ma, Xiaodong; Ma, Jianchao; Fan, Di; Li, Chaofeng; Jiang, Yuanzhong; Luo, Keming

2016-01-01

Higher plants have been shown to experience a juvenile vegetative phase, an adult vegetative phase, and a reproductive phase during its postembryonic development and distinct lateral organ morphologies have been observed at the different development stages. Populus euphratica, commonly known as a desert poplar, has developed heteromorphic leaves during its development. The TCP family genes encode a group of plant-specific transcription factors involved in several aspects of plant development. In particular, TCPs have been shown to influence leaf size and shape in many herbaceous plants. However, whether these functions are conserved in woody plants remains unknown. In the present study, we carried out genome-wide identification of TCP genes in P. euphratica and P. trichocarpa, and 33 and 36 genes encoding putative TCP proteins were found, respectively. Phylogenetic analysis of the poplar TCPs together with Arabidopsis TCPs indicated a biased expansion of the TCP gene family via segmental duplications. In addition, our results have also shown a correlation between different expression patterns of several P. euphratica TCP genes and leaf shape variations, indicating their involvement in the regulation of leaf shape development. PMID:27605130

Cloning, expression and N-terminal myristoylation of CpCPK1, a calcium-dependent protein kinase from zucchini (Cucurbita pepo L.).

PubMed

Ellard-Ivey, M; Hopkins, R B; White, T J; Lomax, T L

1999-01-01

We have isolated a full-length cDNA clone (CpCDPK1) encoding a calcium-dependent protein kinase (CDPK) gene from zucchini (Cucurbita pepo L.). The predicted amino acid sequence of the cDNA shows a remarkably high degree of similarity to members of the CDPK gene family from Arabidopsis thaliana, especially AtCPK1 and AtCPK2. Northern analysis of steady-state mRNA levels for CpCPK1 in etiolated and light-grown zucchini seedlings shows that the transcript is most abundant in etiolated hypocotyls and overall expression is suppressed by light. As described for other members of the CDPK gene family from different species, the CpCPK1 clone has a putative N-terminal myristoylation sequence. In this study, site-directed mutagenesis and an in vitro coupled transcription/translation system were used to demonstrate that the protein encoded by this cDNA is specifically myristoylated by a plant N-myristoyl transferase. This is the first demonstration of myristoylation of a CDPK protein which may contribute to the mechanism by which this protein is localized to the plasma membrane.

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

PubMed Central

Simonin, F; Gavériaux-Ruff, C; Befort, K; Matthes, H; Lannes, B; Micheletti, G; Mattéi, M G; Charron, G; Bloch, B; Kieffer, B

1995-01-01

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors. Images Fig. 3 Fig. 4 PMID:7624359

SensiPath: computer-aided design of sensing-enabling metabolic pathways.

PubMed

Delépine, Baudoin; Libis, Vincent; Carbonell, Pablo; Faulon, Jean-Loup

2016-07-08

Genetically-encoded biosensors offer a wide range of opportunities to develop advanced synthetic biology applications. Circuits with the ability of detecting and quantifying intracellular amounts of a compound of interest are central to whole-cell biosensors design for medical and environmental applications, and they also constitute essential parts for the selection and regulation of high-producer strains in metabolic engineering. However, the number of compounds that can be detected through natural mechanisms, like allosteric transcription factors, is limited; expanding the set of detectable compounds is therefore highly desirable. Here, we present the SensiPath web server, accessible at http://sensipath.micalis.fr SensiPath implements a strategy to enlarge the set of detectable compounds by screening for multi-step enzymatic transformations converting non-detectable compounds into detectable ones. The SensiPath approach is based on the encoding of reactions through signature descriptors to explore sensing-enabling metabolic pathways, which are putative biochemical transformations of the target compound leading to known effectors of transcription factors. In that way, SensiPath enlarges the design space by broadening the potential use of biosensors in synthetic biology applications. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

A resource for characterizing genome-wide binding and putative target genes of transcription factors expressed during secondary growth and wood formation in Populus

Treesearch

Lijun Liu; Trevor Ramsay; Matthew S. Zinkgraf; David Sundell; Nathaniel Robert Street; Vladimir Filkov; Andrew Groover

2015-01-01

Identifying transcription factor target genes is essential for modeling the transcriptional networks underlying developmental processes. Here we report a chromatin immunoprecipitation sequencing (ChIP-seq) resource consisting of genome-wide binding regions and associated putative target genes for four Populus homeodomain transcription factors...

Arabidopsis transcriptional response to extracellular Ca2+ depletion involves a transient rise in cytosolic Ca2+.

PubMed

Wang, Jing; Tergel, Tergel; Chen, Jianhua; Yang, Ju; Kang, Yan; Qi, Zhi

2015-02-01

Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca(2+) levels caused by increased acid deposition and massive timber harvesting. Little is known about the genetic and cellular mechanism of plants' responses to Ca(2+) depletion. In this study, transcriptional profiling analysis helped identify multiple extracellular Ca(2+) ([Ca(2+) ]ext ) depletion-responsive genes in Arabidopsis thaliana L., many of which are involved in response to other environmental stresses. Interestingly, a group of genes encoding putative cytosolic Ca(2+) ([Ca(2+) ]cyt ) sensors were significantly upregulated, implying that [Ca(2+) ]cyt has a role in sensing [Ca(2+) ]ext depletion. Consistent with this observation, [Ca(2+) ]ext depletion stimulated a transient rise in [Ca(2+) ]cyt that was negatively influenced by [K(+) ]ext , suggesting the involvement of a membrane potential-sensitive component. The [Ca(2+) ]cyt response to [Ca(2+) ]ext depletion was significantly desensitized after the initial treatment, which is typical of a receptor-mediated signaling event. The response was insensitive to an animal Ca(2+) sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. Gd(3+) , an inhibitor of Ca(2+) channels, suppressed the [Ca(2+) ]ext -triggered rise in [Ca(2+) ]cyt and downstream changes in gene expression. Taken together, this study demonstrates that [Ca(2+) ]cyt plays an important role in the putative receptor-mediated cellular and transcriptional response to [Ca(2+) ]ext depletion of plant cells. © 2014 Institute of Botany, Chinese Academy of Sciences.

Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

PubMed

Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

2000-08-01

The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

Molecular cloning of ADIR, a novel interferon responsive gene encoding a protein related to the torsins.

PubMed

Dron, Michel; Meritet, Jean François; Dandoy-Dron, Françoise; Meyniel, Jean-Philippe; Maury, Chantal; Tovey, Michael G

2002-03-01

The expression of the previously uncharacterized gene Adir (for ATP dependent interferon responsive gene) was increased by 5- to 15-fold in tissue of the oral cavity or in spleen and liver of mice treated orally or intraperitoneally with IFN-alpha, and in mouse cells treated in vitro with IFN-alpha or IFN-gamma. The level of Adir mRNA was also increased 20- to 40-fold in the brains of animals infected with encephalomyocarditis virus. Adir is expressed ubiquitously in mouse tissues as 1.9-, 2.4-, and 3.5-kb mRNA transcripts encoding a 385-amino-acid protein with a conserved ATP binding domain containing typical nucleotide and Mg(2+) binding sites. We also characterized the human ortholog, ADIR, which is located on chromosome 1q25-q31 and contains six exons encoding a 397-amino-acid protein with 80% homology to the mouse protein. A single 2.3-kb mRNA was detected in all human tissues examined, except for placenta, which also contained a 1.25-kb tissue-specific transcript generated by alternative splicing and encoding a putative 336-amino-acid protein. Although ADIR exhibits low homology to DYT1 and TOR1B, the deduced ADIR protein sequences are highly homologous to torsin A and torsin B and more distantly related to members of the Clp/HSP100 family of proteins, suggesting that ADIR, like torsins, is related to the AAA chaperone-like family of ATPases. An ADIR-EGFP fusion protein expressed in HeLa cells was shown to be associated with the endoplasmic reticulum.

Genetic analysis reveals the identity of the photoreceptor for phototaxis in hormogonium filaments of Nostoc punctiforme.

PubMed

Campbell, Elsie L; Hagen, Kari D; Chen, Rui; Risser, Douglas D; Ferreira, Daniela P; Meeks, John C

2015-02-15

In cyanobacterial Nostoc species, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm that Nostoc punctiforme hormogonia are positively phototactic to white light over a wide range of intensities. N. punctiforme contains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome-methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis in N. punctiforme hormogonia; here these genes are designated ptx (for phototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of the ptx genes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpA and gvpC), which also are expressed only in the hormogonium filaments of N. punctiforme. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization of the gene encoding component C3 of the complement system from the spider Loxosceles laeta venom glands: Phylogenetic implications.

PubMed

Myamoto, D T; Pidde-Queiroz, G; Pedroso, A; Gonçalves-de-Andrade, R M; van den Berg, C W; Tambourgi, D V

2016-09-01

A transcriptome analysis of the venom glands of the spider Loxosceles laeta, performed by our group, in a previous study (Fernandes-Pedrosa et al., 2008), revealed a transcript with a sequence similar to the human complement component C3. Here we present the analysis of this transcript. cDNA fragments encoding the C3 homologue (Lox-C3) were amplified from total RNA isolated from the venom glands of L. laeta by RACE-PCR. Lox-C3 is a 5178 bps cDNA sequence encoding a 190kDa protein, with a domain configuration similar to human C3. Multiple alignments of C3-like proteins revealed two processing sites, suggesting that Lox-C3 is composed of three chains. Furthermore, the amino acids consensus sequences for the thioester was found, in addition to putative sequences responsible for FB binding. The phylogenetic analysis showed that Lox-C3 belongs to the same group as two C3 isoforms from the spider Hasarius adansoni (Family Salcitidae), showing 53% homology with these. This is the first characterization of a Loxosceles cDNA sequence encoding a human C3 homologue, and this finding, together with our previous finding of the expression of a FB-like molecule, suggests that this spider species also has a complement system. This work will help to improve our understanding of the innate immune system in these spiders and the ancestral structure of C3. Copyright © 2016 Elsevier GmbH. All rights reserved.

Role and Regulation of the Flp/Tad Pilus in the Virulence of Pectobacterium atrosepticum SCRI1043 and Pectobacterium wasabiae SCC3193

PubMed Central

Nykyri, Johanna; Mattinen, Laura; Niemi, Outi; Adhikari, Satish; Kõiv, Viia; Somervuo, Panu; Fang, Xin; Auvinen, Petri; Mäe, Andres; Palva, E. Tapio; Pirhonen, Minna

2013-01-01

In this study, we characterized a putative Flp/Tad pilus-encoding gene cluster, and we examined its regulation at the transcriptional level and its role in the virulence of potato pathogenic enterobacteria of the genus Pectobacterium. The Flp/Tad pilus-encoding gene clusters in Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium aroidearum were compared to previously characterized flp/tad gene clusters, including that of the well-studied Flp/Tad pilus model organism Aggregatibacter actinomycetemcomitans, in which this pilus is a major virulence determinant. Comparative analyses revealed substantial protein sequence similarity and open reading frame synteny between the previously characterized flp/tad gene clusters and the cluster in Pectobacterium, suggesting that the predicted flp/tad gene cluster in Pectobacterium encodes a Flp/Tad pilus-like structure. We detected genes for a novel two-component system adjacent to the flp/tad gene cluster in Pectobacterium, and mutant analysis demonstrated that this system has a positive effect on the transcription of selected Flp/Tad pilus biogenesis genes, suggesting that this response regulator regulate the flp/tad gene cluster. Mutagenesis of either the predicted regulator gene or selected Flp/Tad pilus biogenesis genes had a significant impact on the maceration ability of the bacterial strains in potato tubers, indicating that the Flp/Tad pilus-encoding gene cluster represents a novel virulence determinant in Pectobacterium. Soft-rot enterobacteria in the genera Pectobacterium and Dickeya are of great agricultural importance, and an investigation of the virulence of these pathogens could facilitate improvements in agricultural practices, thus benefiting farmers, the potato industry and consumers. PMID:24040039

Role and regulation of the Flp/Tad pilus in the virulence of Pectobacterium atrosepticum SCRI1043 and Pectobacterium wasabiae SCC3193.

PubMed

Nykyri, Johanna; Mattinen, Laura; Niemi, Outi; Adhikari, Satish; Kõiv, Viia; Somervuo, Panu; Fang, Xin; Auvinen, Petri; Mäe, Andres; Palva, E Tapio; Pirhonen, Minna

2013-01-01

In this study, we characterized a putative Flp/Tad pilus-encoding gene cluster, and we examined its regulation at the transcriptional level and its role in the virulence of potato pathogenic enterobacteria of the genus Pectobacterium. The Flp/Tad pilus-encoding gene clusters in Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium aroidearum were compared to previously characterized flp/tad gene clusters, including that of the well-studied Flp/Tad pilus model organism Aggregatibacter actinomycetemcomitans, in which this pilus is a major virulence determinant. Comparative analyses revealed substantial protein sequence similarity and open reading frame synteny between the previously characterized flp/tad gene clusters and the cluster in Pectobacterium, suggesting that the predicted flp/tad gene cluster in Pectobacterium encodes a Flp/Tad pilus-like structure. We detected genes for a novel two-component system adjacent to the flp/tad gene cluster in Pectobacterium, and mutant analysis demonstrated that this system has a positive effect on the transcription of selected Flp/Tad pilus biogenesis genes, suggesting that this response regulator regulate the flp/tad gene cluster. Mutagenesis of either the predicted regulator gene or selected Flp/Tad pilus biogenesis genes had a significant impact on the maceration ability of the bacterial strains in potato tubers, indicating that the Flp/Tad pilus-encoding gene cluster represents a novel virulence determinant in Pectobacterium. Soft-rot enterobacteria in the genera Pectobacterium and Dickeya are of great agricultural importance, and an investigation of the virulence of these pathogens could facilitate improvements in agricultural practices, thus benefiting farmers, the potato industry and consumers.

Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases.

PubMed

Parveen, Iffat; Wang, Mei; Zhao, Jianping; Chittiboyina, Amar G; Tabanca, Nurhayat; Ali, Abbas; Baerson, Scott R; Techen, Natascha; Chappell, Joe; Khan, Ikhlas A; Pan, Zhiqiang

2015-11-01

Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

Expression of ZmMET1, a gene encoding a DNA methyltransferase from maize, is associated not only with DNA replication in actively proliferating cells, but also with altered DNA methylation status in cold-stressed quiescent cells.

PubMed

Steward, N; Kusano, T; Sano, H

2000-09-01

A cDNA fragment encoding part of a DNA methyltransferase was isolated from maize. The putative amino acid sequence identically matched that deduced from a genomic sequence in the database (accession no. AF063403), and the corresponding gene was designated as ZmMET1. Bacterially expressed ZmMET1 actively methylated DNA in vitro. Transcripts of ZmMET1 could be shown to exclusively accumulate in actively proliferating cells of the meristems of mesocotyls and root apices, suggesting ZmMET1 expression to be associated with DNA replication. This was confirmed by simultaneous decrease of transcripts of ZmMET1 and histone H3, a marker for DNA replication, in seedlings exposed to wounding, desiccation and salinity, all of which suppress cell division. Cold stress also depressed both transcripts in root tissues. In contrast, however, accumulation of ZmMET1 transcripts in shoot mesocotyls was not affected by cold stress, whereas those for H3 sharply decreased. Such a differential accumulation of ZmMET1 transcripts was consistent with ZmMET1 protein levels as revealed by western blotting. Expression of ZmMET1 is thus coexistent, but not completely dependent on DNA replication. Southern hybridization analysis with a methylation-sensitive restriction enzyme revealed that cold treatment induced demethylation of DNA in the Ac/Ds transposon region, but not in other genes, and that such demethylation primarily occurred in roots. These results suggested that the methylation level was decreased selectively by cold treatment, and that ZmMET1 may, at least partly, prevent such demethylation.

Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Yangrae; Ohm, Robin A.; Grigoriev, Igor V.

Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. To identify molecular determinants of pathogenicity, we created non-pathogenic mutants of a transcription factor-encoding gene, AbPf2. The frequency and timing of germination and appressorium formation on host plants were similar between the non-pathogenic abpf2 mutants and wild-type A. brassicicola. The mutants were also similar in vitro to wild-type A. brassicicola in terms of vegetative growth, conidium production, and responses to a phytoalexin, reactive oxygen species and osmolites. The hyphae of the mutants grew slowly but did not cause disease symptoms on the surface of host plants. Transcripts of the AbPf2more » gene increased exponentially soon after wild-type conidia contacted their host plants . A small amount of AbPf2 protein, as monitored using GFP fusions, was present in young, mature conidia. The protein level decreased during saprophytic growth, but increased and was located primarily in fungal nuclei during pathogenesis. Levels of the proteins and transcripts sharply decreased following colonization of host tissues beyond the initial infection site. When expression of the transcription factor was induced in the wild-type during early pathogenesis, 106 fungal genes were also induced in the wild-type but not in the abpf2 mutants. Notably, 33 of the 106 genes encoded secreted proteins, including eight putative effector proteins. Plants inoculated with abpf2 mutants expressed higher levels of genes associated with photosynthesis, the pentose phosphate pathway and primary metabolism, but lower levels of defense-related genes. Our results suggest that AbPf2 is an important regulator of pathogenesis, but does not affect other cellular processes in A. brassicicola.« less

Analysis of the Transcriptome of Erigeron breviscapus Uncovers Putative Scutellarin and Chlorogenic Acids Biosynthetic Genes and Genetic Markers

PubMed Central

Zhang, Jia-Jin; Shu, Li-Ping; Zhang, Wei; Long, Guang-Qiang; Liu, Tao; Meng, Zheng-Gui; Chen, Jun-Wen; Yang, Sheng-Chao

2014-01-01

Background Erigeron breviscapus (Vant.) Hand-Mazz. is a famous medicinal plant. Scutellarin and chlorogenic acids are the primary active components in this herb. However, the mechanisms of biosynthesis and regulation for scutellarin and chlorogenic acids in E. breviscapus are considerably unknown. In addition, genomic information of this herb is also unavailable. Principal Findings Using Illumina sequencing on GAIIx platform, a total of 64,605,972 raw sequencing reads were generated and assembled into 73,092 non-redundant unigenes. Among them, 44,855 unigenes (61.37%) were annotated in the public databases Nr, Swiss-Prot, KEGG, and COG. The transcripts encoding the known enzymes involved in flavonoids and in chlorogenic acids biosynthesis were discovered in the Illumina dataset. Three candidate cytochrome P450 genes were discovered which might encode flavone 6-hydroase converting apigenin to scutellarein. Furthermore, 4 unigenes encoding the homologues of maize P1 (R2R3-MYB transcription factors) were defined, which might regulate the biosynthesis of scutellarin. Additionally, a total of 11,077 simple sequence repeat (SSR) were identified from 9,255 unigenes. Of SSRs, tri-nucleotide motifs were the most abundant motif. Thirty-six primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism. The result revealed that 34 (94.40%) primer pairs were successfully amplified and 19 (52.78%) primer pairs exhibited polymorphisms. Conclusion Using next generation sequencing (NGS) technology, this study firstly provides abundant genomic data for E. breviscapus. The candidate genes involved in the biosynthesis and transcriptional regulation of scutellarin and chlorogenic acids were obtained in this study. Additionally, a plenty of genetic makers were generated by identification of SSRs, which is a powerful tool for molecular breeding and genetics applications in this herb. PMID:24956277

CsrA Represses Translation of sdiA, Which Encodes the N-Acylhomoserine-l-Lactone Receptor of Escherichia coli, by Binding Exclusively within the Coding Region of sdiA mRNA ▿ †

PubMed Central

Yakhnin, Helen; Baker, Carol S.; Berezin, Igor; Evangelista, Michael A.; Rassin, Alisa; Romeo, Tony; Babitzke, Paul

2011-01-01

The RNA binding protein CsrA is the central component of a conserved global regulatory system that activates or represses gene expression posttranscriptionally. In every known example of CsrA-mediated translational control, CsrA binds to the 5′ untranslated region of target transcripts, thereby repressing translation initiation and/or altering the stability of the RNA. Furthermore, with few exceptions, repression by CsrA involves binding directly to the Shine-Dalgarno sequence and blocking ribosome binding. sdiA encodes the quorum-sensing receptor for N-acyl-l-homoserine lactone in Escherichia coli. Because sdiA indirectly stimulates transcription of csrB, which encodes a small RNA (sRNA) antagonist of CsrA, we further explored the relationship between sdiA and the Csr system. Primer extension analysis revealed four putative transcription start sites within 85 nucleotides of the sdiA initiation codon. Potential σ70-dependent promoters were identified for each of these primer extension products. In addition, two CsrA binding sites were predicted in the initially translated region of sdiA. Expression of chromosomally integrated sdiA′-′lacZ translational fusions containing the entire promoter and CsrA binding site regions indicates that CsrA represses sdiA expression. The results from gel shift and footprint studies demonstrate that tight binding of CsrA requires both of these sites. Furthermore, the results from toeprint and in vitro translation experiments indicate that CsrA represses translation of sdiA by directly competing with 30S ribosomal subunit binding. Thus, this represents the first example of CsrA preventing translation by interacting solely within the coding region of an mRNA target. PMID:21908661

Analysis of the transcriptome of Erigeron breviscapus uncovers putative scutellarin and chlorogenic acids biosynthetic genes and genetic markers.

PubMed

Jiang, Ni-Hao; Zhang, Guang-Hui; Zhang, Jia-Jin; Shu, Li-Ping; Zhang, Wei; Long, Guang-Qiang; Liu, Tao; Meng, Zheng-Gui; Chen, Jun-Wen; Yang, Sheng-Chao

2014-01-01

Erigeron breviscapus (Vant.) Hand-Mazz. is a famous medicinal plant. Scutellarin and chlorogenic acids are the primary active components in this herb. However, the mechanisms of biosynthesis and regulation for scutellarin and chlorogenic acids in E. breviscapus are considerably unknown. In addition, genomic information of this herb is also unavailable. Using Illumina sequencing on GAIIx platform, a total of 64,605,972 raw sequencing reads were generated and assembled into 73,092 non-redundant unigenes. Among them, 44,855 unigenes (61.37%) were annotated in the public databases Nr, Swiss-Prot, KEGG, and COG. The transcripts encoding the known enzymes involved in flavonoids and in chlorogenic acids biosynthesis were discovered in the Illumina dataset. Three candidate cytochrome P450 genes were discovered which might encode flavone 6-hydroase converting apigenin to scutellarein. Furthermore, 4 unigenes encoding the homologues of maize P1 (R2R3-MYB transcription factors) were defined, which might regulate the biosynthesis of scutellarin. Additionally, a total of 11,077 simple sequence repeat (SSR) were identified from 9,255 unigenes. Of SSRs, tri-nucleotide motifs were the most abundant motif. Thirty-six primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism. The result revealed that 34 (94.40%) primer pairs were successfully amplified and 19 (52.78%) primer pairs exhibited polymorphisms. Using next generation sequencing (NGS) technology, this study firstly provides abundant genomic data for E. breviscapus. The candidate genes involved in the biosynthesis and transcriptional regulation of scutellarin and chlorogenic acids were obtained in this study. Additionally, a plenty of genetic makers were generated by identification of SSRs, which is a powerful tool for molecular breeding and genetics applications in this herb.

Role of an Iron-Dependent Transcriptional Regulator in the Pathogenesis and Host Response to Infection with Streptococcus pneumoniae

PubMed Central

Gupta, Radha; Bhatty, Minny; Swiatlo, Edwin; Nanduri, Bindu

2013-01-01

Iron is a critical cofactor for many enzymes and is known to regulate gene expression in many bacterial pathogens. Streptococcus pneumoniae normally inhabits the upper respiratory mucosa but can also invade and replicate in lungs and blood. These anatomic sites vary considerably in both the quantity and form of available iron. The genome of serotype 4 pneumococcal strain TIGR4 encodes a putative iron-dependent transcriptional regulator (IDTR). A mutant deleted at idtr (Δidtr) exhibited growth kinetics similar to parent strain TIGR4 in vitro and in mouse blood for up to 48 hours following infection. However, Δidtr was significantly attenuated in a murine model of sepsis. IDTR down-regulates the expression of ten characterized and putative virulence genes in nasopharyngeal colonization and pneumonia. The host cytokine response was significantly suppressed in sepsis with Δidtr. Since an exaggerated inflammatory response is associated with a poor prognosis in sepsis, the decreased inflammatory response could explain the increased survival with Δidtr. Our results suggest that IDTR, which is dispensable for pneumococcal growth in vitro, is associated with regulation of pneumococcal virulence in specific host environments. Additionally, IDTR ultimately modulates the host cytokine response and systemic inflammation that contributes to morbidity and mortality of invasive pneumococcal disease. PMID:23437050

Tenebrio molitor antifreeze protein gene identification and regulation.

PubMed

Qin, Wensheng; Walker, Virginia K

2006-02-15

The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes.

PubMed

Noh, Ju Young; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Seo, Gi Won; Kim, Dong Hyun; Patnaik, Hongray Howrelia; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Han, Yeon Soo

2014-01-25

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5'-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3'. The 'helix-short helix-helix' motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor. Copyright © 2013 Elsevier B.V. All rights reserved.

Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene.

PubMed

Soria-Guerra, Ruth Elena; Rosales-Mendoza, Sergio; Márquez-Mercado, Crisóforo; López-Revilla, Rubén; Castillo-Collazo, Rosalba; Alpuche-Solís, Angel Gabriel

2007-07-01

A current priority of vaccinology is the development of multicomponent vaccines that protect against several pathogens. The diphtheria-pertussis-tetanus (DPT) vaccine prevents the symptoms of three serious and often fatal diseases due to the exotoxins produced by Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani. We are attempting to develop an edible DPT multicomponent vaccine in plants, based on the fusion of protective exotoxin epitopes encoded by synthetic genes. By means of Agrobacterium mediated transformation we generated transgenic tomatoes with a plant-optimised synthetic gene encoding a novel polypeptide containing two adjuvant and six DPT immunoprotective exotoxin epitopes joined by peptide linkers. In transformed tomato plants, integration of the synthetic DPT (sDPT) gene detected by PCR was confirmed by Southern blot, and specific transcripts of the expected molecular size were detected by RT-PCR. Expression of the putative polypeptide encoded by the sDPT gene was detected by immunoassay with specific antibodies to the diphtheria, pertussis and tetanus exotoxins. The sDPT gene is therefore integrated, transcribed and translated as the expected recombinant sDPT multiepitope polypeptide in transgenic tomatoes that constitute a potential edible vaccine.

Analysis of the hierarchy of quorum-sensing regulation in Pseudomonas aeruginosa.

PubMed

Wagner, Victoria E; Li, Luen-Luen; Isabella, Vincent M; Iglewski, Barbara H

2007-01-01

Quorum-sensing in Pseudomonas aeruginosa is known to regulate several aspects of pathogenesis, including virulence factor production, biofilm development, and antimicrobial resistance. Recent high-throughput analysis has revealed the existence of several layers of regulation within the QS-circuit. To address this complexity, mutations in genes encoding known or putative transcriptional regulators that were also identified as being regulated by the las and/or rhl QS systems were screened for their contribution in mediating several phenotypes, for example motility, secreted virulence products, and pathogenic capacity in a lettuce leaf model. These studies have further elucidated the potential contribution to virulence of these genes within the QS regulon.

Identification of the Sensory Neuron Specific Regulatory Region for the Mouse Gene Encoding the Voltage Gated Sodium Channel Nav1.8

PubMed Central

Puhl, Henry L.; Ikeda, Stephen R.

2008-01-01

Voltage-gated sodium channels (VGSC) are critical membrane components that participate in the electrical activity of excitable cells. The type one VGSC family includes the tetrodotoxin insensitive sodium channel, Nav1.8, encoded by the Scn10a gene. Nav1.8 expression is restricted to small and medium diameter nociceptive sensory neurons of the dorsal root (DRG) and cranial sensory ganglia. In order to understand the stringent transcriptional regulation of the Scn10a gene, the sensory neuron specific promoter was functionally identified. While identifying the mRNA 5’ end, alternative splicing within the 5’ UTR was observed to create heterogeneity in the RNA transcript. Four kilobases of upstream genomic DNA was cloned and the presence of tissue specific promoter activity was tested by microinjection and adenoviral infection of fluorescent protein reporter constructs into primary mouse and rat neurons, and cell lines. The region contained many putative transcription factor binding sites and strong homology with the predicted rat ortholog. Homology to the predicted human ortholog was limited to the proximal end and several conserved cis elements were noted. Two regulatory modules were identified by microinjection of reporter constructs into DRG and superior cervical ganglia neurons: a neuron specific proximal promoter region between −1.6 and −0.2kb of the transcription start site cluster, and a distal sensory neuron switch region beyond −1.6kb that restricted fluorescent protein expression to a subset of primary sensory neurons. PMID:18466327

Comparative analysis of the mating-type loci from Neurospora crassa and Sordaria macrospora: identification of novel transcribed ORFs.

PubMed

Pöggeler, S; Kück, U

2000-03-01

The mating-type locus controls mating and sexual development in filamentous ascomycetes. In the heterothallic ascomycete Neurospora crassa, the genes that confer mating behavior comprise dissimilar DNA sequences (idiomorphs) in the mat a and mat A mating partners. In the homothallic fungus Sordaria macrospora, sequences corresponding to both idiomorphs are located contiguously in the mating-type locus, which contains one chimeric gene, Smt A-3, that includes sequences which are similar to sequences found at the mat A and mat a mating-type idiomorphs in N. crassa. In this study, we describe the comparative transcriptional analysis of the chimeric mating-type region of S. macrospora and the corresponding region of the N. crassa mat a idiomorph. By means of RT-PCR experiments, we identified novel intervening sequences in the mating-type loci of both ascomycetes and, hence, concluded that an additional ORF, encoding a putative polypeptide of 79 amino acids, is present in the N. crassa mat a idiomorph. Furthermore, our analysis revealed co-transcription of the novel gene with the mat a-1 gene in N. crassa. The same mode of transcription was found in the corresponding mating-type region of S. macrospora, where the chimeric Smt A-3 gene is co-transcribed with the mat a-specific Smt a-1 gene. Analysis of a Smt A-3 cDNA revealed optional splicing of two introns. We believe that this is the first report of co-transcription of protein-encoding nuclear genes in filamentous fungi. Possible functions of the novel ORFs in regulating mating-type gene expression are discussed.

Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila.

PubMed

Andrews, Hillary K; Giagtzoglou, Nikolaos; Yamamoto, Shinya; Schulze, Karen L; Bellen, Hugo J

2009-06-01

The adult Drosophila external sensory organ (ESO), comprising the hair, socket, neuron, sheath and glia cells, arises through the asymmetric division of sensory organ precursor cells (SOPs). In a mosaic screen designed to identify new components in ESO development, we isolated mutations in sequoia, which encodes a putative zinc-finger transcription factor that has previously been shown to have a role in dendritogenesis. Here, we show that adult clones mutant for seq exhibit a loss of hair cells and a gain of socket cells. We propose that the seq mutant phenotype arises, in part, owing to the loss of several crucial transcription factors known to be important in peripheral nervous system development such as D-Pax2, Prospero and Hamlet. Thus, Sequoia is a new upstream regulator of genes that orchestrates cell fate specification during development of the adult ESO lineage.

Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila

PubMed Central

Andrews, Hillary K; Giagtzoglou, Nikolaos; Yamamoto, Shinya; Schulze, Karen L; Bellen, Hugo J

2009-01-01

The adult Drosophila external sensory organ (ESO), comprising the hair, socket, neuron, sheath and glia cells, arises through the asymmetric division of sensory organ precursor cells (SOPs). In a mosaic screen designed to identify new components in ESO development, we isolated mutations in sequoia, which encodes a putative zinc-finger transcription factor that has previously been shown to have a role in dendritogenesis. Here, we show that adult clones mutant for seq exhibit a loss of hair cells and a gain of socket cells. We propose that the seq mutant phenotype arises, in part, owing to the loss of several crucial transcription factors known to be important in peripheral nervous system development such as D-Pax2, Prospero and Hamlet. Thus, Sequoia is a new upstream regulator of genes that orchestrates cell fate specification during development of the adult ESO lineage. PMID:19444309

The Aquaporin Channel Repertoire of the Tardigrade Milnesium tardigradum

PubMed Central

Grohme, Markus A.; Mali, Brahim; Wełnicz, Weronika; Michel, Stephanie; Schill, Ralph O.; Frohme, Marcus

2013-01-01

Limno-terrestrial tardigrades are small invertebrates that are subjected to periodic drought of their micro-environment. They have evolved to cope with these unfavorable conditions by anhydrobiosis, an ametabolic state of low cellular water. During drying and rehydration, tardigrades go through drastic changes in cellular water content. By our transcriptome sequencing effort of the limno-terrestrial tardigrade Milnesium tardigradum and by a combination of cloning and targeted sequence assembly, we identified transcripts encoding eleven putative aquaporins. Analysis of these sequences proposed 2 classical aquaporins, 8 aquaglyceroporins and a single potentially intracellular unorthodox aquaporin. Using quantitative real-time PCR we analyzed aquaporin transcript expression in the anhydrobiotic context. We have identified additional unorthodox aquaporins in various insect genomes and have identified a novel common conserved structural feature in these proteins. Analysis of the genomic organization of insect aquaporin genes revealed several conserved gene clusters. PMID:23761966

The poly(C)-binding proteins: a multiplicity of functions and a search for mechanisms.

PubMed Central

Makeyev, Aleksandr V; Liebhaber, Stephen A

2002-01-01

The poly(C) binding proteins (PCBPs) are encoded at five dispersed loci in the mouse and human genomes. These proteins, which can be divided into two groups, hnRNPs K/J and the alphaCPs (alphaCP1-4), are linked by a common evolutionary history, a shared triple KH domain configuration, and by their poly(C) binding specificity. Given these conserved characteristics it is remarkable to find a substantial diversity in PCBP functions. The roles of these proteins in mRNA stabilization, translational activation, and translational silencing suggest a complex and diverse set of post-transcriptional control pathways. Their additional putative functions in transcriptional control and as structural components of important DNA-protein complexes further support their remarkable structural and functional versatility. Clearly the identification of additional binding targets and delineation of corresponding control mechanisms and effector pathways will establish highly informative models for further exploration. PMID:12003487

The poly(C)-binding proteins: a multiplicity of functions and a search for mechanisms.

PubMed

Makeyev, Aleksandr V; Liebhaber, Stephen A

2002-03-01

The poly(C) binding proteins (PCBPs) are encoded at five dispersed loci in the mouse and human genomes. These proteins, which can be divided into two groups, hnRNPs K/J and the alphaCPs (alphaCP1-4), are linked by a common evolutionary history, a shared triple KH domain configuration, and by their poly(C) binding specificity. Given these conserved characteristics it is remarkable to find a substantial diversity in PCBP functions. The roles of these proteins in mRNA stabilization, translational activation, and translational silencing suggest a complex and diverse set of post-transcriptional control pathways. Their additional putative functions in transcriptional control and as structural components of important DNA-protein complexes further support their remarkable structural and functional versatility. Clearly the identification of additional binding targets and delineation of corresponding control mechanisms and effector pathways will establish highly informative models for further exploration.

Identification and Characterization of Cyprinid Herpesvirus-3 (CyHV-3) Encoded MicroRNAs

PubMed Central

Donohoe, Owen H.; Henshilwood, Kathy; Way, Keith; Hakimjavadi, Roya; Stone, David M.; Walls, Dermot

2015-01-01

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional gene regulation. Some viruses encode their own miRNAs and these are increasingly being recognized as important modulators of viral and host gene expression. Cyprinid herpesvirus 3 (CyHV-3) is a highly pathogenic agent that causes acute mass mortalities in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) worldwide. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. Deep sequencing of small RNA fractions prepared from in vitro CyHV-3 infections led to the identification of potential miRNAs and miRNA–offset RNAs (moRNAs) derived from some bioinformatically predicted pre-miRNAs. DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. The evidence also suggested the presence of an additional four high-probability and two putative viral pre-miRNAs. MiRNAs from the two confirmed pre-miRNAs were also detected in gill tissue from CyHV-3-infected carp. We also present evidence that one confirmed miRNA can regulate the expression of a putative CyHV-3-encoded dUTPase. Candidate homologues of some CyHV-3 pre-miRNAs were identified in CyHV-1 and CyHV-2. This is the first report of miRNA and moRNA genes encoded by members of the Alloherpesviridae family, a group distantly related to the Herpesviridae family. The discovery of these novel CyHV-3 genes may help further our understanding of the biology of this economically important virus and their encoded miRNAs may have potential as biomarkers for the diagnosis of latent CyHV-3. PMID:25928140

5' diversity of human hepatic PXR (NR1I2) transcripts and identification of the major transcription initiation site.

PubMed

Kurose, Kouichi; Koyano, Satoru; Ikeda, Shinobu; Tohkin, Masahiro; Hasegawa, Ryuichi; Sawada, Jun-Ichi

2005-05-01

The human pregnane X receptor (PXR) is a crucial regulator of the genes encoding several major cytochrome P450 enzymes and transporters, such as CYP3A4 and MDR1, but its own transcriptional regulation remains unclear. To elucidate the transcriptional mechanisms of human PXR gene, we first endeavored to identify the transcription initiation site of human PXR using 5'-RACE. Five types of 5'-variable transcripts (a, b, c, d, and e) with common exon 2 sequence were found, and comparison of these sequences with the genomic sequence suggested that their 5' diversity is derived from initiation by alternative promoters and alternative splicing. None of the exons found in our study contain any new in-frame coding regions. Newly identified introns IVS-a and IVS-b were found to have CT-AC splice sites that do not follow the GT-AG rule of conventional donor and acceptor splice sites. Of the five types of 5' variable transcripts identified, RT-PCR showed that type-a was the major transcript type. Four transcription initiation sites (A-D) for type-a transcript were identified by 5'-RACE using GeneRacer RACE Ready cDNA (human liver) constructed by the oligo-capping method. Putative TATA boxes were located approximately 30 bp upstream from the transcriptional start sites of the major transcript (C) and the longest minor transcript (A) expressed in the human liver. These results indicate that the initiation of transcription of human PXR is more complex than previously reported.

Transcription of TP0126, Treponema pallidum Putative OmpW Homolog, Is Regulated by the Length of a Homopolymeric Guanosine Repeat

PubMed Central

Brandt, Stephanie L.; Ke, Wujian; Reid, Tara B.; Molini, Barbara J.; Iverson-Cabral, Stefanie; Ciccarese, Giulia; Drago, Francesco; Lukehart, Sheila A.; Centurion-Lara, Arturo

2015-01-01

An effective mechanism for introduction of phenotypic diversity within a bacterial population exploits changes in the length of repetitive DNA elements located within gene promoters. This phenomenon, known as phase variation, causes rapid activation or silencing of gene expression and fosters bacterial adaptation to new or changing environments. Phase variation often occurs in surface-exposed proteins, and in Treponema pallidum subsp. pallidum, the syphilis agent, it was reported to affect transcription of three putative outer membrane protein (OMP)-encoding genes. When the T. pallidum subsp. pallidum Nichols strain genome was initially annotated, the TP0126 open reading frame was predicted to include a poly(G) tract and did not appear to have a predicted signal sequence that might suggest the possibility of its being an OMP. Here we show that the initial annotation was incorrect, that this poly(G) is instead located within the TP0126 promoter, and that it varies in length in vivo during experimental syphilis. Additionally, we show that TP0126 transcription is affected by changes in the poly(G) length consistent with regulation by phase variation. In silico analysis of the TP0126 open reading frame based on the experimentally identified transcriptional start site shortens this hypothetical protein by 69 amino acids, reveals a predicted cleavable signal peptide, and suggests structural homology with the OmpW family of porins. Circular dichroism of recombinant TP0126 supports structural homology to OmpW. Together with the evidence that TP0126 is fully conserved among T. pallidum subspecies and strains, these data suggest an important role for TP0126 in T. pallidum biology and syphilis pathogenesis. PMID:25802057

LBH Gene Transcription Regulation by the Interplay of an Enhancer Risk Allele and DNA Methylation in Rheumatoid Arthritis.

PubMed

Hammaker, Deepa; Whitaker, John W; Maeshima, Keisuke; Boyle, David L; Ekwall, Anna-Karin H; Wang, Wei; Firestein, Gary S

2016-11-01

To identify nonobvious therapeutic targets for rheumatoid arthritis (RA), we performed an integrative analysis incorporating multiple "omics" data and the Encyclopedia of DNA Elements (ENCODE) database for potential regulatory regions. This analysis identified the limb bud and heart development (LBH) gene, which has risk alleles associated with RA/celiac disease and lupus, and can regulate cell proliferation in RA. We identified a novel LBH transcription enhancer with an RA risk allele (rs906868 G [Ref]/T) 6 kb upstream of the LBH gene with a differentially methylated locus. The confluence of 3 regulatory elements, rs906868, an RA differentially methylated locus, and a putative enhancer, led us to investigate their effects on LBH regulation in fibroblast-like synoviocytes (FLS). We cloned the 1.4-kb putative enhancer with either the rs906868 Ref allele or single-nucleotide polymorphism (SNP) variant into reporter constructs. The constructs were methylated in vitro and transfected into cultured FLS by nucleofection. We found that both variants increased transcription, thereby confirming the region's enhancer function. Unexpectedly, the transcriptional activity of the Ref risk allele was significantly lower than that of the SNP variant and is consistent with low LBH levels as a risk factor for aggressive FLS behavior. Using RA FLS lines with a homozygous Ref or SNP allele, we confirmed that homozygous Ref lines expressed lower LBH messenger RNA levels than did the SNP lines. Methylation significantly reduced enhancer activity for both alleles, indicating that enhancer function is dependent on its methylation status. This study shows how the interplay between genetics and epigenetics can affect expression of LBH in RA. © 2016, American College of Rheumatology.

Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

PubMed Central

Dai, Xin; Tian, Yan; Li, Jinting; Su, Xiaoyun; Wang, Xuewei; Zhao, Shengguo; Liu, Li; Luo, Yingfeng; Liu, Di; Zheng, Huajun; Wang, Jiaqi; Dong, Zhiyang

2014-01-01

The bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ∼1% and ∼0.1% of the total non-rRNAs, respectively. The majority (∼98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (∼82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen. PMID:25501482

Analyzing the soybean transcriptome during autoregulation of mycorrhization identifies the transcription factors GmNF-YA1a/b as positive regulators of arbuscular mycorrhization.

PubMed

Schaarschmidt, Sara; Gresshoff, Peter M; Hause, Bettina

2013-06-18

Similarly to the legume-rhizobia symbiosis, the arbuscular mycorrhiza interaction is controlled by autoregulation representing a feedback inhibition involving the CLAVATA1-like receptor kinase NARK in shoots. However, little is known about signals and targets down-stream of NARK. To find NARK-related transcriptional changes in mycorrhizal soybean (Glycine max) plants, we analyzed wild-type and two nark mutant lines interacting with the arbuscular mycorrhiza fungus Rhizophagus irregularis. Affymetrix GeneChip analysis of non-inoculated and partially inoculated plants in a split-root system identified genes with potential regulation by arbuscular mycorrhiza or NARK. Most transcriptional changes occur locally during arbuscular mycorrhiza symbiosis and independently of NARK. RT-qPCR analysis verified nine genes as NARK-dependently regulated. Most of them have lower expression in roots or shoots of wild type compared to nark mutants, including genes encoding the receptor kinase GmSIK1, proteins with putative function as ornithine acetyl transferase, and a DEAD box RNA helicase. A predicted annexin named GmAnnx1a is differentially regulated by NARK and arbuscular mycorrhiza in distinct plant organs. Two putative CCAAT-binding transcription factor genes named GmNF-YA1a and GmNF-YA1b are down-regulated NARK-dependently in non-infected roots of mycorrhizal wild-type plants and functional gene analysis confirmed a positive role for these genes in the development of an arbuscular mycorrhiza symbiosis. Our results indicate GmNF-YA1a/b as positive regulators in arbuscular mycorrhiza establishment, whose expression is down-regulated by NARK in the autoregulated root tissue thereby diminishing subsequent infections. Genes regulated independently of arbuscular mycorrhization by NARK support an additional function of NARK in symbioses-independent mechanisms.

Transcription activation mediated by a cyclic AMP receptor protein from Thermus thermophilus HB8.

PubMed

Shinkai, Akeo; Kira, Satoshi; Nakagawa, Noriko; Kashihara, Aiko; Kuramitsu, Seiki; Yokoyama, Shigeyuki

2007-05-01

The extremely thermophilic bacterium Thermus thermophilus HB8, which belongs to the phylum Deinococcus-Thermus, has an open reading frame encoding a protein belonging to the cyclic AMP (cAMP) receptor protein (CRP) family present in many bacteria. The protein named T. thermophilus CRP is highly homologous to the CRP family proteins from the phyla Firmicutes, Actinobacteria, and Cyanobacteria, and it forms a homodimer and interacts with cAMP. CRP mRNA and intracellular cAMP were detected in this strain, which did not drastically fluctuate during cultivation in a rich medium. The expression of several genes was altered upon disruption of the T. thermophilus CRP gene. We found six CRP-cAMP-dependent promoters in in vitro transcription assays involving DNA fragments containing the upstream regions of the genes exhibiting decreased expression in the CRP disruptant, indicating that the CRP is a transcriptional activator. The consensus T. thermophilus CRP-binding site predicted upon nucleotide sequence alignment is 5'-(C/T)NNG(G/T)(G/T)C(A/C)N(A/T)NNTCACAN(G/C)(G/C)-3'. This sequence is unique compared with the known consensus binding sequences of CRP family proteins. A putative -10 hexamer sequence resides at 18 to 19 bp downstream of the predicted T. thermophilus CRP-binding site. The CRP-regulated genes found in this study comprise clustered regularly interspaced short palindromic repeat (CRISPR)-associated (cas) ones, and the genes of a putative transcriptional regulator, a protein containing the exonuclease III-like domain of DNA polymerase, a GCN5-related acetyltransferase homolog, and T. thermophilus-specific proteins of unknown function. These results suggest a role for cAMP signal transduction in T. thermophilus and imply the T. thermophilus CRP is a cAMP-responsive regulator.

Transcriptome Changes Associated with Delayed Flower Senescence on Transgenic Petunia by Inducing Expression of etr1-1, a Mutant Ethylene Receptor

PubMed Central

Lin, Jing; Liu, Gang; Zhang, Zhen; Chang, Youhong; Reid, Michael S.; Jiang, Cai-Zhong

2013-01-01

Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr1-1), a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr1-1 gene was over-expressed under the control of a chemically-inducible promoter, which would allow expression of etr1-1 to be initiated at the desired time and stage of development. Here, we showed that transgenic plants grew and developed normally without a chemical inducer. Semi-quantitative RT-PCR demonstrated that the abundance of transcripts of Arabidopsis etr1-1 gene was substantially induced in flowers with 30 μM dexamethasone (DEX). Consequently, t he life of the flowers was almost doubled and the peak of ethylene production was delayed. We compared gene expression changes of petals with DEX to those without DEX at 24 h and 48 h by microarray. Our results indicated that transcripts of many putative genes encoding transcription factors were down-regulated by etr1-1 induced expression at the early stage. In addition, putative genes involved in gibberellin biosynthesis, response to jasmonic acid/gibberellins stimulus, cell wall modification, ethylene biosynthesis, and cell death were down-regulated associating with etr1-1 induced expression. We investigated time-course gene expression profiles and found two profiles which displayed totally opposite expression patterns under these two treatments. In these profiles, ‘the regulation of transcription’ was predominant in GO categories. Taking all results together, we concluded those transcription factors down-regulated at early stage might exert a major role in regulating the senescence process which were consequently characterized by cell wall modification and cell death. PMID:23874385

Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins.

PubMed

Hamady, Zaed Z R; Farrar, Mark D; Whitehead, Terence R; Holland, Keith T; Lodge, J Peter A; Carding, Simon R

2008-10-01

The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.

Metagenomic insights into the carbohydrate-active enzymes carried by the microorganisms adhering to solid digesta in the rumen of cows.

PubMed

Wang, Lingling; Hatem, Ayat; Catalyurek, Umit V; Morrison, Mark; Yu, Zhongtang

2013-01-01

The ruminal microbial community is a unique source of enzymes that underpin the conversion of cellulosic biomass. In this study, the microbial consortia adherent on solid digesta in the rumen of Jersey cattle were subjected to an activity-based metagenomic study to explore the genetic diversity of carbohydrolytic enzymes in Jersey cows, with a particular focus on cellulases and xylanases. Pyrosequencing and bioinformatic analyses of 120 carbohydrate-active fosmids identified genes encoding 575 putative Carbohydrate-Active Enzymes (CAZymes) and proteins putatively related to transcriptional regulation, transporters, and signal transduction coupled with polysaccharide degradation and metabolism. Most of these genes shared little similarity to sequences archived in databases. Genes that were predicted to encode glycoside hydrolases (GH) involved in xylan and cellulose hydrolysis (e.g., GH3, 5, 9, 10, 39 and 43) were well represented. A new subfamily (S-8) of GH5 was identified from contigs assigned to Firmicutes. These subfamilies of GH5 proteins also showed significant phylum-dependent distribution. A number of polysaccharide utilization loci (PULs) were found, and two of them contained genes encoding Sus-like proteins and cellulases that have not been reported in previous metagenomic studies of samples from the rumens of cows or other herbivores. Comparison with the large metagenomic datasets previously reported of other ruminant species (or cattle breeds) and wallabies showed that the rumen microbiome of Jersey cows might contain differing CAZymes. Future studies are needed to further explore how host genetics and diets affect the diversity and distribution of CAZymes and utilization of plant cell wall materials.

Identification of functional elements and regulatory circuits by Drosophila modENCODE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V.

2010-12-22

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- andmore » tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation. Several years after the complete genetic sequencing of many species, it is still unclear how to translate genomic information into a functional map of cellular and developmental programs. The Encyclopedia of DNA Elements (ENCODE) (1) and model organism ENCODE (modENCODE) (2) projects use diverse genomic assays to comprehensively annotate the Homo sapiens (human), Drosophila melanogaster (fruit fly), and Caenorhabditis elegans (worm) genomes, through systematic generation and computational integration of functional genomic data sets. Previous genomic studies in flies have made seminal contributions to our understanding of basic biological mechanisms and genome functions, facilitated by genetic, experimental, computational, and manual annotation of the euchromatic and heterochromatic genome (3), small genome size, short life cycle, and a deep knowledge of development, gene function, and chromosome biology. The functions of {approx}40% of the protein and nonprotein-coding genes [FlyBase 5.12 (4)] have been determined from cDNA collections (5, 6), manual curation of gene models (7), gene mutations and comprehensive genome-wide RNA interference screens (8-10), and comparative genomic analyses (11, 12). The Drosophila modENCODE project has generated more than 700 data sets that profile transcripts, histone modifications and physical nucleosome properties, general and specific transcription factors (TFs), and replication programs in cell lines, isolated tissues, and whole organisms across several developmental stages (Fig. 1). Here, we computationally integrate these data sets and report (i) improved and additional genome annotations, including full-length proteincoding genes and peptides as short as 21 amino acids; (ii) noncoding transcripts, including 132 candidate structural RNAs and 1608 nonstructural transcripts; (iii) additional Argonaute (Ago)-associated small RNA genes and pathways, including new microRNAs (miRNAs) encoded within protein-coding exons and endogenous small interfering RNAs (siRNAs) from 3-inch untranslated regions; (iv) chromatin 'states' defined by combinatorial patterns of 18 chromatin marks that are associated with distinct functions and properties; (v) regions of high TF occupancy and replication activity with likely epigenetic regulation; (vi)mixed TF and miRNA regulatory networks with hierarchical structure and enriched feed-forward loops; (vii) coexpression- and co-regulation-based functional annotations for nearly 3000 genes; (viii) stage- and tissue-specific regulators; and (ix) predictive models of gene expression levels and regulator function.« less

GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes

PubMed Central

Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

2014-01-01

Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12–ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex. PMID:25046113

The Putative C2H2 Transcription Factor MtfA Is a Novel Regulator of Secondary Metabolism and Morphogenesis in Aspergillus nidulans

PubMed Central

Ramamoorthy, Vellaisamy; Dhingra, Sourabh; Kincaid, Alexander; Shantappa, Sourabha; Feng, Xuehuan; Calvo, Ana M.

2013-01-01

Secondary metabolism in the model fungus Aspergillus nidulans is controlled by the conserved global regulator VeA, which also governs morphological differentiation. Among the secondary metabolites regulated by VeA is the mycotoxin sterigmatocystin (ST). The presence of VeA is necessary for the biosynthesis of this carcinogenic compound. We identified a revertant mutant able to synthesize ST intermediates in the absence of VeA. The point mutation occurred at the coding region of a gene encoding a novel putative C2H2 zinc finger domain transcription factor that we denominated mtfA. The A. nidulans mtfA gene product localizes at nuclei independently of the illumination regime. Deletion of the mtfA gene restores mycotoxin biosynthesis in the absence of veA, but drastically reduced mycotoxin production when mtfA gene expression was altered, by deletion or overexpression, in A. nidulans strains with a veA wild-type allele. Our study revealed that mtfA regulates ST production by affecting the expression of the specific ST gene cluster activator aflR. Importantly, mtfA is also a regulator of other secondary metabolism gene clusters, such as genes responsible for the synthesis of terrequinone and penicillin. As in the case of ST, deletion or overexpression of mtfA was also detrimental for the expression of terrequinone genes. Deletion of mtfA also decreased the expression of the genes in the penicillin gene cluster, reducing penicillin production. However, in this case, over-expression of mtfA enhanced the transcription of penicillin genes, increasing penicillin production more than 5 fold with respect to the control. Importantly, in addition to its effect on secondary metabolism, mtfA also affects asexual and sexual development in A. nidulans. Deletion of mtfA results in a reduction of conidiation and sexual stage. We found mtfA putative orthologs conserved in other fungal species. PMID:24066102

Two modes of control of pilA, the gene encoding type 1 pilin in Escherichia coli.

PubMed Central

Orndorff, P E; Spears, P A; Schauer, D; Falkow, S

1985-01-01

Type 1 piliation in Escherichia coli is subject to metastable regulation at the transcriptional level (B. I. Eisenstein, Science 214:337-339, 1981). However, the genes controlling in this fashion are not known. We present evidence that the pilA gene, encoding the structural subunit of type 1 pili, is subject to metastable transcriptional regulation. A pilA'-lacZ fusion, constructed in vitro on a recombinant plasmid, was used in conjunction with a recBC sbcB mutant of E. coli K-12 to introduce the fusion into the chromosomal region encoding Pil. This fusion was found to be subject to metastable transcriptional control. The rate of switching from the Lac+ to the Lac- phenotype was 4 X 10(-4) per cell per generation and 6.2 X 10(-4) in the opposite direction. A ca. 10-fold difference in beta-galactosidase activity was observed between phenotypically "ON" (Lac+) and "OFF" (Lac-) populations. P1 transduction experiments showed that the element determining the ON or OFF phenotype was tightly linked to pilA. In addition to the metastable regulation of pilA, a second type of transcriptional regulation was effected by the product of a gene, hyp, adjacent to pilA. By using a recombinant plasmid containing just a pilA'-lacZ fusion and the putative pilA promoter, we found that a lesion in hyp conferred a beta-galactosidase activity about fivefold higher than that of a strain possessing the parental hyp gene. Mutants constructed to have a pilA'-lacZ fusion and a hyp::Tn5-132 mutation in the chromosome exhibited a frequency of switching from Lac+ to Lac- and vice versa indistinguishable from that of the parental strain. However, in the ON mode, hyp::Tn5-132 mutants showed a twofold-higher beta-galactosidase activity. Thus, hyp does not appear to affect metastable variation but does affect the level of transcription of the pilA gene in the ON (transcribed) mode. Images PMID:3930469

Genome-Wide Search for Genes Required for Bifidobacterial Growth under Iron-Limitation

PubMed Central

Lanigan, Noreen; Bottacini, Francesca; Casey, Pat G.; O'Connell Motherway, Mary; van Sinderen, Douwe

2017-01-01

Bacteria evolved over millennia in the presence of the vital micronutrient iron. Iron is involved in numerous processes within the cell and is essential for nearly all living organisms. The importance of iron to the survival of bacteria is obvious from the large variety of mechanisms by which iron may be acquired from the environment. Random mutagenesis and global gene expression profiling led to the identification of a number of genes, which are essential for Bifidobacterium breve UCC2003 survival under iron-restrictive conditions. These genes encode, among others, Fe-S cluster-associated proteins, a possible ferric iron reductase, a number of cell wall-associated proteins, and various DNA replication and repair proteins. In addition, our study identified several presumed iron uptake systems which were shown to be essential for B. breve UCC2003 growth under conditions of either ferric and/or ferrous iron chelation. Of these, two gene clusters encoding putative iron-uptake systems, bfeUO and sifABCDE, were further characterised, indicating that sifABCDE is involved in ferrous iron transport, while the bfeUO-encoded transport system imports both ferrous and ferric iron. Transcription studies showed that bfeUO and sifABCDE constitute two separate transcriptional units that are induced upon dipyridyl-mediated iron limitation. In the anaerobic gastrointestinal environment ferrous iron is presumed to be of most relevance, though a mutation in the sifABCDE cluster does not affect B. breve UCC2003's ability to colonise the gut of a murine model. PMID:28620359

Vru (Sub0144) controls expression of proven and putative virulence determinants and alters the ability of Streptococcus uberis to cause disease in dairy cattle

PubMed Central

Egan, Sharon A.; Ward, Philip N.; Watson, Michael; Field, Terence R.

2012-01-01

The regulation and control of gene expression in response to differing environmental stimuli is crucial for successful pathogen adaptation and persistence. The regulatory gene vru of Streptococcus uberis encodes a stand-alone response regulator with similarity to the Mga of group A Streptococcus. Mga controls expression of a number of important virulence determinants. Experimental intramammary challenge of dairy cattle with a mutant of S. uberis carrying an inactivating lesion in vru showed reduced ability to colonize the mammary gland and an inability to induce clinical signs of mastitis compared with the wild-type strain. Analysis of transcriptional differences of gene expression in the mutant, determined by microarray analysis, identified a number of coding sequences with altered expression in the absence of Vru. These consisted of known and putative virulence determinants, including Lbp (Sub0145), SclB (Sub1095), PauA (Sub1785) and hasA (Sub1696). PMID:22383474

Soybean SAT1 (Symbiotic Ammonium Transporter 1) encodes a bHLH transcription factor involved in nodule growth and NH4+ transport

PubMed Central

Chiasson, David M.; Loughlin, Patrick C.; Mazurkiewicz, Danielle; Mohammadidehcheshmeh, Manijeh; Fedorova, Elena E.; Okamoto, Mamoru; McLean, Elizabeth; Glass, Anthony D. M.; Smith, Sally E.; Bisseling, Ton; Tyerman, Stephen D.; Day, David A.; Kaiser, Brent N.

2014-01-01

Glycine max symbiotic ammonium transporter 1 was first documented as a putative ammonium (NH4+) channel localized to the symbiosome membrane of soybean root nodules. We show that Glycine max symbiotic ammonium transporter 1 is actually a membrane-localized basic helix–loop–helix (bHLH) DNA-binding transcription factor now renamed Glycine max bHLH membrane 1 (GmbHLHm1). In yeast, GmbHLHm1 enters the nucleus and transcriptionally activates a unique plasma membrane NH4+ channel Saccharomyces cerevisiae ammonium facilitator 1. Ammonium facilitator 1 homologs are present in soybean and other plant species, where they often share chromosomal microsynteny with bHLHm1 loci. GmbHLHm1 is important to the soybean rhizobium symbiosis because loss of activity results in a reduction of nodule fitness and growth. Transcriptional changes in nodules highlight downstream signaling pathways involving circadian clock regulation, nutrient transport, hormone signaling, and cell wall modification. Collectively, these results show that GmbHLHm1 influences nodule development and activity and is linked to a novel mechanism for NH4+ transport common to both yeast and plants. PMID:24707045

Soybean SAT1 (Symbiotic Ammonium Transporter 1) encodes a bHLH transcription factor involved in nodule growth and NH4+ transport.

PubMed

Chiasson, David M; Loughlin, Patrick C; Mazurkiewicz, Danielle; Mohammadidehcheshmeh, Manijeh; Fedorova, Elena E; Okamoto, Mamoru; McLean, Elizabeth; Glass, Anthony D M; Smith, Sally E; Bisseling, Ton; Tyerman, Stephen D; Day, David A; Kaiser, Brent N

2014-04-01

Glycine max symbiotic ammonium transporter 1 was first documented as a putative ammonium (NH4(+)) channel localized to the symbiosome membrane of soybean root nodules. We show that Glycine max symbiotic ammonium transporter 1 is actually a membrane-localized basic helix-loop-helix (bHLH) DNA-binding transcription factor now renamed Glycine max bHLH membrane 1 (GmbHLHm1). In yeast, GmbHLHm1 enters the nucleus and transcriptionally activates a unique plasma membrane NH4(+) channel Saccharomyces cerevisiae ammonium facilitator 1. Ammonium facilitator 1 homologs are present in soybean and other plant species, where they often share chromosomal microsynteny with bHLHm1 loci. GmbHLHm1 is important to the soybean rhizobium symbiosis because loss of activity results in a reduction of nodule fitness and growth. Transcriptional changes in nodules highlight downstream signaling pathways involving circadian clock regulation, nutrient transport, hormone signaling, and cell wall modification. Collectively, these results show that GmbHLHm1 influences nodule development and activity and is linked to a novel mechanism for NH4(+) transport common to both yeast and plants.

The LysR-type transcription factor PacR is a global regulator of photosynthetic carbon assimilation in Anabaena.

PubMed

Picossi, Silvia; Flores, Enrique; Herrero, Antonia

2015-09-01

Cyanobacteria perform water-splitting photosynthesis and are important primary producers impacting the carbon and nitrogen cycles at global scale. They fix CO2 through ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) and have evolved a distinct CO2 concentrating mechanism (CCM) that builds high CO2 concentrations in the vicinity of RuBisCo favouring its carboxylase activity. Filamentous cyanobacteria such as Anabaena fix CO2 in photosynthetic vegetative cells, which donate photosynthate to heterocysts that rely on a heterotrophic metabolism to fix N2 . CCM elements are induced in response to inorganic carbon limitation, a cue that exposes the photosynthetic apparatus to photodamage by over-reduction. An Anabaena mutant lacking the LysR-type transcription factor All3953 grew poorly and dies under high light. The rbcL operon encoding RuBisCo was induced upon carbon limitation in the wild type but not in the mutant. ChIP-Seq analysis was used to globally identify All3953 targets under carbon limitation. Targets include, besides rbcL, genes encoding CCM elements, photorespiratory pathway- photosystem- and electron transport-related components, and factors, including flavodiiron proteins, with a demonstrated or putative function in photoprotection. Quantitative reverse transcription polymerase chain reaction analysis of selected All3953 targets showed regulation in the wild type but not in the mutant. All3953 (PacR) is a global regulator of carbon assimilation in an oxygenic photoautotroph. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos

PubMed Central

Famiglietti, Amber L.; Wei, Zheng; Beres, Thomas M.; Milac, Adina L.; Tran, Duy T.; Patel, Divya; Angerer, Robert C.; Angerer, Lynne M.

2017-01-01

Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered that morpholino-mediated knockdown of SpGalnt13 transcripts, results in a deficiency of embryonic skeleton and neurons, suggesting that mucin-type O-glycans play essential roles during embryonic development in S. purpuratus. PMID:28448610

[Exon-intron structure of the fet5+ gene of Schizosaccharomyces pombe and physical mapping of genome encompassing regions].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N

1998-01-01

Plasmid pYUK3 bearing the fet5+ gene of Schizosaccharomyces pombe was isolated from a genomic library of the fission yeast, and a detailed physical map of the whole genomic insert (ca. 9.6 Kbp) was constructed. The primary structure of the fet5+ gene and its flanking regions is established. The gene contains a single 45-bp intron in its distal part. A typical TATA-box (TATAAG) was found in the 5'-noncoding region ca. 50 bp upstream of the putative start of transcription, and the 3'-noncoding region contains AT-rich palindromes, which are probably involved in termination of the fet5+ transcription. A previously unidentified gene of Sz. pombe encoding a protein with some similarity to one of the transcriptional activators from the TBP (TATA-binding protein) group of SPT factors of transcription was found in the vicinity of the fet5+ gene. Taking into account that cDNA of the fet5(+)-gene was isolated as a suppressor of the genetic-defect of nuclear RNA polymerases I-III (Bioorg. Khim., 1997, vol. 23, No 3, pp. 234-237), this vicinity may be the first evidence of possible clustering, in the genome of the fission yeast, of genes participating in transcription regulation.

The head-regeneration transcriptome of the planarian Schmidtea mediterranea.

PubMed

Sandmann, Thomas; Vogg, Matthias C; Owlarn, Suthira; Boutros, Michael; Bartscherer, Kerstin

2011-08-16

Planarian flatworms can regenerate their head, including a functional brain, within less than a week. Despite the enormous potential of these animals for medical research and regenerative medicine, the mechanisms of regeneration and the molecules involved remain largely unknown. To identify genes that are differentially expressed during early stages of planarian head regeneration, we generated a de novo transcriptome assembly from more than 300 million paired-end reads from planarian fragments regenerating the head at 16 different time points. The assembly yielded 26,018 putative transcripts, including very long transcripts spanning multiple genomic supercontigs, and thousands of isoforms. Using short-read data from two platforms, we analyzed dynamic gene regulation during the first three days of head regeneration. We identified at least five different temporal synexpression classes, including genes specifically induced within a few hours after injury. Furthermore, we characterized the role of a conserved Runx transcription factor, smed-runt-like1. RNA interference (RNAi) knockdown and immunofluorescence analysis of the regenerating visual system indicated that smed-runt-like1 encodes a transcriptional regulator of eye morphology and photoreceptor patterning. Transcriptome sequencing of short reads allowed for the simultaneous de novo assembly and differential expression analysis of transcripts, demonstrating highly dynamic regulation during head regeneration in planarians.

The head-regeneration transcriptome of the planarian Schmidtea mediterranea

PubMed Central

2011-01-01

Background Planarian flatworms can regenerate their head, including a functional brain, within less than a week. Despite the enormous potential of these animals for medical research and regenerative medicine, the mechanisms of regeneration and the molecules involved remain largely unknown. Results To identify genes that are differentially expressed during early stages of planarian head regeneration, we generated a de novo transcriptome assembly from more than 300 million paired-end reads from planarian fragments regenerating the head at 16 different time points. The assembly yielded 26,018 putative transcripts, including very long transcripts spanning multiple genomic supercontigs, and thousands of isoforms. Using short-read data from two platforms, we analyzed dynamic gene regulation during the first three days of head regeneration. We identified at least five different temporal synexpression classes, including genes specifically induced within a few hours after injury. Furthermore, we characterized the role of a conserved Runx transcription factor, smed-runt-like1. RNA interference (RNAi) knockdown and immunofluorescence analysis of the regenerating visual system indicated that smed-runt-like1 encodes a transcriptional regulator of eye morphology and photoreceptor patterning. Conclusions Transcriptome sequencing of short reads allowed for the simultaneous de novo assembly and differential expression analysis of transcripts, demonstrating highly dynamic regulation during head regeneration in planarians. PMID:21846378

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed Central

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-01-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis. PMID:8892842

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-11-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.

Glucose transporter 8 (GLUT8) from the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae).

PubMed

Chen, Mei-Er; Holmes, Steven P; Pietrantonio, Patricia V

2006-06-01

We have cloned the fire ant glucose transporter 8 (GLUT8) cDNA providing the first molecular characterization of a GLUT8 in insects. Glucose is a poly-alcohol and, due to its high hydrophilicity, cannot move across cell membranes. GLUT8 is a putative facilitative transporter for the cellular import and export of glucose. The complete 2,974-bp cDNA encodes a 501-residue protein with a predicted molecular mass of 54.8 kDa. Transcripts were detected in the brain, midgut, hindgut, Malpighian tubule, fat body, ovary, and testis. The highest transcriptional expression was found in fat body. Northern blot analysis revealed different transcript sizes in mated queen brains, alate female ovaries, and male testes. We propose that four other sequences obtained from insect genome projects from the honey bee Apis mellifera (ENSAPMP00000006624), the malaria mosquito Anopheles gambiae (EAA11842), and the fruit fly Drosophila melanogaster (AAQ23604 and AAM52591) are likely the orthologues of the fire ant GLUT8. Phylogenetic relationships in insect glucose transporters are presented.

Characterizing the developmental transcriptome of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) through comparative genomic analysis with Drosophila melanogaster utilizing modENCODE datasets.

PubMed

Geib, Scott M; Calla, Bernarda; Hall, Brian; Hou, Shaobin; Manoukis, Nicholas C

2014-10-28

The oriental fruit fly, Bactrocera dorsalis, is an important pest of fruit and vegetable crops throughout Asia, and is considered a high risk pest for establishment in the mainland United States. It is a member of the family Tephritidae, which are the most agriculturally important family of flies, and can be considered an out-group to well-studied members of the family Drosophilidae. Despite their importance as pests and their relatedness to Drosophila, little information is present on B. dorsalis transcripts and proteins. The objective of this paper is to comprehensively characterize the transcripts present throughout the life history of B. dorsalis and functionally annotate and analyse these transcripts relative to the presence, expression, and function of orthologous sequences present in Drosophila melanogaster. We present a detailed transcriptome assembly of B. dorsalis from egg through adult stages containing 20,666 transcripts across 10,799 unigene components. Utilizing data available through Flybase and the modENCODE project, we compared expression patterns of these transcripts to putative orthologs in D. melanogaster in terms of timing, abundance, and function. In addition, temporal expression patterns in B. dorsalis were characterized between stages, to establish the constitutive or stage-specific expression patterns of particular transcripts. A fully annotated transcriptome assembly is made available through NCBI, in addition to corresponding expression data. Through characterizing the transcriptome of B. dorsalis through its life history and comparing the transcriptome of B. dorsalis to the model organism D. melanogaster, a database has been developed that can be used as the foundation to functional genomic research in Bactrocera flies and help identify orthologous genes between B. dorsalis and D. melanogaster. This data provides the foundation for future functional genomic research that will focus on improving our understanding of the physiology and biology of this species at the molecular level. This knowledge can also be applied towards developing improved methods for control, survey, and eradication of this important pest.

Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury.

PubMed Central

Witzgall, R; O'Leary, E; Gessner, R; Ouellette, A J; Bonventre, J V

1993-01-01

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins. Images PMID:8382778

Genome-wide organization and expression profiling of the NAC transcription factor family in potato (Solanum tuberosum L.).

PubMed

Singh, Anil Kumar; Sharma, Vishal; Pal, Awadhesh Kumar; Acharya, Vishal; Ahuja, Paramvir Singh

2013-08-01

NAC [no apical meristem (NAM), Arabidopsis thaliana transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins belong to one of the largest plant-specific transcription factor (TF) families and play important roles in plant development processes, response to biotic and abiotic cues and hormone signalling. Our genome-wide analysis identified 110 StNAC genes in potato encoding for 136 proteins, including 14 membrane-bound TFs. The physical map positions of StNAC genes on 12 potato chromosomes were non-random, and 40 genes were found to be distributed in 16 clusters. The StNAC proteins were phylogenetically clustered into 12 subgroups. Phylogenetic analysis of StNACs along with their Arabidopsis and rice counterparts divided these proteins into 18 subgroups. Our comparative analysis has also identified 36 putative TNAC proteins, which appear to be restricted to Solanaceae family. In silico expression analysis, using Illumina RNA-seq transcriptome data, revealed tissue-specific, biotic, abiotic stress and hormone-responsive expression profile of StNAC genes. Several StNAC genes, including StNAC072 and StNAC101that are orthologs of known stress-responsive Arabidopsis RESPONSIVE TO DEHYDRATION 26 (RD26) were identified as highly abiotic stress responsive. Quantitative real-time polymerase chain reaction analysis largely corroborated the expression profile of StNAC genes as revealed by the RNA-seq data. Taken together, this analysis indicates towards putative functions of several StNAC TFs, which will provide blue-print for their functional characterization and utilization in potato improvement.

Gene silencing reveals multiple functions of Na+/K+-ATPase in the salmon louse (Lepeophtheirus salmonis).

PubMed

Komisarczuk, Anna Z; Kongshaug, Heidi; Nilsen, Frank

2018-02-01

Na + /K + -ATPase has a key function in a variety of physiological processes including membrane excitability, osmoregulation, regulation of cell volume, and transport of nutrients. While knowledge about Na + /K + -ATPase function in osmoregulation in crustaceans is extensive, the role of this enzyme in other physiological and developmental processes is scarce. Here, we report characterization, transcriptional distribution and likely functions of the newly identified L. salmonis Na + /K + -ATPase (LsalNa + /K + -ATPase) α subunit in various developmental stages. The complete mRNA sequence was identified, with 3003 bp open reading frame encoding a putative protein of 1001 amino acids. Putative protein sequence of LsalNa + /K + -ATPase revealed all typical features of Na + /K + -ATPase and demonstrated high sequence identity to other invertebrate and vertebrate species. Quantitative RT-PCR analysis revealed higher LsalNa + /K + -ATPase transcript level in free-living stages in comparison to parasitic stages. In situ hybridization analysis of copepodids and adult lice revealed LsalNa + /K + -ATPase transcript localization in a wide variety of tissues such as nervous system, intestine, reproductive system, and subcuticular and glandular tissue. RNAi mediated knock-down of LsalNa + /K + -ATPase caused locomotion impairment, and affected reproduction and feeding. Morphological analysis of dsRNA treated animals revealed muscle degeneration in larval stages, severe changes in the oocyte formation and maturation in females and abnormalities in tegmental glands. Thus, the study represents an important foundation for further functional investigation and identification of physiological pathways in which Na + /K + -ATPase is directly or indirectly involved. Copyright © 2018 Elsevier Inc. All rights reserved.

Global Microarray Analysis of Carbohydrate Use in Alkaliphilic Hemicellulolytic Bacterium Bacillus sp. N16-5

PubMed Central

Song, Yajian; Xue, Yanfen; Ma, Yanhe

2013-01-01

The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose) and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose) using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources. PMID:23326578

Expression of arsenic resistance genes in the obligate anaerobe Bacteroides vulgatus ATCC 8482, a gut microbiome bacterium

PubMed Central

Li, Jiaojiao; Mandal, Goutam; Rosen, Barry P.

2016-01-01

The response of the obligate anaerobe Bacteroides vulgatus ATCC 8482, a common human gut microbiota, to arsenic was determined. B. vulgatus ATCC 8482 is highly resistant to pentavalent As(V) and methylarsenate (MAs(V)). It is somewhat more sensitive to trivalent inorganic As(III) but 100-fold more sensitive to methylarsenite (MAs(III)) than to As(III). B. vulgatus ATCC 8482 has eight continuous genes in its genome that we demonstrate form an arsenical-inducible transcriptional unit. The first gene of this ars operon, arsR, encodes a putative ArsR As(III)-responsive transcriptional repressor. The next three genes encode proteins of unknown function. The remaining genes, arsDABC, have well-characterized roles in detoxification of inorganic arsenic, but there are no known genes for MAs(III) resistance. Expression of each gene after exposure to trivalent and pentavalent inorganic and methylarsenicals was analyzed. MAs(III) was the most effective inducer. The arsD gene was the most highly expressed of the ars operon genes. These results demonstrate that this anaerobic microbiome bacterium has arsenic-responsive genes that confer resistance to inorganic arsenic and may be responsible for the organism's ability to maintain its prevalence in the gut following dietary exposure to inorganic arsenic. PMID:27040269

QTLs Regulating the Contents of Antioxidants, Phenolics, and Flavonoids in Soybean Seeds Share a Common Genomic Region.

PubMed

Li, Man-Wah; Muñoz, Nacira B; Wong, Chi-Fai; Wong, Fuk-Ling; Wong, Kwong-Sen; Wong, Johanna Wing-Hang; Qi, Xinpeng; Li, Kwan-Pok; Ng, Ming-Sin; Lam, Hon-Ming

2016-01-01

Soybean seeds are a rich source of phenolic compounds, especially isoflavonoids, which are important nutraceuticals. Our study using 14 wild- and 16 cultivated-soybean accessions shows that seeds from cultivated soybeans generally contain lower total antioxidants compared to their wild counterparts, likely an unintended consequence of domestication or human selection. Using a recombinant inbred population resulting from a wild and a cultivated soybean parent and a bin map approach, we have identified an overlapping genomic region containing major quantitative trait loci (QTLs) that regulate the seed contents of total antioxidants, phenolics, and flavonoids. The QTL for seed antioxidant content contains 14 annotated genes based on the Williams 82 reference genome (Gmax1.01). None of these genes encodes functions that are related to the phenylpropanoid pathway of soybean. However, we found three putative Multidrug And Toxic Compound Extrusion (MATE) transporter genes within this QTL and one adjacent to it (GmMATE1-4). Moreover, we have identified non-synonymous changes between GmMATE1 and GmMATE2, and that GmMATE3 encodes an antisense transcript that expresses in pods. Whether the polymorphisms in GmMATE proteins are major determinants of the antioxidant contents, or whether the antisense transcripts of GmMATE3 play important regulatory roles, awaits further functional investigations.

Zinc-Responsive Regulation of Alternative Ribosomal Protein Genes in Streptomyces coelicolor Involves Zur and σR▿ †

PubMed Central

Owen, Gillian A.; Pascoe, Ben; Kallifidas, Dimitris; Paget, Mark S. B.

2007-01-01

Streptomyces coelicolor contains paralogous versions of seven ribosomal proteins (S14, S18, L28, L31, L32, L33, and L36), which differ in their potential to bind structural zinc. The paralogues are termed C+ or C− on the basis of the presence or absence of putative cysteine ligands. Here, mutational studies suggest that the C− version of L31 can functionally replace its C+ paralogue only when expressed at an artificially elevated level. We show that the level of expression of four transcriptional units encoding C− proteins is elevated under conditions of zinc deprivation. Zur controls the expression of three transcriptional units (including rpmG2, rpmE2, rpmB2, rpsN2, rpmF2, and possibly rpsR2). Zur also controls the expression of the znuACB operon, which is predicted to encode a high-affinity zinc transport system. Surprisingly, the zinc-responsive control of the rpmG3-rpmJ2 operon is dictated by σR, a sigma factor that was previously shown to control the response to disulfide stress in S. coelicolor. The induction of σR activity during zinc limitation establishes an important link between thiol-disulfide metabolism and zinc homeostasis. PMID:17400736

Zinc-responsive regulation of alternative ribosomal protein genes in Streptomyces coelicolor involves zur and sigmaR.

PubMed

Owen, Gillian A; Pascoe, Ben; Kallifidas, Dimitris; Paget, Mark S B

2007-06-01

Streptomyces coelicolor contains paralogous versions of seven ribosomal proteins (S14, S18, L28, L31, L32, L33, and L36), which differ in their potential to bind structural zinc. The paralogues are termed C(+) or C(-) on the basis of the presence or absence of putative cysteine ligands. Here, mutational studies suggest that the C(-) version of L31 can functionally replace its C(+) paralogue only when expressed at an artificially elevated level. We show that the level of expression of four transcriptional units encoding C(-) proteins is elevated under conditions of zinc deprivation. Zur controls the expression of three transcriptional units (including rpmG2, rpmE2, rpmB2, rpsN2, rpmF2, and possibly rpsR2). Zur also controls the expression of the znuACB operon, which is predicted to encode a high-affinity zinc transport system. Surprisingly, the zinc-responsive control of the rpmG3-rpmJ2 operon is dictated by sigma(R), a sigma factor that was previously shown to control the response to disulfide stress in S. coelicolor. The induction of sigma(R) activity during zinc limitation establishes an important link between thiol-disulfide metabolism and zinc homeostasis.

A Genetic Locus Necessary for Rhamnose Uptake and Catabolism in Rhizobium leguminosarum bv. trifolii

PubMed Central

Richardson, Jason S.; Hynes, Michael F.; Oresnik, Ivan J.

2004-01-01

Rhizobium leguminosarum bv. trifolii mutants unable to catabolize the methyl-pentose rhamnose are unable to compete effectively for nodule occupancy. In this work we show that the locus responsible for the transport and catabolism of rhamnose spans 10,959 bp. Mutations in this region were generated by transposon mutagenesis, and representative mutants were characterized. The locus contains genes coding for an ABC-type transporter, a putative dehydrogenase, a probable isomerase, and a sugar kinase necessary for the transport and subsequent catabolism of rhamnose. The regulation of these genes, which are inducible by rhamnose, is carried out in part by a DeoR-type negative regulator (RhaR) that is encoded within the same transcript as the ABC-type transporter but is separated from the structural genes encoding the transporter by a terminator-like sequence. RNA dot blot analysis demonstrated that this terminator-like sequence is correlated with transcript attenuation only under noninducing conditions. Transport assays utilizing tritiated rhamnose demonstrated that uptake of rhamnose was inducible and dependent upon the presence of the ABC transporter at this locus. Phenotypic analyses of representative mutants from this locus provide genetic evidence that the catabolism of rhamnose differs from previously described methyl-pentose catabolic pathways. PMID:15576793

Flower-predominant expression of a gene encoding a novel class I chitinase in rice (Oryza sativa L.).

PubMed

Takakura, Y; Ito, T; Saito, H; Inoue, T; Komari, T; Kuwata, S

2000-04-01

A flower-predominant cDNA for a gene, termed OsChia 1;175, was isolated from a cDNA library of rice pistils. Northern blot and RT-PCR analyses revealed that the OsChia 1;175 gene is highly expressed in floral organs (pistils, stamens and lodicules at the heading stage) but not or at an extremely low level in vegetative organs. OsChia 1;175 encodes a protein that consists of 340 amino acid residues, and the putative mature protein shows 52% to 63% amino acid identity to class I chitinases of rice or other plants. The phylogenetic tree shows that the OsChia 1;175 protein is a new type of plant class I chitinase in rice. The expression of OsChia 1;175 in vegetative organs is not induced by several chemicals, UV, and wounding. The soluble putative mature OsChia 1;175 protein expressed in Escherichia coli exhibited chitinase activity in the assay with colloidal chitin as a substrate. Genomic Southern analysis revealed that the OsChia 1;175 gene was organized as a low-copy gene family. The rice genomic library was screened and a genome clone corresponding to OsChia 1;175 was isolated. The transcription start sites of the OsChia 1;175 gene were mapped by primer extension analysis. The 1.2 kb putative promoter region of the OsChia 1;175 gene was fused to the GUS (beta-glucuronidase) gene, and this chimeric gene was introduced to rice by Agrobacterium-mediated transformation. The flower-predominant gene expression was identified also in the transgenic rice plants. The high promoter activity was detected in the stigmas, styles, stamens and lodicules in transgenic plants. The possible functions of OsChia 1;175 are discussed.

Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes.

PubMed

Diallinas, G; Gorfinkiel, L; Arst, H N; Cecchetto, G; Scazzocchio, C

1995-04-14

In Aspergillus nidulans, loss-of-function mutations in the uapA and azgA genes, encoding the major uric acid-xanthine and hypoxanthine-adenine-guanine permeases, respectively, result in impaired utilization of these purines as sole nitrogen sources. The residual growth of the mutant strains is due to the activity of a broad specificity purine permease. We have identified uapC, the gene coding for this third permease through the isolation of both gain-of-function and loss-of-function mutations. Uptake studies with wild-type and mutant strains confirmed the genetic analysis and showed that the UapC protein contributes 30% and 8-10% to uric acid and hypoxanthine transport rates, respectively. The uapC gene was cloned, its expression studied, its sequence and transcript map established, and the sequence of its putative product analyzed. uapC message accumulation is: (i) weakly induced by 2-thiouric acid; (ii) repressed by ammonium; (iii) dependent on functional uaY and areA regulatory gene products (mediating uric acid induction and nitrogen metabolite repression, respectively); (iv) increased by uapC gain-of-function mutations which specifically, but partially, suppress a leucine to valine mutation in the zinc finger of the protein coded by the areA gene. The putative uapC gene product is a highly hydrophobic protein of 580 amino acids (M(r) = 61,251) including 12-14 putative transmembrane segments. The UapC protein is highly similar (58% identity) to the UapA permease and significantly similar (23-34% identity) to a number of bacterial transporters. Comparisons of the sequences and hydropathy profiles of members of this novel family of transporters yield insights into their structure, functionally important residues, and possible evolutionary relationships.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-06-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed Central

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-01-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

Genetic transformation of Dichanthium annulatum (Forssk)--an apomictic tropical forage grass.

PubMed

Dalton, S J; Bettany, A J E; Bhat, V; Gupta, M G; Bailey, K; Timms, E; Morris, P

2003-06-01

Eleven Dichanthium annulatum (Forssk) plants were regenerated from embryogenic callus co-transformed with two plasmids encoding either the hygromycin phosphotransferase gene (hph) or the beta-glucuronidase (GUS) gene (uidA). Analysis of these putative transformants showed that three plants were transformed with the hph gene, showed the presence of the hph transcript and expressed hygromycin resistance after transfer to soil. Two of these also contained the uidA gene but did not express GUS and were shown to be the same transformation event. All three of the transformants set seed. Hygromycin resistance varied from 68-100% in the progeny of the three transformants. Transgene transmission appeared to have been mainly through apomixis.

Mapping of the Tuple1 gene to mouse chromosome 16A-B1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mattei, M.G.; Halford, S.; Scambler, P.J.

The human TUPLE1 gene encodes a putative transcriptional regulator and maps to chromosome 22, and therefore may play a role in Di-George syndrome (DGS), relo-cardio-facial syndrome (VCFS), or a related pathology. The murine TUPLE1 gene has also been cloned and shows strong sequence similarity to TUPLE1. Comparative mapping is useful in the study of chromosome evolution and is sometimes able to indicate possible mouse mutations that are potential models of human genetic disorders. As TIPLE1 is a candidate gene for the haploinsufficient phenotype in DGS, we mapped TUPLE1 to mouse chromosome 16A-B1. 6 refs., 1 fig.

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop

PubMed Central

Ligerot, Yasmine; de Saint Germain, Alexandre; Troadec, Christelle; Citerne, Sylvie; Pillot, Jean-Paul; Prigge, Michael; Aubert, Grégoire; Bendahmane, Abdelhafid; Estelle, Mark; Debellé, Frédéric

2017-01-01

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner. PMID:29220348

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop.

PubMed

Ligerot, Yasmine; de Saint Germain, Alexandre; Waldie, Tanya; Troadec, Christelle; Citerne, Sylvie; Kadakia, Nikita; Pillot, Jean-Paul; Prigge, Michael; Aubert, Grégoire; Bendahmane, Abdelhafid; Leyser, Ottoline; Estelle, Mark; Debellé, Frédéric; Rameau, Catherine

2017-12-01

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner.

An Alternative σ Factor, σ8, Controls Avermectin Production and Multiple Stress Responses in Streptomyces avermitilis.

PubMed

Sun, Di; Wang, Qian; Chen, Zhi; Li, Jilun; Wen, Ying

2017-01-01

Alternative σ factors in bacteria redirect RNA polymerase to recognize alternative promoters, thereby facilitating coordinated gene expression necessary for adaptive responses. The gene sig8 ( sav_741 ) in Streptomyces avermitilis encodes an alternative σ factor, σ 8 , highly homologous to σ B in Streptomyces coelicolor . Studies reported here demonstrate that σ 8 is an important regulator of both avermectin production and stress responses in S. avermitilis . σ 8 inhibited avermectin production by indirectly repressing expression of cluster-situated activator gene aveR , and by directly initiating transcription of its downstream gene sav_742 , which encodes a direct repressor of ave structural genes. σ 8 had no effect on cell growth or morphological differentiation under normal growth conditions. Growth of a sig8- deletion mutant was less than that of wild-type strain on YMS plates following treatment with heat, H 2 O 2 , diamide, NaCl, or KCl. sig8 transcription was strongly induced by these environmental stresses, indicating response by σ 8 itself. A series of σ 8 -dependent genes responsive to heat, oxidative and osmotic stress were identified by EMSAs, qRT-PCR and in vitro transcription experiments. These findings indicate that σ 8 plays an important role in mediating protective responses to various stress conditions by activating transcription of its target genes. Six σ 8 -binding promoter sequences were determined and consensus binding sequence BGVNVH-N 15 -GSNNHH (B: C, T or G, V: A, C or G, S: C or G, H: A, C or T, N: any nucleotide) was identified, leading to prediction of the σ 8 regulon. The list consists of 940 putative σ 8 target genes, assignable to 17 functional groups, suggesting the wide range of cellular functions controlled by σ 8 in S. avermitilis .

Lhx6 and Lhx8 promote palate development through negative regulation of a cell cycle inhibitor gene, p57Kip2

PubMed Central

Cesario, Jeffry M.; Landin Malt, Andre; Deacon, Lindsay J.; Sandberg, Magnus; Vogt, Daniel; Tang, Zuojian; Zhao, Yangu; Brown, Stuart; Rubenstein, John L.; Jeong, Juhee

2015-01-01

Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives. Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57Kip2 (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6−/−;Lhx8−/− mutants. p57Kip2 has been linked to Beckwith–Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57Kip2 by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor-binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57Kip2 via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors. Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57Kip2 expression in the prospective palate area to allow adequate levels of cell proliferation and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development. PMID:26071365

Functional signaling and gene regulatory networks between the oocyte and the surrounding cumulus cells.

PubMed

Biase, Fernando H; Kimble, Katelyn M

2018-05-10

The maturation and successful acquisition of developmental competence by an oocyte, the female gamete, during folliculogenesis is highly dependent on molecular interactions with somatic cells. Most of the cellular interactions identified, thus far, are modulated by growth factors, ions or metabolites. We hypothesized that this interaction is also modulated at the transcriptional level, which leads to the formation of gene regulatory networks between the oocyte and cumulus cells. We tested this hypothesis by analyzing transcriptome data from single oocytes and the surrounding cumulus cells collected from antral follicles employing an analytical framework to determine interdependencies at the transcript level. We overlapped our transcriptome data with putative protein-protein interactions and identified hundreds of ligand-receptor pairs that can transduce paracrine signaling between an oocyte and cumulus cells. We determined that 499 ligand-encoding genes expressed in oocytes and cumulus cells are functionally associated with transcription regulation (FDR < 0.05). Ligand-encoding genes with specific expression in oocytes or cumulus cells were enriched for biological functions that are likely associated with the coordinated formation of transzonal projections from cumulus cells that reach the oocyte's membrane. Thousands of gene pairs exhibit significant linear co-expression (absolute correlation > 0.85, FDR < 1.8 × 10 - 5 ) patterns between oocytes and cumulus cells. Hundreds of co-expressing genes showed clustering patterns associated with biological functions (FDR < 0.5) necessary for a coordinated function between the oocyte and cumulus cells during folliculogenesis (i.e. regulation of transcription, translation, apoptosis, cell differentiation and transport). Our analyses revealed a complex and functional gene regulatory circuit between the oocyte and surrounding cumulus cells. The regulatory profile of each cumulus-oocyte complex is likely associated with the oocytes' developmental potential to derive an embryo.

The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA.

PubMed

Mars, Ruben A T; Mendonça, Karoline; Denham, Emma L; van Dijl, Jan Maarten

2015-10-01

One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress. Copyright © 2015 Elsevier B.V. All rights reserved.

EARLY BUD-BREAK 1 (EBB1) is a regulator of release from seasonal dormancy in poplar trees

PubMed Central

Yordanov, Yordan S.; Ma, Cathleen; Strauss, Steven H.; Busov, Victor B.

2014-01-01

Trees from temperate latitudes transition between growth and dormancy to survive dehydration and freezing stress during winter months. We used activation tagging to isolate a dominant mutation affecting release from dormancy and identified the corresponding gene EARLY BUD-BREAK 1 (EBB1). We demonstrate through positioning of the tag, expression analysis, and retransformation experiments that EBB1 encodes a putative APETALA2/Ethylene responsive factor transcription factor. Transgenic up-regulation of the gene caused early bud-flush, whereas down-regulation delayed bud-break. Native EBB1 expression was highest in actively growing apices, undetectable during the dormancy period, but rapidly increased before bud-break. The EBB1 transcript was localized in the L1/L2 layers of the shoot meristem and leaf primordia. EBB1-overexpressing transgenic plants displayed enlarged shoot meristems, open and poorly differentiated buds, and a higher rate of cell division in the apex. Transcriptome analyses of the EBB1 transgenics identified 971 differentially expressed genes whose expression correlated with the EBB1 expression changes in the transgenic plants. Promoter analysis among the differentially expressed genes for the presence of a canonical EBB1-binding site identified 65 putative target genes, indicative of a broad regulatory context of EBB1 function. Our results suggest that EBB1 has a major and integrative role in reactivation of meristem activity after winter dormancy. PMID:24951507

Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Hao, E-mail: hao.hu1@uqconnect.edu.au; Yu, Ting, E-mail: t.yu2@uq.edu.au; Arpiainen, Satu, E-mail: Satu.Juhila@orion.fi

2015-11-15

Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3 kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5′-Luc constructs – down to − 160 bp from the TSS – showed p53 responsivenessmore » in p53 overexpressed C3A cells. However, a further deletion from − 160 to − 74 bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene – a well-known p53 activator – increased the expression of the p53 responsive positive control and the CYP2A6-5′-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5′-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6. - Highlights: • CYP2A6 is an immediate target gene of p53. • Six putative p53REs located on 3 kb proximate CYP2A6 promoter region. • The region − 160 bp from TSS is highly homologous with the p53 consensus sequence. • P53 specifically bind to the p53RE on the − 160 bp region. • HNF4α may interact with p53 in regulating CYP2A6 expression.« less

Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

PubMed

Graentzdoerffer, Andrea; Rauh, David; Pich, Andreas; Andreesen, Jan R

2003-01-01

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

SEMI-ROLLED LEAF1 Encodes a Putative Glycosylphosphatidylinositol-Anchored Protein and Modulates Rice Leaf Rolling by Regulating the Formation of Bulliform Cells1[W][OA

PubMed Central

Xiang, Jing-Jing; Zhang, Guang-Heng; Qian, Qian; Xue, Hong-Wei

2012-01-01

Leaf rolling is an important agronomic trait in rice (Oryza sativa) breeding and moderate leaf rolling maintains the erectness of leaves and minimizes shadowing between leaves, leading to improved photosynthetic efficiency and grain yields. Although a few rolled-leaf mutants have been identified and some genes controlling leaf rolling have been isolated, the molecular mechanisms of leaf rolling still need to be elucidated. Here we report the isolation and characterization of SEMI-ROLLED LEAF1 (SRL1), a gene involved in the regulation of leaf rolling. Mutants srl1-1 (point mutation) and srl1-2 (transferred DNA insertion) exhibit adaxially rolled leaves due to the increased numbers of bulliform cells at the adaxial cell layers, which could be rescued by complementary expression of SRL1. SRL1 is expressed in various tissues and is expressed at low levels in bulliform cells. SRL1 protein is located at the plasma membrane and predicted to be a putative glycosylphosphatidylinositol-anchored protein. Moreover, analysis of the gene expression profile of cells that will become epidermal cells in wild type but probably bulliform cells in srl1-1 by laser-captured microdissection revealed that the expression of genes encoding vacuolar H+-ATPase (subunits A, B, C, and D) and H+-pyrophosphatase, which are increased during the formation of bulliform cells, were up-regulated in srl1-1. These results provide the transcript profile of rice leaf cells that will become bulliform cells and demonstrate that SRL1 regulates leaf rolling through inhibiting the formation of bulliform cells by negatively regulating the expression of genes encoding vacuolar H+-ATPase subunits and H+-pyrophosphatase, which will help to understand the mechanism regulating leaf rolling. PMID:22715111

Sequence analysis and expression of the M1 and M2 matrix protein genes of hirame rhabdovirus (HIRRV)

USGS Publications Warehouse

Nishizawa, T.; Kurath, G.; Winton, J.R.

1997-01-01

We have cloned and sequenced a 2318 nucleotide region of the genomic RNA of hirame rhabdovirus (HIRRV), an important viral pathogen of Japanese flounder Paralichthys olivaceus. This region comprises approximately two-thirds of the 3' end of the nucleocapsid protein (N) gene and the complete matrix protein (M1 and M2) genes with the associated intergenic regions. The partial N gene sequence was 812 nucleotides in length with an open reading frame (ORF) that encoded the carboxyl-terminal 250 amino acids of the N protein. The M1 and M2 genes were 771 and 700 nucleotides in length, respectively, with ORFs encoding proteins of 227 and 193 amino acids. The M1 gene sequence contained an additional small ORF that could encode a highly basic, arginine-rich protein of 25 amino acids. Comparisons of the N, M1, and M2 gene sequences of HIRRV with the corresponding sequences of the fish rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) or viral hemorrhagic septicemia virus (VHSV) indicated that HIRRV was more closely related to IHNV than to VHSV, but was clearly distinct from either. The putative consensus gene termination sequence for IHNV and VHSV, AGAYAG(A)(7), was present in the N-M1, M1-M2, and M2-G intergenic regions of HIRRV as were the putative transcription initiation sequences YGGCAC and AACA. An Escherichia coli expression system was used to produce recombinant proteins from the M1 and M2 genes of HIRRV. These were the same size as the authentic M1 and M2 proteins and reacted with anti-HIRRV rabbit serum in western blots. These reagents can be used for further study of the fish immune response and to test novel control methods.

Bile Stress Response in Listeria monocytogenes LO28: Adaptation, Cross-Protection, and Identification of Genetic Loci Involved in Bile Resistance

PubMed Central

Begley, Máire; Gahan, Cormac G. M.; Hill, Colin

2002-01-01

Bile is one of many barriers that Listeria monocytogenes must overcome in the human gastrointestinal tract in order to infect and cause disease. We demonstrated that stationary-phase cultures of L. monocytogenes LO28 were able to tolerate concentrations of bovine, porcine, and human bile and bile acids well in excess of those encountered in vivo. Strain LO28 was relatively bile resistant compared with other clinical isolates of L. monocytogenes, as well as with Listeria innocua, Salmonella enterica serovar Typhimurium LT2, and Lactobacillus sakei. While exponential-phase L. monocytogenes LO28 cells were exquisitely sensitive to unconjugated bile acids, prior adaptation to sublethal levels of bile acids or heterologous stresses, such as acid, heat, salt, or sodium dodecyl sulfate (SDS), significantly enhanced bile resistance. This adaptive response was independent of protein synthesis, and in the cases of bile and SDS adaptation, occurred in seconds. In order to identify genetic loci involved in the bile tolerance phenotype of L. monocytogenes LO28, transposon (Tn917) and plasmid (pORI19) integration banks were screened for bile-sensitive mutants. The disrupted genes included a homologue of the capA locus required for capsule formation in Bacillus anthracis; a gene encoding the transcriptional regulator ZurR; a homologue of an Escherichia coli gene, lytB, involved in isoprenoid biosynthesis; a gene encoding a homologue of the Bacillus subtilis membrane protein YxiO; and a gene encoding an amino acid transporter with a putative role in pH homeostasis, gadE. Interestingly, all of the identified loci play putative roles in maintenance of the cell envelope or in stress responses. PMID:12450822

Function and Regulation of the Formate Dehydrogenase Genes of the Methanogenic Archaeon Methanococcus maripaludis

PubMed Central

Wood, Gwendolyn E.; Haydock, Andrew K.; Leigh, John A.

2003-01-01

Methanococcus maripaludis is a mesophilic species of Archaea capable of producing methane from two substrates: hydrogen plus carbon dioxide and formate. To study the latter, we identified the formate dehydrogenase genes of M. maripaludis and found that the genome contains two gene clusters important for formate utilization. Phylogenetic analysis suggested that the two formate dehydrogenase gene sets arose from duplication events within the methanococcal lineage. The first gene cluster encodes homologs of formate dehydrogenase α (FdhA) and β (FdhB) subunits and a putative formate transporter (FdhC) as well as a carbonic anhydrase analog. The second gene cluster encodes only FdhA and FdhB homologs. Mutants lacking either fdhA gene exhibited a partial growth defect on formate, whereas a double mutant was completely unable to grow on formate as a sole methanogenic substrate. Investigation of fdh gene expression revealed that transcription of both gene clusters is controlled by the presence of H2 and not by the presence of formate. PMID:12670979

Molecular characterization of a tyrosine-specific protein phosphatase encoded by a stress-responsive gene in Arabidopsis.

PubMed Central

Xu, Q; Fu, H H; Gupta, R; Luan, S

1998-01-01

Protein tyrosine kinases and phosphatases play a vital role in the regulation of cell growth and differentiation in animal systems. However, none of these enzymes has been characterized from higher plants. In this study, we isolated a cDNA encoding a putative protein tyrosine phosphatase (PTPase) from Arabidopsis (referred to as AtPTP1). The expression level of AtPTP1 is highly sensitive to environmental stresses. High-salt conditions increased AtPTP1 mRNA levels, whereas cold treatment rapidly eliminated the AtPTP1 transcript. The recombinant AtPTP1 protein specifically hydrolyzed phosphotyrosine, but not phosphoserine/threonine, in protein substrates. Site-directed mutagenesis defined two highly conserved amino acids, cysteine-265 and aspartate-234, as being essential for the phosphatase activity of the AtPTP1 protein, suggesting a common catalytic mechanism for PTPases from all eukaryotic systems. In summary, we have identified AtPTP1 as a tyrosine-specific protein phosphatase that may function in stress responses of higher plants. PMID:9596642

Low Sensitivity of Listeria monocytogenes to Quaternary Ammonium Compounds

PubMed Central

Mereghetti, L.; Quentin, R.; Marquet-Van Der Mee, N.; Audurier, A.

2000-01-01

Ninety-seven epidemiologically unrelated strains of Listeria monocytogenes were investigated for their sensitivities to quaternary ammonium compounds (benzalkonium chloride and cetrimide). The MICs for seven serogroup 1/2 strains were high. Three came from the environment and four came from food; none were isolated from human or animal samples. All 97 strains carried the mdrL gene, which encodes a multidrug efflux pump, and the orfA gene, a putative transcriptional repressor of mdrL. The absence of plasmids in four of the seven resistant strains and the conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid borne. Moreover, PCR amplification and Southern blot hybridization experiments failed to find genes phylogenetically related to the qacA and smr genes, encoding multidrug efflux systems previously described for the genus Staphylococcus. The high association between nontypeability by phages and the loss of sensitivity to quaternary ammonium compounds are suggestive of an intrinsic resistance due to modifications in the cell wall. PMID:11055967

X-chromosome-counting mechanisms that determine nematode sex.

PubMed

Nicoll, M; Akerib, C C; Meyer, B J

1997-07-10

Sex is determined in Caenorhabditis elegans by an X-chromosome-counting mechanism that reliably distinguishes the twofold difference in X-chromosome dose between males (1X) and hermaphrodites (2X). This small quantitative difference is translated into the 'on/off' response of the target gene, xol-1, a switch that specifies the male fate when active and the hermaphrodite fate when inactive. Specific regions of X contain counted signal elements whose combined dose sets the activity of xol-1. Here we ascribe the dose effects of one region to a discrete, protein-encoding gene, fox-1. We demonstrate that the dose-sensitive signal elements on chromosome X control xol-1 through two different molecular mechanisms. One involves the transcriptional repression of xol-1 in XX animals. The other uses the putative RNA-binding protein encoded by fox-1 to reduce the level of xol-1 protein. These two mechanisms of repression act together to ensure the fidelity of the X-chromosome counting process.

Single nucleotide resolution RNA-seq uncovers new regulatory mechanisms in the opportunistic pathogen Streptococcus agalactiae.

PubMed

Rosinski-Chupin, Isabelle; Sauvage, Elisabeth; Sismeiro, Odile; Villain, Adrien; Da Cunha, Violette; Caliot, Marie-Elise; Dillies, Marie-Agnès; Trieu-Cuot, Patrick; Bouloc, Philippe; Lartigue, Marie-Frédérique; Glaser, Philippe

2015-05-30

Streptococcus agalactiae, or Group B Streptococcus, is a leading cause of neonatal infections and an increasing cause of infections in adults with underlying diseases. In an effort to reconstruct the transcriptional networks involved in S. agalactiae physiology and pathogenesis, we performed an extensive and robust characterization of its transcriptome through a combination of differential RNA-sequencing in eight different growth conditions or genetic backgrounds and strand-specific RNA-sequencing. Our study identified 1,210 transcription start sites (TSSs) and 655 transcript ends as well as 39 riboswitches and cis-regulatory regions, 39 cis-antisense non-coding RNAs and 47 small RNAs potentially acting in trans. Among these putative regulatory RNAs, ten were differentially expressed in response to an acid stress and two riboswitches sensed directly or indirectly the pH modification. Strikingly, 15% of the TSSs identified were associated with the incorporation of pseudo-templated nucleotides, showing that reiterative transcription is a pervasive process in S. agalactiae. In particular, 40% of the TSSs upstream genes involved in nucleotide metabolism show reiterative transcription potentially regulating gene expression, as exemplified for pyrG and thyA encoding the CTP synthase and the thymidylate synthase respectively. This comprehensive map of the transcriptome at the single nucleotide resolution led to the discovery of new regulatory mechanisms in S. agalactiae. It also provides the basis for in depth analyses of transcriptional networks in S. agalactiae and of the regulatory role of reiterative transcription following variations of intra-cellular nucleotide pools.

First comparative insight into the architecture of COI mitochondrial minicircle molecules of dicyemids reveals marked inter-species variation.

PubMed

Catalano, Sarah R; Whittington, Ian D; Donnellan, Stephen C; Bertozzi, Terry; Gillanders, Bronwyn M

2015-07-01

Dicyemids, poorly known parasites of benthic cephalopods, are one of the few phyla in which mitochondrial (mt) genome architecture departs from the typical ~16 kb circular metazoan genome. In addition to a putative circular genome, a series of mt minicircles that each comprises the mt encoded units (I-III) of the cytochrome c oxidase complex have been reported. Whether the structure of the mt minicircles is a consistent feature among dicyemid species is unknown. Here we analyse the complete cytochrome c oxidase subunit I (COI) minicircle molecule, containing the COI gene and an associated non-coding region (NCR), for ten dicyemid species, allowing for first time comparisons between species of minicircle architecture, NCR function and inferences of minicircle replication. Divergence in COI nucleotide sequences between dicyemid species was high (average net divergence = 31.6%) while within species diversity was lower (average net divergence = 0.2%). The NCR and putative 5' section of the COI gene were highly divergent between dicyemid species (average net nucleotide divergence of putative 5' COI section = 61.1%). No tRNA genes were found in the NCR, although palindrome sequences with the potential to form stem-loop structures were identified in some species, which may play a role in transcription or other biological processes.

Sequence analysis and gene expression of putative exo- and endo-glucanases from oil palm (Elaeis guineensis) during fungal infection.

PubMed

Yeoh, Keat-Ai; Othman, Abrizah; Meon, Sariah; Abdullah, Faridah; Ho, Chai-Ling

2012-10-15

Glucanases are enzymes that hydrolyze a variety β-d-glucosidic linkages. Plant β-1,3-glucanases are able to degrade fungal cell walls; and promote the release of cell-wall derived fungal elicitors. In this study, three full-length cDNA sequences encoding oil palm (Elaeis guineensis) glucanases were analyzed. Sequence analyses of the cDNA sequences suggested that EgGlc1-1 is a putative β-d-glucan exohydolase belonging to glycosyl hydrolase (GH) family 3 while EgGlc5-1 and EgGlc5-2 are putative glucan endo-1,3-β-glucosidases belonging to GH family 17. The transcript abundance of these genes in the roots and leaves of oil palm seedlings treated with Ganoderma boninense and Trichoderma harzianum was profiled to investigate the involvement of these glucanases in oil palm during fungal infection. The gene expression of EgGlc1-1 in the root of oil palm seedlings was increased by T. harzianum but suppressed by G. boninense; while the gene expression of both EgGlc5-1 and EgGlc5-2 in the roots of oil palm seedlings was suppressed by G. boninense or/and T. harzianum. Copyright © 2012 Elsevier GmbH. All rights reserved.

Ginger and turmeric expressed sequence tags identify signature genes for rhizome identity and development and the biosynthesis of curcuminoids, gingerols and terpenoids

PubMed Central

2013-01-01

Background Ginger (Zingiber officinale) and turmeric (Curcuma longa) accumulate important pharmacologically active metabolites at high levels in their rhizomes. Despite their importance, relatively little is known regarding gene expression in the rhizomes of ginger and turmeric. Results In order to identify rhizome-enriched genes and genes encoding specialized metabolism enzymes and pathway regulators, we evaluated an assembled collection of expressed sequence tags (ESTs) from eight different ginger and turmeric tissues. Comparisons to publicly available sorghum rhizome ESTs revealed a total of 777 gene transcripts expressed in ginger/turmeric and sorghum rhizomes but apparently absent from other tissues. The list of rhizome-specific transcripts was enriched for genes associated with regulation of tissue growth, development, and transcription. In particular, transcripts for ethylene response factors and AUX/IAA proteins appeared to accumulate in patterns mirroring results from previous studies regarding rhizome growth responses to exogenous applications of auxin and ethylene. Thus, these genes may play important roles in defining rhizome growth and development. Additional associations were made for ginger and turmeric rhizome-enriched MADS box transcription factors, their putative rhizome-enriched homologs in sorghum, and rhizomatous QTLs in rice. Additionally, analysis of both primary and specialized metabolism genes indicates that ginger and turmeric rhizomes are primarily devoted to the utilization of leaf supplied sucrose for the production and/or storage of specialized metabolites associated with the phenylpropanoid pathway and putative type III polyketide synthase gene products. This finding reinforces earlier hypotheses predicting roles of this enzyme class in the production of curcuminoids and gingerols. Conclusion A significant set of genes were found to be exclusively or preferentially expressed in the rhizome of ginger and turmeric. Specific transcription factors and other regulatory genes were found that were common to the two species and that are excellent candidates for involvement in rhizome growth, differentiation and development. Large classes of enzymes involved in specialized metabolism were also found to have apparent tissue-specific expression, suggesting that gene expression itself may play an important role in regulating metabolite production in these plants. PMID:23410187

Gene Expression Patterns during Light and Dark Infection of Prochlorococcus by Cyanophage

PubMed Central

Chisholm, Sallie W.

2016-01-01

Cyanophage infecting the marine cyanobacteria Prochlorococcus and Synechococcus require light and host photosystem activity for optimal reproduction. Many cyanophages encode multiple photosynthetic electron transport (PET) proteins, which are presumed to maintain electron flow and produce ATP and NADPH for nucleotide biosynthesis and phage genome replication. However, evidence suggests phage augment NADPH production via the pentose phosphate pathway (PPP), thus calling into question the need for NADPH production by PET. Genes implicated in cyclic PET have since been identified in cyanophage genomes. It remains an open question which mode of PET, cyclic or linear, predominates in infected cyanobacteria, and thus whether the balance is towards producing ATP or NADPH. We sequenced transcriptomes of a cyanophage (P-HM2) and its host (Prochlorococcus MED4) throughout infection in the light or in the dark, and analyzed these data in the context of phage replication and metabolite measurements. Infection was robust in the light, but phage were not produced in the dark. Host gene transcripts encoding high-light inducible proteins and two terminal oxidases (plastoquinol terminal oxidase and cytochrome c oxidase)—implicated in protecting the photosynthetic membrane from light stress—were the most enriched in light but not dark infection. Among the most diminished transcripts in both light and dark infection was ferredoxin–NADP+ reductase (FNR), which uses the electron acceptor NADP+ to generate NADPH in linear photosynthesis. The phage gene for CP12, which putatively inhibits the Calvin cycle enzyme that receives NADPH from FNR, was highly expressed in light infection. Therefore, both PET production of NADPH and its consumption by carbon fixation are putatively repressed during phage infection in light. Transcriptomic evidence is thus consistent with cyclic photophosphorylation using oxygen as the terminal electron acceptor as the dominant mode of PET under infection, with ATP from PET and NADPH from the PPP producing the energy and reducing equivalents for phage nucleotide biosynthesis and replication. PMID:27788196

Tanscriptomic Study of the Soybean-Fusarium virguliforme Interaction Revealed a Novel Ankyrin-Repeat Containing Defense Gene, Expression of Whose during Infection Led to Enhanced Resistance to the Fungal Pathogen in Transgenic Soybean Plants

PubMed Central

Ngaki, Micheline N.; Wang, Bing; Sahu, Binod B.; Srivastava, Subodh K.; Farooqi, Mohammad S.; Kambakam, Sekhar; Swaminathan, Sivakumar

2016-01-01

Fusarium virguliforme causes the serious disease sudden death syndrome (SDS) in soybean. Host resistance to this pathogen is partial and is encoded by a large number of quantitative trait loci, each conditioning small effects. Breeding SDS resistance is therefore challenging and identification of single-gene encoded novel resistance mechanisms is becoming a priority to fight this devastating this fungal pathogen. In this transcriptomic study we identified a few putative soybean defense genes, expression of which is suppressed during F. virguliforme infection. The F. virguliforme infection-suppressed genes were broadly classified into four major classes. The steady state transcript levels of many of these genes were suppressed to undetectable levels immediately following F. virguliforme infection. One of these classes contains two novel genes encoding ankyrin repeat-containing proteins. Expression of one of these genes, GmARP1, during F. virguliforme infection enhances SDS resistance among the transgenic soybean plants. Our data suggest that GmARP1 is a novel defense gene and the pathogen presumably suppress its expression to establish compatible interaction. PMID:27760122

A novel family of integrases associated with prophages and genomic islands integrated within the tRNA-dihydrouridine synthase A (dusA) gene

PubMed Central

Farrugia, Daniel N.; Elbourne, Liam D. H.; Mabbutt, Bridget C.; Paulsen, Ian T.

2015-01-01

Genomic islands play a key role in prokaryotic genome plasticity. Genomic islands integrate into chromosomal loci such as transfer RNA genes and protein coding genes, whilst retaining various cargo genes that potentially bestow novel functions on the host organism. A gene encoding a putative integrase was identified at a single site within the 5′ end of the dusA gene in the genomes of over 200 bacteria. This integrase was discovered to be a component of numerous genomic islands, which appear to share a target site within the dusA gene. dusA encodes the tRNA-dihydrouridine synthase A enzyme, which catalyses the post-transcriptional reduction of uridine to dihydrouridine in tRNA. Genomic islands encoding homologous dusA-associated integrases were found at a much lower frequency within the related dusB and dusC genes, and non-dus genes. Excision of these dusA-associated islands from the chromosome as circularized intermediates was confirmed by polymerase chain reaction. Analysis of the dusA-associated islands indicated that they were highly diverse, with the integrase gene representing the only universal common feature. PMID:25883135

Protein encoding genes in an ancient plant: analysis of codon usage, retained genes and splice sites in a moss, Physcomitrella patens

PubMed Central

Rensing, Stefan A; Fritzowsky, Dana; Lang, Daniel; Reski, Ralf

2005-01-01

Background The moss Physcomitrella patens is an emerging plant model system due to its high rate of homologous recombination, haploidy, simple body plan, physiological properties as well as phylogenetic position. Available EST data was clustered and assembled, and provided the basis for a genome-wide analysis of protein encoding genes. Results We have clustered and assembled Physcomitrella patens EST and CDS data in order to represent the transcriptome of this non-seed plant. Clustering of the publicly available data and subsequent prediction resulted in a total of 19,081 non-redundant ORF. Of these putative transcripts, approximately 30% have a homolog in both rice and Arabidopsis transcriptome. More than 130 transcripts are not present in seed plants but can be found in other kingdoms. These potential "retained genes" might have been lost during seed plant evolution. Functional annotation of these genes reveals unequal distribution among taxonomic groups and intriguing putative functions such as cytotoxicity and nucleic acid repair. Whereas introns in the moss are larger on average than in the seed plant Arabidopsis thaliana, position and amount of introns are approximately the same. Contrary to Arabidopsis, where CDS contain on average 44% G/C, in Physcomitrella the average G/C content is 50%. Interestingly, moss orthologs of Arabidopsis genes show a significant drift of codon fraction usage, towards the seed plant. While averaged codon bias is the same in Physcomitrella and Arabidopsis, the distribution pattern is different, with 15% of moss genes being unbiased. Species-specific, sensitive and selective splice site prediction for Physcomitrella has been developed using a dataset of 368 donor and acceptor sites, utilizing a support vector machine. The prediction accuracy is better than those achieved with tools trained on Arabidopsis data. Conclusion Analysis of the moss transcriptome displays differences in gene structure, codon and splice site usage in comparison with the seed plant Arabidopsis. Putative retained genes exhibit possible functions that might explain the peculiar physiological properties of mosses. Both the transcriptome representation (including a BLAST and retrieval service) and splice site prediction have been made available on , setting the basis for assembly and annotation of the Physcomitrella genome, of which draft shotgun sequences will become available in 2005. PMID:15784153

PcFKH1, a novel regulatory factor from the forkhead family, controls the biosynthesis of penicillin in Penicillium chrysogenum.

PubMed

Domínguez-Santos, Rebeca; García-Estrada, Carlos; Kosalková, Katarina; Prieto, Carlos; Santamarta, Irene; Martín, Juan-Francisco

2015-08-01

Penicillin biosynthesis in Penicillium chrysogenum (re-identified as Penicillium rubens) is a good example of a biological process subjected to complex global regulatory networks and serves as a model to study fungal secondary metabolism. The winged-helix family of transcription factors recently described, which includes the forkhead type of proteins, is a key type of regulatory proteins involved in this process. In yeasts and humans, forkhead transcription factors are involved in different processes (cell cycle regulation, cell death control, pre-mRNA processing and morphogenesis); one member of this family of proteins has been identified in the P. chrysogenum genome (Pc18g00430). In this work, we have characterized this novel transcription factor (named PcFKH1) by generating knock-down mutants and overexpression strains. Results clearly indicate that PcFKH1 positively controls antibiotic biosynthesis through the specific interaction with the promoter region of the penDE gene, thus regulating penDE mRNA levels. PcFKH1 also binds to the pcbC promoter, but with low affinity. In addition, it also controls other ancillary genes of the penicillin biosynthetic process, such as phlA (encoding phenylacetyl CoA ligase) and ppt (encoding phosphopantetheinyl transferase). PcFKH1 also plays a role in conidiation and spore pigmentation, but it does not seem to be involved in hyphal morphology or cell division in the improved laboratory reference strain Wisconsin 54-1255. A genome-wide analysis of processes putatively coregulated by PcFKH1 and PcRFX1 (another winged-helix transcription factor) in P. chrysogenum provided evidence of the global effect of these transcription factors in P. chrysogenum metabolism. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Identification of Germ Plasm-Associated Transcripts by Microarray Analysis of Xenopus Vegetal Cortex RNA

PubMed Central

Cuykendall, Tawny N.; Houston, Douglas W.

2011-01-01

RNA localization is a common mechanism for regulating cell structure and function. Localized RNAs in Xenopus oocytes are critical for early development, including germline specification by the germ plasm. Despite the importance of these localized RNAs, only approximately 25 have been identified and fewer are functionally characterized. Using microarrays, we identified a large set of localized RNAs from the vegetal cortex. Overall, our results indicate a minimum of 275 localized RNAs in oocytes, or 2–3% of maternal transcripts, which are in general agreement with previous findings. We further validated vegetal localization for 24 candidates and further characterized three genes expressed in the germ plasm. We identified novel germ plasm expression for reticulon 3.1, exd2 (a novel exonuclease-domain encoding gene), and a putative noncoding RNA. Further analysis of these and other localized RNAs will likely identify new functions of germ plasm and facilitate the identification of cis-acting RNA localization elements. PMID:20503379

A direct molecular link between the autism candidate gene RORa and the schizophrenia candidate MIR137

NASA Astrophysics Data System (ADS)

Devanna, Paolo; Vernes, Sonja C.

2014-02-01

Retinoic acid-related orphan receptor alpha gene (RORa) and the microRNA MIR137 have both recently been identified as novel candidate genes for neuropsychiatric disorders. RORa encodes a ligand-dependent orphan nuclear receptor that acts as a transcriptional regulator and miR-137 is a brain enriched small non-coding RNA that interacts with gene transcripts to control protein levels. Given the mounting evidence for RORa in autism spectrum disorders (ASD) and MIR137 in schizophrenia and ASD, we investigated if there was a functional biological relationship between these two genes. Herein, we demonstrate that miR-137 targets the 3'UTR of RORa in a site specific manner. We also provide further support for MIR137 as an autism candidate by showing that a large number of previously implicated autism genes are also putatively targeted by miR-137. This work supports the role of MIR137 as an ASD candidate and demonstrates a direct biological link between these previously unrelated autism candidate genes.

A cyanase is transcriptionally regulated by arginine and involved in cyanate decomposition in Sordaria macrospora.

PubMed

Elleuche, Skander; Pöggeler, Stefanie

2008-11-01

Cyanase degrades toxic cyanate to NH3 and CO2 in a bicarbonate-dependent reaction. High concentrations of cyanate are fairly toxic to organisms. Here, we characterize a eukaryotic cyanase for the first time. We have isolated the cyn1 gene encoding a cyanase from the filamentous ascomycete Sordaria macrospora and functionally characterized the cyn1 product after heterologous expression in Escherichia coli. Site-directed mutagenesis confirmed a predicted catalytic centre of three conserved amino-acids. A Deltacyn1 knockout in S. macrospora was totally devoid of cyanase activity and showed an increased sensitivity to exogenously supplied cyanate in an arginine-depleted medium, defects in ascospore germination, but no other obvious morphological phenotype. By means of real-time PCR we have demonstrated that the transcriptional level of cyn1 is markedly elevated in the presence of cyanate and down-regulated by addition of arginine. The putative functions of cyanase in fungi are discussed.

Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

PubMed

de Vries, R P; van de Vondervoort, P J I; Hendriks, L; van de Belt, M; Visser, J

2002-09-01

The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

Genes regulated by AoXlnR, the xylanolytic and cellulolytic transcriptional regulator, in Aspergillus oryzae.

PubMed

Noguchi, Yuji; Sano, Motoaki; Kanamaru, Kyoko; Ko, Taro; Takeuchi, Michio; Kato, Masashi; Kobayashi, Tetsuo

2009-11-01

XlnR is a Zn(II)2Cys6 transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus. Overexpression of the aoxlnR gene in Aspergillus oryzae (A. oryzae xlnR gene) resulted in elevated xylanolytic and cellulolytic activities in the culture supernatant, in which nearly 40 secreted proteins were detected by two-dimensional electrophoresis. DNA microarray analysis to identify the transcriptional targets of AoXlnR led to the identification of 75 genes that showed more than fivefold increase in their expression in the AoXlnR overproducer than in the disruptant. Of these, 32 genes were predicted to encode a glycoside hydrolase, highlighting the biotechnological importance of AoXlnR in biomass degradation. The 75 genes included the genes previously identified as AoXlnR targets (xynF1, xynF3, xynG2, xylA, celA, celB, celC, and celD). Thirty-six genes were predicted to be extracellular, which was consistent with the number of proteins secreted, and 61 genes possessed putative XlnR-binding sites (5'-GGCTAA-3', 5'-GGCTAG-3', and 5'-GGCTGA-3') in their promoter regions. Functional annotation of the genes revealed that AoXlnR regulated the expression of hydrolytic genes for degradation of beta-1,4-xylan, arabinoxylan, cellulose, and xyloglucan and of catabolic genes for the conversion of D-xylose to xylulose-5-phosphate. In addition, genes encoding glucose-6-phosphate 1-dehydrogenase and L-arabinitol-4- dehydrogenase involved in D-glucose and L-arabinose catabolism also appeared to be targets of AoXlnR.

Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

PubMed Central

Burnside, Kellie; Lembo, Annalisa; de los Reyes, Melissa; Iliuk, Anton; BinhTran, Nguyen-Thao; Connelly, James E.; Lin, Wan-Jung; Schmidt, Byron Z.; Richardson, Anthony R.; Fang, Ferric C.; Tao, Weiguo Andy; Rajagopal, Lakshmi

2010-01-01

Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence. PMID:20552019

A Major Facilitator Superfamily protein encoded by TcMucK gene is not required for cuticle pigmentation, growth and development in Tribolium castaneum.

PubMed

Mun, Seulgi; Noh, Mi Young; Osanai-Futahashi, Mizuko; Muthukrishnan, Subbaratnam; Kramer, Karl J; Arakane, Yasuyuki

2014-06-01

Insect cuticle pigmentation and sclerotization (tanning) are vital physiological processes for insect growth, development and survival. We have previously identified several colorless precursor molecules as well as enzymes involved in their biosynthesis and processing to yield the mature intensely colored body cuticle pigments. A recent study indicated that the Bombyx mori (silkmoth) gene, BmMucK, which encodes a protein orthologous to a Culex pipiens quiquefasciatus (Southern house mosquito) cis,cis, muconate transporter, is a member of the "Major Facilitator Superfamily" (MFS) of transporter proteins and is associated with the appearance of pigmented body segments of naturally occurring body color mutants of B. mori. While RNA interference of the BmMucK gene failed to result in any observable phenotype, RNAi using a dsRNA for an orthologous gene from the red flour beetle, Tribolium castaneum, was reported to result in molting defects and darkening of the cuticle and some body parts, leading to the suggestion that orthologs of MucK genes may differ in their functions among insects. To verify the role and essentiality of the ortholog of this gene in development and body pigmentation function in T. castaneum we obtained cDNAs for the orthologous gene (TcMucK) from RNA isolated from the GA-1 wild-type strain of T. castaneum. The sequence of a 1524 nucleotides-long cDNA for TcMucK which encodes the putatively full-length protein, was assembled from two overlapping RT-PCR fragments and the expression profile of this gene during development was analyzed by real-time PCR. This cDNA encodes a 55.8 kDa protein consisting of 507 amino acid residues and includes 11 putative transmembrane segments. Transcripts of TcMucK were detected throughout all of the developmental stages analyzed. The function of this gene was explored by injection of two different double-stranded RNAs targeting different regions of the TcMucK gene (dsTcMucKs) into young larvae to down-regulate transcripts during subsequent stages of insect development until the adult stage. RNA interference of TcMucK had no observable effects on larval, pupal or adult pigmentation. In addition, it did not affect larval-larval, larval-pupal and pupal-adult molting or survival. Thus, in contrast to the results of Zhao et al. (2012), our study demonstrates that TcMucK is not essential for growth, development or cuticle pigmentation of T. castaneum. Copyright © 2014 Elsevier Ltd. All rights reserved.

MicroRNAs: regulators of gene expression and cell differentiation

PubMed Central

Shivdasani, Ramesh A.

2006-01-01

The existence and roles of a class of abundant regulatory RNA molecules have recently come into sharp focus. Micro-RNAs (miRNAs) are small (approximately 22 bases), non–protein-coding RNAs that recognize target sequences of imperfect complementarity in cognate mRNAs and either destabilize them or inhibit protein translation. Although mechanisms of miRNA biogenesis have been elucidated in some detail, there is limited appreciation of their biological functions. Reported examples typically focus on miRNA regulation of a single tissue-restricted transcript, often one encoding a transcription factor, that controls a specific aspect of development, cell differentiation, or physiology. However, computational algorithms predict up to hundreds of putative targets for individual miRNAs, single transcripts may be regulated by multiple miRNAs, and miRNAs may either eliminate target gene expression or serve to finetune transcript and protein levels. Theoretical considerations and early experimental results hence suggest diverse roles for miRNAs as a class. One appealing possibility, that miRNAs eliminate low-level expression of unwanted genes and hence refine unilineage gene expression, may be especially amenable to evaluation in models of hematopoiesis. This review summarizes current understanding of miRNA mechanisms, outlines some of the important outstanding questions, and describes studies that attempt to define miRNA functions in hematopoiesis. PMID:16882713

Diversity and Evolutionary History of Iron Metabolism Genes in Diatoms

PubMed Central

Groussman, Ryan D.; Parker, Micaela S.; Armbrust, E. Virginia

2015-01-01

Ferroproteins arose early in Earth’s history, prior to the emergence of oxygenic photosynthesis and the subsequent reduction of bioavailable iron. Today, iron availability limits primary productivity in about 30% of the world’s oceans. Diatoms, responsible for nearly half of oceanic primary production, have evolved molecular strategies for coping with variable iron concentrations. Our understanding of the evolutionary breadth of these strategies has been restricted by the limited number of species for which molecular sequence data is available. To uncover the diversity of strategies marine diatoms employ to meet cellular iron demands, we analyzed 367 newly released marine microbial eukaryotic transcriptomes, which include 47 diatom species. We focused on genes encoding proteins previously identified as having a role in iron management: iron uptake (high-affinity ferric reductase, multi-copper oxidase, and Fe(III) permease); iron storage (ferritin); iron-induced protein substitutions (flavodoxin/ferredoxin, and plastocyanin/cytochrome c6) and defense against reactive oxygen species (superoxide dismutases). Homologs encoding the high-affinity iron uptake system components were detected across the four diatom Classes suggesting an ancient origin for this pathway. Ferritin transcripts were also detected in all Classes, revealing a more widespread utilization of ferritin throughout diatoms than previously recognized. Flavodoxin and plastocyanin transcripts indicate possible alternative redox metal strategies. Predicted localization signals for ferredoxin identify multiple examples of gene transfer from the plastid to the nuclear genome. Transcripts encoding four superoxide dismutase metalloforms were detected, including a putative nickel-coordinating isozyme. Taken together, our results suggest that the majority of iron metabolism genes in diatoms appear to be vertically inherited with functional diversity achieved via possible neofunctionalization of paralogs. This refined view of iron use strategies in diatoms elucidates the history of these adaptations, and provides potential molecular markers for determining the iron nutritional status of different diatom species in environmental samples. PMID:26052941

Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae.

PubMed

Nemchinov, Lev G; Shao, Jonathan; Lee, Maya N; Postnikova, Olga A; Samac, Deborah A

2017-01-01

Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common disease of alfalfa (Medicago sativa L). Little is known about host-pathogen interactions and host defense mechanisms. Here, individual resistant and susceptible plants were selected from cultivars Maverick and ZG9830 and used for transcript profiling at 24 and 72 hours after inoculation (hai) with the isolate PssALF3. Bioinformatic analysis revealed a number of differentially expressed genes (DEGs) in resistant and susceptible genotypes. Although resistant plants from each cultivar produced a hypersensitive response, transcriptome analyses indicated that they respond differently at the molecular level. The number of DEGs was higher in resistant plants of ZG9830 at 24 hai than in Maverick, suggesting that ZG9830 plants had a more rapid effector triggered immune response. Unique up-regulated genes in resistant ZG9830 plants included genes encoding putative nematode resistance HSPRO2-like proteins, orthologs for the rice Xa21 and soybean Rpg1-b resistance genes, and TIR-containing R genes lacking both NBS and LRR domains. The suite of R genes up-regulated in resistant Maverick plants had an over-representation of R genes in the CC-NBS-LRR family including two genes for atypical CCR domains and a putative ortholog of the Arabidopsis RPM1 gene. Resistance in both cultivars appears to be mediated primarily by WRKY family transcription factors and expression of genes involved in protein phosphorylation, regulation of transcription, defense response including synthesis of isoflavonoids, and oxidation-reduction processes. These results will further the identification of mechanisms involved in resistance to facilitate selection of parent populations and development of commercial varieties.

Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial stem blight caused by Pseudomonas syringae pv. syringae

PubMed Central

Shao, Jonathan; Lee, Maya N.; Postnikova, Olga A.; Samac, Deborah A.

2017-01-01

Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common disease of alfalfa (Medicago sativa L). Little is known about host-pathogen interactions and host defense mechanisms. Here, individual resistant and susceptible plants were selected from cultivars Maverick and ZG9830 and used for transcript profiling at 24 and 72 hours after inoculation (hai) with the isolate PssALF3. Bioinformatic analysis revealed a number of differentially expressed genes (DEGs) in resistant and susceptible genotypes. Although resistant plants from each cultivar produced a hypersensitive response, transcriptome analyses indicated that they respond differently at the molecular level. The number of DEGs was higher in resistant plants of ZG9830 at 24 hai than in Maverick, suggesting that ZG9830 plants had a more rapid effector triggered immune response. Unique up-regulated genes in resistant ZG9830 plants included genes encoding putative nematode resistance HSPRO2-like proteins, orthologs for the rice Xa21 and soybean Rpg1-b resistance genes, and TIR-containing R genes lacking both NBS and LRR domains. The suite of R genes up-regulated in resistant Maverick plants had an over-representation of R genes in the CC-NBS-LRR family including two genes for atypical CCR domains and a putative ortholog of the Arabidopsis RPM1 gene. Resistance in both cultivars appears to be mediated primarily by WRKY family transcription factors and expression of genes involved in protein phosphorylation, regulation of transcription, defense response including synthesis of isoflavonoids, and oxidation-reduction processes. These results will further the identification of mechanisms involved in resistance to facilitate selection of parent populations and development of commercial varieties. PMID:29244864

The SBP-Box Gene VpSBP11 from Chinese Wild Vitis Is Involved in Floral Transition and Affects Leaf Development.

PubMed

Hou, Hongmin; Yan, Xiaoxiao; Sha, Ting; Yan, Qin; Wang, Xiping

2017-07-13

Flowering occurs in angiosperms during a major developmental transition from vegetative growth to the reproductive phase. Squamosa promoter binding protein (SBP)-box genes have been found to play critical roles in regulating flower and fruit development, but their roles in grapevine have remained unclear. To better understand the functions of the grape SBP-box genes in both vegetative and reproductive growth phases, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP11 , was obtained from Chinese wild grapevine Vitis pseudoreticulata Wen Tsai Wang (W. T. Wang) clone 'Baihe-35-1'. VpSBP11 encoded a putative polypeptide of 170 amino acids with a highly conserved SBP-domain with two zinc-binding sites of the Cx2C-x3-H-x11-C-x6-H (C2HCH) type and a nuclear localization signal. We confirmed that the VpSBP11 protein was targeted to the nucleus and possessed transcriptional activation activity by subcellular localization and trans -activation assay. Over-expression of VpSBP11 in Arabidopsis thaliana was shown to activate the FUL gene, and subsequently the AP1 and LFY genes, all of which were floral meristem identity genes, and to cause earlier flowering than in wild type (WT) plants. The pattern of vegetative growth was also different between the transgenic and WT plants. For example, in the VpSBP11 over-expressing transgenic plants, the number of rosette leaves was less than that of WT; the petiole was significantly elongated; and the rosette and cauline leaves curled upwards or downwards. These results were consistent with VpSBP11 acting as a transcription factor during the transition from the vegetative stage to the reproductive stage.

Characterization of Five Novel Brevibacillus Bacteriophages and Genomic Comparison of Brevibacillus Phages

PubMed Central

Berg, Jordan A.; Merrill, Bryan D.; Crockett, Justin T.; Esplin, Kyle P.; Evans, Marlee R.; Heaton, Karli E.; Hilton, Jared A.; Hyde, Jonathan R.; McBride, Morgan S.; Schouten, Jordan T.; Simister, Austin R.; Thurgood, Trever L.; Ward, Andrew T.; Breakwell, Donald P.; Hope, Sandra; Grose, Julianne H.

2016-01-01

Brevibacillus laterosporus is a spore-forming bacterium that causes a secondary infection in beehives following European Foulbrood disease. To better understand the contributions of Brevibacillus bacteriophages to the evolution of their hosts, five novel phages (Jenst, Osiris, Powder, SecTim467, and Sundance) were isolated and characterized. When compared with the five Brevibacillus phages currently in NCBI, these phages were assigned to clusters based on whole genome and proteome synteny. Powder and Osiris, both myoviruses, were assigned to the previously described Jimmer-like cluster. SecTim467 and Jenst, both siphoviruses, formed a novel phage cluster. Sundance, a siphovirus, was assigned as a singleton phage along with the previously isolated singleton, Emery. In addition to characterizing the basic relationships between these phages, several genomic features were observed. A motif repeated throughout phages Jenst and SecTim467 was frequently upstream of genes predicted to function in DNA replication, nucleotide metabolism, and transcription, suggesting transcriptional co-regulation. In addition, paralogous gene pairs that encode a putative transcriptional regulator were identified in four Brevibacillus phages. These paralogs likely evolved to bind different DNA sequences due to variation at amino acid residues predicted to bind specific nucleotides. Finally, a putative transposable element was identified in SecTim467 and Sundance that carries genes homologous to those found in Brevibacillus chromosomes. Remnants of this transposable element were also identified in phage Jenst. These discoveries provide a greater understanding of the diversity of phages, their behavior, and their evolutionary relationships to one another and to their host. In addition, they provide a foundation with which further Brevibacillus phages can be compared. PMID:27304881

Transcript Assembly and Quantification by RNA-Seq Reveals Differentially Expressed Genes between Soft-Endocarp and Hard-Endocarp Hawthorns

PubMed Central

Zhang, Feng; Liu, Zhongchi; Li, Xiaoming; Li, Wenran; Ma, Yue; Li, He; Liu, Yuexue; Zhang, Zhihong

2013-01-01

Hawthorn (Crataegus spp.) is an important pome with a long history as a fruit, an ornamental, and a source of medicine. Fruits of hawthorn are marked by hard stony endocarps, but a hawthorn germplasm with soft and thin endocarp was found in Liaoning province of China. To elucidate the molecular mechanism underlying the soft endocarp of hawthorn, we conducted a de novo assembly of the fruit transcriptome of Crataegus pinnatifida and compared gene expression profiles between the soft-endocarp and the hard-endocarp hawthorn varieties. De novo assembly yielded 52,673 putative unigenes, 20.4% of which are longer than 1,000 bp. Among the high-quality unique sequences, 35,979 (68.3%) had at least one significant match to an existing gene model. A total of 1,218 genes, represented 2.31% total putative unigenes, were differentially expressed between the soft-endocarp hawthorn and the hard-endocarp hawthorn. Among these differentially expressed genes, a number of lignin biosynthetic pathway genes were down-regulated while almost all the flavonoid biosynthetic pathway genes were strongly up-regulated, concomitant with the formation of soft endocarp. In addition, we have identified some MYB and NAC transcription factors that could potentially control lignin and flavonoid biosynthesis. The altered expression levels of the genes encoding lignin biosynthetic enzymes, MYB and NAC transcription factors were confirmed by quantitative RT-PCR. This is the first transcriptome analysis of Crataegus genus. The high quality ESTs generated in this study will aid future gene cloning from hawthorn. Our study provides important insights into the molecular mechanisms underlying soft endocarp formation in hawthorn. PMID:24039819

Identification of estrogen-responsive genes using a genome-wide analysis of promoter elements for transcription factor binding sites.

PubMed

Kamalakaran, Sitharthan; Radhakrishnan, Senthil K; Beck, William T

2005-06-03

We developed a pipeline to identify novel genes regulated by the steroid hormone-dependent transcription factor, estrogen receptor, through a systematic analysis of upstream regions of all human and mouse genes. We built a data base of putative promoter regions for 23,077 human and 19,984 mouse transcripts from National Center for Biotechnology Information annotation and 8793 human and 6785 mouse promoters from the Data Base of Transcriptional Start Sites. We used this data base of putative promoters to identify potential targets of estrogen receptor by identifying estrogen response elements (EREs) in their promoters. Our program correctly identified EREs in genes known to be regulated by estrogen in addition to several new genes whose putative promoters contained EREs. We validated six genes (KIAA1243, NRIP1, MADH9, NME3, TPD52L, and ABCG2) to be estrogen-responsive in MCF7 cells using reverse transcription PCR. To allow for extensibility of our program in identifying targets of other transcription factors, we have built a Web interface to access our data base and programs. Our Web-based program for Promoter Analysis of Genome, PAGen@UIC, allows a user to identify putative target genes for vertebrate transcription factors through the analysis of their upstream sequences. The interface allows the user to search the human and mouse promoter data bases for potential target genes containing one or more listed transcription factor binding sites (TFBSs) in their upstream elements, using either regular expression-based consensus or position weight matrices. The data base can also be searched for promoters harboring user-defined TFBSs given as a consensus or a position weight matrix. Furthermore, the user can retrieve putative promoter sequences for any given gene together with identified TFBSs located on its promoter. Orthologous promoters are also analyzed to determine conserved elements.

Scallop DMT functions as a Ca2+ transporter.

PubMed

Toyohara, Haruhiko; Yamamoto, Sayuri; Hosoi, Masatomi; Takagi, Masaya; Hayashi, Isao; Nakao, Kenji; Kaneko, Shuji

2005-05-09

We identified a DMT (divalent metal transporter) homologous protein that functions as a Ca(2+) transporter. Scallop DMT cDNA encodes a 539-amino-acid protein with 12 putative membrane-spanning domains and has a consensus transport motif in the fourth extracellular loop. Since its mRNA is significantly expressed in the gill and intestine, it is assumed that scallop DMT transports Ca(2+) from seawater by the gill and from food by the intestine. Scallop DMT lacks the iron-responsive element commonly found in iron-regulatory proteins, suggesting that it is free of the post-transcriptional regulation from intracellular Fe(2+) concentration. Scallop DMT distinctly functions as a Ca(2+) transporter unlike other DMTs, however, it also transports Fe(2+) and Cd(2+) similar to them.

Identification and expression analysis of a novel R-type lectin from the coleopteran beetle, Tenebrio molitor.

PubMed

Kim, Dong Hyun; Patnaik, Bharat Bhusan; Seo, Gi Won; Kang, Seong Min; Lee, Yong Seok; Lee, Bok Luel; Han, Yeon Soo

2013-11-01

We have identified novel ricin-type (R-type) lectin by sequencing of random clones from cDNA library of the coleopteran beetle, Tenebrio molitor. The cDNA sequence is comprised of 495 bp encoding a protein of 164 amino acid residues and shows 49% identity with galectin of Tribolium castaneum. Bioinformatics analysis shows that the amino acid residues from 35 to 162 belong to ricin-type beta-trefoil structure. The transcript was significantly upregulated after early hours of injection with peptidoglycans derived from Gram (+) and Gram (-) bacteria, beta-1, 3 glucan from fungi and an intracellular pathogen, Listeria monocytogenes suggesting putative function in innate immunity. Copyright © 2013 Elsevier Inc. All rights reserved.

Transcript profiles of mitochondrial and cytoplasmic manganese superoxide dismutases in Exopalaemon carinicauda under ammonia stress

NASA Astrophysics Data System (ADS)

Ren, Hai; Li, Jian; Li, Jitao; Liu, Ping; Liang, Zhongxiu; Wu, Jianhua

2015-05-01

Superoxide dismutase (SOD) is one of the most important antioxidant defense enzymes, and is considered as the first line against oxidative stress. In this study, we cloned a mitochondrial manganese (Mn) SOD ( mMnSOD) cDNA from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) methods. The full-length cDNA for mMnSOD was 1 014-bp long, containing a 5'-untranslated region (UTR) of 37-bp, a 3'-UTR of 321-bp with a poly (A) tail, and included a 657-bp open reading frame encoding a protein of 218 amino acids with a 16-amino-acid signal peptide. The protein had a calculated molecular weight of 23.87 kDa and a theoretical isoelectric point of 6.75. The mMnSOD sequence included two putative N-glycosylation sites (NHT and NLS), the MnSOD signature sequence 180DVWEHAYY187, and four putative Mn binding sites (H48, H96, D180, and H184). Sequence comparison showed that the mMnSOD deduced amino acid sequence of E. carinicauda shared 97%, 95%, 89%, 84%, 82%, 72%, and 69% identity with that of Macrobrachium rosenbergii, Macrobrachium nipponense, Fenneropeneaus chinensis, Callinectes sapidus, Perisesarma bidens, Danio rerio, and Homo sapiens, resectively. Quantitative real-time RT-PCR analysis showed that mMnSOD transcripts were present in all E. carinicauda tissues examined, with the highest levels in the hepatopancreas. During an ammonia stress treatment, the transcript levels of mMnSOD and cMnSOD were up-regulated at 12 h in hemocytes and at 24 h in the hepatopancreas. As the duration of the ammonia stress treatment extended to 72 h, the transcript levels of mMnSOD and cMnSOD significantly decreased both in hemocytes and hepatopancreas. These findings indicate that the SOD system is induced to respond to acute ammonia stress, and may be involved in environmental stress responses in E. carinicauda.

Trehalose metabolism genes of Aphelenchoides besseyi (Nematoda: Aphelenchoididae) in hypertonic osmotic pressure survival

PubMed Central

Chen, Qiaoli; Li, Danlei; Zhang, Ruizhi; Ling, Yaming

2017-01-01

ABSTRACT Some organisms can survive extreme desiccation caused by hypertonic osmotic pressure by entering a state of suspended animation known as osmobiosis. The free-living mycophagous nematode Aphelenchoides besseyi can be induced to enter osmobiosis by soaking in osmolytes. It is assumed that sugars (in particular trehalose) are instrumental for survival under environmental stress. In A. besseyi, two putative trehalose-6-phosphate synthase genes (TPS) encoding enzymes catalyzing trehalose synthesis, and a putative trehalase gene (TRE) encoding enzymes that catalyze hydrolysis of trehalose were identified and then characterized based on their transcriptome. RT-qPCR analyses showed that each of these genes is expressed as mRNA when A. besseyi is entering in, during and recovering from osmobiosis, but only for certain periods. The changes of TRE activity were consistent with the transcript level changes of the TRE gene, and the trehalose level declined at certain periods when the nematodes were in, as well as recovering from, osmobiosis; this suggested that the hydrolysis of threhalose is essential. The feeding method of RNA interference (RNAi) was used to temporarily knock down the expression of each of the TPS and TRE genes. No obviously different phenotype was observed from any of the genes silenced individually or simultaneously, but the survival under hypertonic osmotic pressure reduced significantly and the recovery was delayed. These results indicated that trehalose metabolism genes should play a role in osmobiosis regulation and function within a restricted time frame. PMID:28396490

The Bordetella bhu Locus Is Required for Heme Iron Utilization

PubMed Central

Vanderpool, Carin K.; Armstrong, Sandra K.

2001-01-01

Bordetella pertussis and Bordetella bronchiseptica are capable of obtaining iron from hemin and hemoglobin. Genes encoding a putative bacterial heme iron acquisition system (bhu, for Bordetella heme utilization) were identified in a B. pertussis genomic sequence database, and the corresponding DNA was isolated from a virulent strain of B. pertussis. A B. pertussis bhuR mutant, predicted to lack the heme outer membrane receptor, was generated by allelic exchange. In contrast to the wild-type strain, bhuR mutant PM5 was incapable of acquiring iron from hemin and hemoglobin; genetic complementation of PM5 with the cloned bhuRSTUV genes restored heme utilization to wild-type levels. In parallel studies, B. bronchiseptica bhu sequences were also identified and a B. bronchiseptica bhuR mutant was constructed and confirmed to be defective in heme iron acquisition. The wild-type B. bronchiseptica parent strain grown under low-iron conditions produced the presumptive BhuR protein, which was absent in the bhuR mutant. Furthermore, production of BhuR by iron-starved B. bronchiseptica was markedly enhanced by culture in hemin-supplemented medium, suggesting that these organisms sense and respond to heme in the environment. Analysis of the genetic region upstream of the bhu cluster identified open reading frames predicted to encode homologs of the Escherichia coli ferric citrate uptake regulators FecI and FecR. These putative Bordetella regulators may mediate heme-responsive positive transcriptional control of the bhu genes. PMID:11418569

Molecular interactions and immune responses between Maize fine streak virus and the leafhopper vector Graminella nigrifrons through differential expression and RNA interference.

PubMed

Chen, Y; Redinbaugh, M G; Michel, A P

2015-06-01

Graminella nigrifrons is the only known vector for Maize fine streak virus (MFSV). In this study, we used real-time quantitative PCR to compare the expression profiles of transcripts that putatively function in the insect immune response: four peptidoglycan recognition proteins (PGRP-SB1, -SD, -LC and LB), Toll, spaetzle, defensin, Dicer-2 (Dcr-2), Argonaut-2 (Ago-2) and Arsenic resistance protein 2 (Ars-2). Except for PGRP-LB and defensin, transcripts involved in humoral pathways were significantly suppressed in G. nigrifrons fed on MFSV-infected maize. The abundance of three RNA interference (RNAi) pathway transcripts (Dcr-2, Ago-2, Ars-2) was significantly lower in nontransmitting relative to transmitting G. nigrifrons. Injection with double-stranded RNA (dsRNA) encoding segments of the PGRP-LC and Dcr-2 transcripts effectively reduced transcript levels by 90 and 75% over 14 and 22 days, respectively. MFSV acquisition and transmission were not significantly affected by injection of either dsRNA. Knock-down of PGRP-LC resulted in significant mortality (greater than 90%) at 27 days postinjection, and resulted in more abnormal moults relative to those injected with Dcr-2 or control dsRNA. The use of RNAi to silence G. nigrifrons transcripts will facilitate the study of gene function and pathogen transmission, and may provide approaches for developing novel targets of RNAi-based pest control. © 2015 The Royal Entomological Society.

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

PubMed Central

Alderwick, Luke J.; Molle, Virginie; Kremer, Laurent; Cozzone, Alain J.; Dafforn, Timothy R.; Besra, Gurdyal S.; Fütterer, Klaus

2006-01-01

Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 Å), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 Å) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors. PMID:16477027

Investigation of DNA sequence recognition by a streptomycete MarR family transcriptional regulator through surface plasmon resonance and X-ray crystallography

PubMed Central

Stevenson, Clare E. M.; Assaad, Aoun; Chandra, Govind; Le, Tung B. K.; Greive, Sandra J.; Bibb, Mervyn J.; Lawson, David M.

2013-01-01

Consistent with their complex lifestyles and rich secondary metabolite profiles, the genomes of streptomycetes encode a plethora of transcription factors, the vast majority of which are uncharacterized. Herein, we use Surface Plasmon Resonance (SPR) to identify and delineate putative operator sites for SCO3205, a MarR family transcriptional regulator from Streptomyces coelicolor that is well represented in sequenced actinomycete genomes. In particular, we use a novel SPR footprinting approach that exploits indirect ligand capture to vastly extend the lifetime of a standard streptavidin SPR chip. We define two operator sites upstream of sco3205 and a pseudopalindromic consensus sequence derived from these enables further potential operator sites to be identified in the S. coelicolor genome. We evaluate each of these through SPR and test the importance of the conserved bases within the consensus sequence. Informed by these results, we determine the crystal structure of a SCO3205-DNA complex at 2.8 Å resolution, enabling molecular level rationalization of the SPR data. Taken together, our observations support a DNA recognition mechanism involving both direct and indirect sequence readout. PMID:23748564

Genomic organization of human fetal specific P-450IIIA7 (cytochrome P-450HFLa)-related gene(s) and interaction of transcriptional regulatory factor with its DNA element in the 5' flanking region.

PubMed

Itoh, S; Yanagimoto, T; Tagawa, S; Hashimoto, H; Kitamura, R; Nakajima, Y; Okochi, T; Fujimoto, S; Uchino, J; Kamataki, T

1992-03-24

P-450IIIA7 is a form of cytochrome P-450 which was isolated from human fetal livers and termed P-450HFLa. This form has been clarified to be expressed during fetal life specifically (Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. and Kamataki, T. (1990) Biochemistry 29, 4430-4433). In the present study, we isolated five independent clones which probably corresponded to the human P-450IIIA7 gene. These clones were completely sequenced, all exons, exon-intron junctions and the 5' flanking region from the cap site to-869. Although the sequences in the coding region were completely identical to P-450IIIA7, it is possible that genomic fragments sequenced in this study encode portions of other P-450IIIA7-related genes since we could not obtain a complete overlapping set of genomic clones. Within its 5' flanking sequence, the putative binding sites of several transcriptional regulatory factors existed. Among them, it was shown that a basic transcription element binding factor (BTEB) actually interacted with the 5' flanking region of this gene.

Deciphering cGMP signatures and cGMP-dependent pathways in plant defence

PubMed Central

Meier, Stuart; Madeo, Laura; Ederli, Luisa; Donaldson, Lara; Gehring, Chris

2009-01-01

The second messenger, 3′,5′-cyclic monophosphate (cGMP), is a critical component of many different processes in plants while guanylyl cyclases that catalyse the formation of cGMP from GTP have remained somewhat elusive in higher plants. Consequently, two major aims are the discovery of novel GCs and the identification of cGMP mediated processes. Recently, we have reported temporal signatures of ozone (O3)-induced hydrogen peroxide (H2O2) and nitric oxide (NO) generation, their effect on cGMP generation, and consequent transcriptional changes of genes diagnostic for stress responses in tobacco. We demonstrated that O3 and NO induced early transcriptional activation of the scavenger encoding proteins, alternative oxidase (AOX1a), glutathione peroxidase (GPX) and the induction of ethylene production through aminocyclopropancarboxylic acid synthase (ACS2) are cGMP-independent. By contrast, the early response of the phenylalanine ammonia lyase gene (PALa) and the late response of the gene encoding the pathogenesis-related protein (PR1a) show critical dependence on cGMP. Here we show differential cGMP responses to virulent and avirulent Pseudomonas syringae strains and propose that host-pathogen recognition and/or down-stream processes are transduced by complex cGMP signatures. This is in accordance with the identification of a growing number of multi-domain molecules in Arabidopsis that are reported to contain putative functional GC catalytic centers. PMID:19794847

MGOUN1 encodes an Arabidopsis type IB DNA topoisomerase required in stem cell regulation and to maintain developmentally regulated gene silencing.

PubMed

Graf, Philipp; Dolzblasz, Alicja; Würschum, Tobias; Lenhard, Michael; Pfreundt, Ulrike; Laux, Thomas

2010-03-01

Maintenance of stem cells in the Arabidopsis thaliana shoot meristem is regulated by signals from the underlying cells of the organizing center, provided through the transcription factor WUSCHEL (WUS). Here, we report the isolation of several independent mutants of MGOUN1 (MGO1) as genetic suppressors of ectopic WUS activity and enhancers of stem cell defects in hypomorphic wus alleles. mgo1 mutants have previously been reported to result in a delayed progression of meristem cells into differentiating organ primordia (Laufs et al., 1998). Genetic analyses indicate that MGO1 functions together with WUS in stem cell maintenance at all stages of shoot and floral meristems. Synergistic interactions of mgo1 with several chromatin mutants suggest that MGO1 affects gene expression together with chromatin remodeling pathways. In addition, the expression states of developmentally regulated genes are randomly switched in mgo1 in a mitotically inheritable way, indicating that MGO1 stabilizes epigenetic states against stochastically occurring changes. Positional cloning revealed that MGO1 encodes a putative type IB topoisomerase, which in animals and yeast has been shown to be required for regulation of DNA coiling during transcription and replication. The specific developmental defects in mgo1 mutants link topoisomerase IB function in Arabidopsis to stable propagation of developmentally regulated gene expression.

Cloning and characterization of a novel NAC family gene CarNAC1 from chickpea (Cicer arietinum L.).

PubMed

Peng, Hui; Yu, Xingwang; Cheng, Huiying; Shi, Qinghua; Zhang, Hua; Li, Jiangui; Ma, Hao

2010-01-01

The plant-specific NAC (for NAM, ATAF1,2 and CUC2) proteins have been found to play important roles in plant development and stress responses. In this study, a NAC gene CarNAC1 (for Cicer arietinum L. NAC gene 1) was isolated from a cDNA library constructed with chickpea seedling leaves treated by polyethylene glycol. CarNAC1 encoded a putative protein with 239 amino acids and contained 3 exons and 2 introns within genomic DNA sequence. CarNAC1 had a conserved NAC domain in the N-terminus and the CarNAC1:GFP (green fluorescent protein) fusion protein was localized in the nucleus of onion epidermal cells. Additionally, CarNAC1 exhibited the trans-activation activity which was mapped to the C-terminus. The CarNAC1 transcript was detected in many chickpea organs including seedling leaves, stems, roots, flowers, and young pods, but less accumulated in young seeds. CarNAC1 was induced by leaf age and showed changes in expression during seed development and germination. Furthermore, the expression of CarNAC1 was strongly induced by drought, salt, cold, wounding, H(2)O(2), ethephon, salicylic acid, indole-3-acetic acid, and gibberellin. Our results suggest that CarNAC1 encodes a novel NAC-domain protein and may be a transcriptional activator involved in plant development and various stress responses.

Bacterial discrimination by Dictyostelid amoebae reveals the complexity of ancient interspecies interactions

PubMed Central

Nasser, Waleed; Santhanam, Balaji; Miranda, Edward Roshan; Parikh, Anup; Juneja, Kavina; Rot, Gregor; Dinh, Chris; Chen, Rui; Zupan, Blaz; Shaulsky, Gad; Kuspa, Adam

2014-01-01

Background Amoebae and bacteria interact within predator/prey and host/pathogen relationships, but the general response of amoeba to bacteria is not well understood. The amoeba Dictyostelium discoideum feeds on, and is colonized by diverse bacterial species including Gram-positive [Gram(+)] and Gram-negative [Gram(−)] bacteria, two major groups of bacteria that differ in structure and macromolecular composition. Results Transcriptional profiling of D. discoideum revealed sets of genes whose expression is enriched in amoebae interacting with different species of bacteria, including sets that appear specific to amoebae interacting with Gram(+), or with Gram(−) bacteria. In a genetic screen utilizing the growth of mutant amoebae on a variety of bacteria as a phenotypic readout, we identified amoebal genes that are only required for growth on Gram(+) bacteria, including one that encodes the cell surface protein gp130, as well as several genes that are only required for growth on Gram(−) bacteria including one that encodes a putative lysozyme, AlyL. These genes are required for parts of the transcriptional response of wild-type amoebae, and this allowed their classification into potential response pathways. Conclusions We have defined genes that are critical for amoebal survival during feeding on Gram(+), or Gram(−), bacteria which we propose form part of a regulatory network that allows D. discoideum to elicit specific cellular responses to different species of bacteria in order to optimize survival. PMID:23664307

Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog

PubMed Central

Willkomm, Dagmar K.; Minnerup, Jens; Hüttenhofer, Alexander; Hartmann, Roland K.

2005-01-01

By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its γ-proteobacterial homologs (∼185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the γ-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the γ-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites. PMID:15814812

The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies.

PubMed

Dostálová, Anna; Votýpka, Jan; Favreau, Amanda J; Barbian, Kent D; Volf, Petr; Valenzuela, Jesus G; Jochim, Ryan C

2011-05-10

Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles. A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal. This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that L. infantum infection can reduce the transcript abundance of trypsin PperTryp3 in the midgut of P. perniciosus.

Methylotrophic Bacillus methanolicus Encodes Two Chromosomal and One Plasmid Born NAD+ Dependent Methanol Dehydrogenase Paralogs with Different Catalytic and Biochemical Properties

PubMed Central

Müller, Jonas E. N.; Kupper, Christiane E.; Schneider, Olha; Vorholt, Julia A.; Ellingsen, Trond E.; Brautaset, Trygve

2013-01-01

Bacillus methanolicus can utilize methanol as the sole carbon source for growth and it encodes an NAD+-dependent methanol dehydrogenase (Mdh), catalyzing the oxidation of methanol to formaldehyde. Recently, the genomes of the B. methanolicus strains MGA3 (ATCC53907) and PB1 (NCIMB13113) were sequenced and found to harbor three different putative Mdh encoding genes, each belonging to the type III Fe-NAD+-dependent alcohol dehydrogenases. In each strain, two of these genes are encoded on the chromosome and one on a plasmid; only one chromosomal act gene encoding the previously described activator protein ACT was found. The six Mdhs and the ACT proteins were produced recombinantly in Escherichia coli, purified, and characterized. All Mdhs required NAD+ as cosubstrate, were catalytically stimulated by ACT, exhibited a broad and different substrate specificity range and displayed both dehydrogenase and reductase activities. All Mdhs catalyzed the oxidation of methanol; however the catalytic activity for methanol was considerably lower than for most other alcohols tested, suggesting that these enzymes represent a novel class of alcohol dehydrogenases. The kinetic constants for the Mdhs were comparable when acting as pure enzymes, but together with ACT the differences were more pronounced. Quantitative PCR experiments revealed major differences with respect to transcriptional regulation of the paralogous genes. Taken together our data indicate that the repertoire of methanol oxidizing enzymes in thermotolerant bacilli is larger than expected with complex mechanisms involved in their regulation. PMID:23527128

Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD+ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties.

PubMed

Krog, Anne; Heggeset, Tonje M B; Müller, Jonas E N; Kupper, Christiane E; Schneider, Olha; Vorholt, Julia A; Ellingsen, Trond E; Brautaset, Trygve

2013-01-01

Bacillus methanolicus can utilize methanol as the sole carbon source for growth and it encodes an NAD(+)-dependent methanol dehydrogenase (Mdh), catalyzing the oxidation of methanol to formaldehyde. Recently, the genomes of the B. methanolicus strains MGA3 (ATCC53907) and PB1 (NCIMB13113) were sequenced and found to harbor three different putative Mdh encoding genes, each belonging to the type III Fe-NAD(+)-dependent alcohol dehydrogenases. In each strain, two of these genes are encoded on the chromosome and one on a plasmid; only one chromosomal act gene encoding the previously described activator protein ACT was found. The six Mdhs and the ACT proteins were produced recombinantly in Escherichia coli, purified, and characterized. All Mdhs required NAD(+) as cosubstrate, were catalytically stimulated by ACT, exhibited a broad and different substrate specificity range and displayed both dehydrogenase and reductase activities. All Mdhs catalyzed the oxidation of methanol; however the catalytic activity for methanol was considerably lower than for most other alcohols tested, suggesting that these enzymes represent a novel class of alcohol dehydrogenases. The kinetic constants for the Mdhs were comparable when acting as pure enzymes, but together with ACT the differences were more pronounced. Quantitative PCR experiments revealed major differences with respect to transcriptional regulation of the paralogous genes. Taken together our data indicate that the repertoire of methanol oxidizing enzymes in thermotolerant bacilli is larger than expected with complex mechanisms involved in their regulation.

Transcriptomics insights into the genetic regulation of root apical meristem exhaustion and determinate primary root growth in Pachycereus pringlei (Cactaceae).

PubMed

Rodriguez-Alonso, Gustavo; Matvienko, Marta; López-Valle, Mayra L; Lázaro-Mixteco, Pedro E; Napsucialy-Mendivil, Selene; Dubrovsky, Joseph G; Shishkova, Svetlana

2018-06-04

Many Cactaceae species exhibit determinate growth of the primary root as a consequence of root apical meristem (RAM) exhaustion. The genetic regulation of this growth pattern is unknown. Here, we de novo assembled and annotated the root apex transcriptome of the Pachycereus pringlei primary root at three developmental stages, with active or exhausted RAM. The assembled transcriptome is robust and comprehensive, and was used to infer a transcriptional regulatory network of the primary root apex. Putative orthologues of Arabidopsis regulators of RAM maintenance, as well as putative lineage-specific transcripts were identified. The transcriptome revealed putative orthologues of most proteins involved in housekeeping processes, hormone signalling, and metabolic pathways. Our results suggest that specific transcriptional programs operate in the root apex at specific developmental time points. Moreover, the transcriptional state of the P. pringlei root apex as the RAM becomes exhausted is comparable to the transcriptional state of cells from the meristematic, elongation, and differentiation zones of Arabidopsis roots along the root axis. We suggest that the transcriptional program underlying the drought stress response is induced during Cactaceae root development, and that lineage-specific transcripts could contribute to RAM exhaustion in Cactaceae.

The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

PubMed Central

Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

1980-01-01

Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

Identification and Characterization of Putative Integron-Like Elements of the Heavy-Metal-Hypertolerant Strains of Pseudomonas spp.

PubMed

Ciok, Anna; Adamczuk, Marcin; Bartosik, Dariusz; Dziewit, Lukasz

2016-11-28

Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas .

Molecular and Mutational Analysis of a Gelsolin-Family Member Encoded by the Flightless I Gene of Drosophila Melanogaster

PubMed Central

de-Couet, H. G.; Fong, KSK.; Weeds, A. G.; McLaughlin, P. J.; Miklos, GLG.

1995-01-01

The flightless locus of Drosophila melanogaster has been analyzed at the genetic, molecular, ultrastructural and comparative crystallographic levels. The gene encodes a single transcript encoding a protein consisting of a leucine-rich amino terminal half and a carboxyterminal half with high sequence similarity to gelsolin. We determined the genomic sequence of the flightless landscape, the breakpoints of four chromosomal rearrangements, and the molecular lesions in two lethal and two viable alleles of the gene. The two alleles that lead to flight muscle abnormalities encode mutant proteins exhibiting amino acid replacements within the S1-like domain of their gelsolin-like region. Furthermore, the deduced intronexon structure of the D. melanogaster gene has been compared with that of the Caenorhabditis elegans homologue. Furthermore, the sequence similarities of the flightless protein with gelsolin allow it to be evaluated in the context of the published crystallographic structure of the S1 domain of gelsolin. Amino acids considered essential for the structural integrity of the core are found to be highly conserved in the predicted flightless protein. Some of the residues considered essential for actin and calcium binding in gelsolin S1 and villin V1 are also well conserved. These data are discussed in light of the phenotypic characteristics of the mutants and the putative functions of the protein. PMID:8582612

Comparison of good- and bad-quality cork: application of high-throughput sequencing of phellogenic tissue.

PubMed

Teixeira, Rita Teresa; Fortes, Ana Margarida; Pinheiro, Carla; Pereira, Helena

2014-09-01

Cork is one of the most valuable non-wood forest products and plays an important role in Mediterranean economies. The production of high-quality cork is dependent on both genome and environment, posing constraints on the industry because an ever-growing amount of bad-quality cork (BQC) development has been observed. In order to identify genes responsible for production of cork of superior quality we performed a comparative analysis using the 454 pyrosequencing approach on phellogenic tissue of good- and bad-quality samples. The transcriptional profiling showed a high number of genes differentially expressed (8.48%) from which 78.8% displayed annotation. Genes more highly represented in BQC are involved in DNA synthesis, RNA processing, proteolysis, and transcription factors related to the abiotic stress response. Putative stomatal/lenticular-associated genes which may be responsible for the disadvantageous higher number of lenticular channels in BQC are also more highly represented. BQC also showed an elevated content of free phenolics. On the other hand, good-quality cork (GQC) can be distinguished by highly expressed genes encoding heat-shock proteins. Together the results provide valuable new information about the molecular events leading to cork formation and provide putative biomarkers associated with cork quality that can be useful in breeding programmes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Function analysis of 5'-UTR of the cellulosomal xyl-doc cluster in Clostridium papyrosolvens.

PubMed

Zou, Xia; Ren, Zhenxing; Wang, Na; Cheng, Yin; Jiang, Yuanyuan; Wang, Yan; Xu, Chenggang

2018-01-01

Anaerobic, mesophilic, and cellulolytic Clostridium papyrosolvens produces an efficient cellulolytic extracellular complex named cellulosome that hydrolyzes plant cell wall polysaccharides into simple sugars. Its genome harbors two long cellulosomal clusters: cip - cel operon encoding major cellulosome components (including scaffolding) and xyl - doc gene cluster encoding hemicellulases. Compared with works on cip - cel operon, there are much fewer studies on xyl - doc mainly due to its rare location in cellulolytic clostridia. Sequence analysis of xyl - doc revealed that it harbors a 5' untranslated region (5'-UTR) which potentially plays a role in the regulation of downstream gene expression. Here, we analyzed the function of 5'-UTR of xyl - doc cluster in C. papyrosolvens in vivo via transformation technology developed in this study. In this study, we firstly developed an electrotransformation method for C. papyrosolvens DSM 2782 before the analysis of 5'-UTR of xyl - doc cluster. In the optimized condition, a field with an intensity of 7.5-9.0 kV/cm was applied to a cuvette (0.2 cm gap) containing a mixture of plasmid and late cell suspended in exponential phase to form a 5 ms pulse in a sucrose-containing buffer. Afterwards, the putative promoter and the 5'-UTR of xyl - doc cluster were determined by sequence alignment. It is indicated that xyl - doc possesses a long conservative 5'-UTR with a complex secondary structure encompassing at least two perfect stem-loops which are potential candidates for controlling the transcriptional termination. In the last step, we employed an oxygen-independent flavin-based fluorescent protein (FbFP) as a quantitative reporter to analyze promoter activity and 5'-UTR function in vivo. It revealed that 5'-UTR significantly blocked transcription of downstream genes, but corn stover can relieve its suppression. In the present study, our results demonstrated that 5'-UTR of the cellulosomal xyl - doc cluster blocks the transcriptional activity of promoter. However, some substrates, such as corn stover, can relieve the effect of depression of 5'-UTR. Thus, it is speculated that 5'-UTR of xyl - doc was a putative riboswitch to regulate the expression of downstream cellulosomal genes, which is helpful to understand the complex regulation of cellulosome.

Pathogen-regulated genes in wheat isogenic lines differing in resistance to brown rust Puccinia triticina.

PubMed

Dmochowska-Boguta, Marta; Alaba, Sylwia; Yanushevska, Yuliya; Piechota, Urszula; Lasota, Elzbieta; Nadolska-Orczyk, Anna; Karlowski, Wojciech M; Orczyk, Waclaw

2015-10-05

Inoculation of wheat plants with Puccinia triticina (Pt) spores activates a wide range of host responses. Compatible Pt interaction with susceptible Thatcher plants supports all stages of the pathogen life cycle. Incompatible interaction with TcLr9 activates defense responses including oxidative burst and micronecrotic reactions associated with the pathogen's infection structures and leads to complete termination of pathogen development. These two contrasting host-pathogen interactions were a foundation for transcriptome analysis of incompatible wheat-Pt interaction. A suppression subtractive hybridization (SSH) library was constructed using cDNA from pathogen-inoculated susceptible Thatcher and resistant TcLr9 isogenic lines. cDNA represented steps of wheat-brown rust interactions: spore germination, haustorium mother cell (HMC) formation and micronecrotic reactions. All ESTs were clustered and validated by similarity search to wheat genome using BLASTn and sim4db tools. qRT-PCR was used to determine transcript levels of selected ESTs after inoculation in both lines. Out of 793 isolated cDNA clones, 183 were classified into 152 contigs. 89 cDNA clones and encoded proteins were functionally annotated and assigned to 5 Gene Ontology categories: catalytic activity 48 clones (54 %), binding 32 clones (36 %), transporter activity 6 clones (7 %), structural molecule activity 2 clones (2 %) and molecular transducer activity 1 clone (1 %). Detailed expression profiles of 8 selected clones were analyzed using the same plant-pathogen system. The strongest induction after pathogen infection and the biggest differences between resistant and susceptible interactions were detected for clones encoding wall-associated kinase (GenBank accession number JG969003), receptor with leucine-rich repeat domain (JG968955), putative serine/threonine protein kinase (JG968944), calcium-mediated signaling protein (JG968925) and 14-3-3 protein (JG968969). The SSH library represents transcripts regulated by pathogen infection during compatible and incompatible interactions of wheat with P. triticina. Annotation of selected clones confirms their putative roles in successive steps of plant-pathogen interactions. The transcripts can be categorized as defense-related due to their involvement in either basal defense or resistance through an R-gene mediated reaction. The possible involvement of selected clones in pathogen recognition and pathogen-induced signaling as well as resistance mechanisms such as cell wall enforcement, oxidative burst and micronecrotic reactions is discussed.

Functional analysis of the theobroma cacao NPR1 gene in arabidopsis

PubMed Central

2010-01-01

Background The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. Results A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. Conclusion Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies. PMID:21078185

Functional analysis of the Theobroma cacao NPR1 gene in Arabidopsis.

PubMed

Shi, Zi; Maximova, Siela N; Liu, Yi; Verica, Joseph; Guiltinan, Mark J

2010-11-15

The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies.

Three cDNAs encoding vitellogenin homologs from Antarctic copepod, Tigriopus kingsejongensis: Cloning and transcriptional analysis in different maturation stages, temperatures, and putative reproductive hormones.

PubMed

Lee, Soo Rin; Lee, Ji-Hyun; Kim, Ah Ran; Kim, Sanghee; Park, Hyun; Baek, Hea Ja; Kim, Hyun-Woo

2016-02-01

Three full-length cDNAs encoding lipoprotein homologs were identified in Tigriopus kingsejongensis, a newly identified copepod from Antarctica. Structural and transcriptional analyses revealed homology with two vitellogenin-like proteins, Tik-Vg1 and Tik-Vg2, which were 1855 and 1795 amino acids in length, respectively, along with a third protein, Tik-MEP, which produced a 1517-residue protein with similarity to a melanin engaging protein (MEP) in insects Phylogenetic analysis showed that Vgs in Maxillopods including two Tik-Vgs belong to the arthropod vitellogenin-like clade, which includes clottable proteins (CPs) in decapod crustaceans and vitellogenins in insects. Tik-MEP clustered together with insect MEPs, which appear to have evolved before the apoB-like and arthropod Vg-like clades. Interestingly, no genes orthologous to those found in the apoB clade were identified in Maxillopoda, suggesting that functions of large lipid transfer proteins (LLTPs) in reproduction and lipid metabolism may be different from those in insect and decapod crustaceans. As suggested by phylogenetic analyses, the two Tik-Vgs belonging to the arthropod Vg-like clade appear to play major roles in oocyte maturation, while Vgs belonging to the apoB clade function primarily in the reproduction of decapod crustaceans. Transcriptional analysis of Tik-Vg expression revealed a 24-fold increase in mature and ovigerous females compared with immature female, whereas expression of Tik-MEP remained low through all reproductive stages. Acute temperature changes did not affect the transcription of Tik-Vg genes, whereas Tik-MEP appeared to be affected by temperature change. Among the three hormones thought to be involved in molting and reproduction in arthropods, only farnesoic acid (FA) induced transcription of the two Tik-Vg genes. Regardless of developmental stage and hormone treatment, Tik-Vg1 and Tik-Vg2 exhibited a strong positive correlation in expression, suggesting that expression of these genes may be regulated by the same transcriptional machinery. Copyright © 2015 Elsevier Inc. All rights reserved.

The switch from fermentation to respiration in Saccharomyces cerevisiae is regulated by the Ert1 transcriptional activator/repressor.

PubMed

Gasmi, Najla; Jacques, Pierre-Etienne; Klimova, Natalia; Guo, Xiao; Ricciardi, Alessandra; Robert, François; Turcotte, Bernard

2014-10-01

In the yeast Saccharomyces cerevisiae, fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration. This adaptation results in massive reprogramming of gene expression. Increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced. The zinc cluster proteins Cat8, Sip4, and Rds2, as well as Adr1, have been shown to mediate this reprogramming of gene expression. In this study, we have characterized the gene YBR239C encoding a putative zinc cluster protein and it was named ERT1 (ethanol regulated transcription factor 1). ChIP-chip analysis showed that Ert1 binds to a limited number of targets in the presence of glucose. The strongest enrichment was observed at the promoter of PCK1 encoding an important gluconeogenic enzyme. With ethanol as the carbon source, enrichment was observed with many additional genes involved in gluconeogenesis and mitochondrial function. Use of lacZ reporters and quantitative RT-PCR analyses demonstrated that Ert1 regulates expression of its target genes in a manner that is highly redundant with other regulators of gluconeogenesis. Interestingly, in the presence of ethanol, Ert1 is a repressor of PDC1 encoding an important enzyme for fermentation. We also show that Ert1 binds directly to the PCK1 and PDC1 promoters. In summary, Ert1 is a novel factor involved in the regulation of gluconeogenesis as well as a key fermentation gene. Copyright © 2014 by the Genetics Society of America.

Genes associated with lignin degradation in the polyphagous white-rot pathogen Heterobasidion irregulare show substrate-specific regulation.

PubMed

Yakovlev, Igor A; Hietala, Ari M; Courty, Pierre-Emmanuel; Lundell, Taina; Solheim, Halvor; Fossdal, Carl Gunnar

2013-07-01

The pathogenic white-rot basidiomycete Heterobasidion irregulare is able to remove lignin and hemicellulose prior to cellulose during the colonization of root and stem xylem of conifer and broadleaf trees. We identified and followed the regulation of expression of genes belonging to families encoding ligninolytic enzymes. In comparison with typical white-rot fungi, the H. irregulare genome has exclusively the short-manganese peroxidase type encoding genes (6 short-MnPs) and thereby a slight contraction in the pool of class II heme-containing peroxidases, but an expansion of the MCO laccases with 17 gene models. Furthermore, the genome shows a versatile set of other oxidoreductase genes putatively involved in lignin oxidation and conversion, including 5 glyoxal oxidases, 19 quinone-oxidoreductases and 12 aryl-alcohol oxidases. Their genetic multiplicity and gene-specific regulation patterns on cultures based on defined lignin, cellulose or Norway spruce lignocellulose substrates suggest divergent specificities and physiological roles for these enzymes. While the short-MnP encoding genes showed similar transcript levels upon fungal growth on heartwood and reaction zone (RZ), a xylem defense tissue rich in phenolic compounds unique to trees, a subset of laccases showed higher gene expression in the RZ cultures. In contrast, other oxidoreductases depending on initial MnP activity showed generally lower transcript levels on RZ than on heartwood. These data suggest that the rate of fungal oxidative conversion of xylem lignin differs between spruce RZ and heartwood. It is conceivable that in RZ part of the oxidoreductase activities of laccases are related to the detoxification of phenolic compounds involved in host-defense. Expression of the several short-MnP enzymes indicated an important role for these enzymes in effective delignification of wood by H. irregulare. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular Cloning and Expression Analysis of Eight PgWRKY Genes in Panax ginseng Responsive to Salt and Hormones.

PubMed

Xiu, Hao; Nuruzzaman, Mohammed; Guo, Xiangqian; Cao, Hongzhe; Huang, Jingjia; Chen, Xianghui; Wu, Kunlu; Zhang, Ru; Huang, Yuzhao; Luo, Junli; Luo, Zhiyong

2016-03-04

Despite the importance of WRKY genes in plant physiological processes, little is known about their roles in Panax ginseng C.A. Meyer. Forty-eight unigenes on this species were previously reported as WRKY transcripts using the next-generation sequencing (NGS) technology. Subsequently, one gene that encodes PgWRKY1 protein belonging to subgroup II-d was cloned and functionally characterized. In this study, eight WRKY genes from the NGS-based transcriptome sequencing dataset designated as PgWRKY2-9 have been cloned and characterized. The genes encoding WRKY proteins were assigned to WRKY Group II (one subgroup II-c, four subgroup II-d, and three subgroup II-e) based on phylogenetic analysis. The cDNAs of the cloned PgWRKYs encode putative proteins ranging from 194 to 358 amino acid residues, each of which includes one WRKYGQK sequence motif and one C₂H₂-type zinc-finger motif. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the eight analyzed PgWRKY genes were expressed at different levels in various organs including leaves, roots, adventitious roots, stems, and seeds. Importantly, the transcription responses of these PgWRKYs to methyl jasmonate (MeJA) showed that PgWRKY2, PgWRKY3, PgWRKY4, PgWRKY5, PgWRKY6, and PgWRKY7 were downregulated by MeJA treatment, while PgWRKY8 and PgWRKY9 were upregulated to varying degrees. Moreover, the PgWRKY genes increased or decreased by salicylic acid (SA), abscisic acid (ABA), and NaCl treatments. The results suggest that the PgWRKYs may be multiple stress-inducible genes responding to both salt and hormones.

Biosynthesis and uptake of glycine betaine as cold-stress response to low temperature in fish pathogen Vibrio anguillarum.

PubMed

Ma, Yue; Wang, Qiyao; Gao, Xiating; Zhang, Yuanxing

2017-01-01

Fish pathogen Vibrio anguillarum, a mesophile bacterium, is usually found in estuarine and marine coastal ecosystems worldwide that pose a constant stress to local organism by its fluctuation in salinity as well as notable temperature change. Though V. anguillarum is able to proliferate while maintain its pathogenicity under low temperature (5-18°C), so far, coldadaption molecular mechanism of the bacteria is unknown. In this study, V. anguillarum was found possessing a putative glycine betaine synthesis system, which is encoded by betABI and synthesizes glycine betaine from its precursor choline. Furthermore, significant up-regulation of the bet gene at the transcriptional level was noted in log phase in response to cold-stress. Moreover, the accumulation of betaine glycine was only found appearing at low growth temperatures, suggesting that response regulation of both synthesis system and transporter system are cold-dependent. Furthermore, in-frame deletion mutation in the two putative ABC transporters and three putative BCCT family transporters associated with glycine betaine uptake could not block cellular accumulation of betaine glycine in V. anguillarum under coldstress, suggesting the redundant feature in V. anguillarum betaine transporter system. These findings confirmed that glycine betaine serves as an effective cold stress protectant and highlighted an underappreciated facet of the acclimatization of V. anguillarum to cold environments.

Characterization of BcMF23a and BcMF23b, two putative pectin methylesterase genes related to pollen development in Brassica campestris ssp. chinensis.

PubMed

Lin, Sue; Huang, Li; Yu, Xiaolin; Xiong, Xingpeng; Yue, Xiaoyan; Liu, Tingting; Liang, Ying; Lv, Meiling; Cao, Jiashu

2017-02-01

Two homologous genes, Brassica campestris Male Fertility 23a (BcMF23a) and Brassica campestris Male Fertility 23b (BcMF23b), encoding putative pectin methylesterases (PMEs) were isolated from Brassica campestris ssp. chinensis (syn. Brassica rapa ssp. chinensis). These two genes sharing high sequence identity with each other were highly expressed in the fertile flower buds but silenced in the sterile ones of genic male sterile line system ('Bcajh97-01A/B'). Results of RT-PCR and in situ hybridization suggested that BcMF23a and BcMF23b were pollen-expressed genes, whose transcripts were first detected at the binucleate pollen and maintained throughout to the mature pollen grains. Western blot indicated that both of the putative BcMF23a and BcMF23b proteins are approximately 40 kDa, which exhibited extracellular localization revealed by transient expression analysis in the onion epidermal cells. The promoter of BcMF23a was active specifically in pollen during the late pollen developmental stages, while, in addition to the pollen, BcMF23b promoter drove an extra gene expression in the valve margins, abscission layer at the base of the first true leaves, taproot and lateral roots in seedlings.

Relating genes to function: identifying enriched transcription factors using the ENCODE ChIP-Seq significance tool.

PubMed

Auerbach, Raymond K; Chen, Bin; Butte, Atul J

2013-08-01

Biological analysis has shifted from identifying genes and transcripts to mapping these genes and transcripts to biological functions. The ENCODE Project has generated hundreds of ChIP-Seq experiments spanning multiple transcription factors and cell lines for public use, but tools for a biomedical scientist to analyze these data are either non-existent or tailored to narrow biological questions. We present the ENCODE ChIP-Seq Significance Tool, a flexible web application leveraging public ENCODE data to identify enriched transcription factors in a gene or transcript list for comparative analyses. The ENCODE ChIP-Seq Significance Tool is written in JavaScript on the client side and has been tested on Google Chrome, Apple Safari and Mozilla Firefox browsers. Server-side scripts are written in PHP and leverage R and a MySQL database. The tool is available at http://encodeqt.stanford.edu. abutte@stanford.edu Supplementary material is available at Bioinformatics online.

A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions

PubMed Central

Shimizu, Kiminori; Paul, Sanjoy; Ohba, Ayumi; Gonoi, Tohru; Watanabe, Akira; Gomi, Katsuya

2017-01-01

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B). PMID:28052140

Regulation of autotrophic CO2 fixation in the archaeon Thermoproteus neutrophilus.

PubMed

Ramos-Vera, W Hugo; Labonté, Valérie; Weiss, Michael; Pauly, Julia; Fuchs, Georg

2010-10-01

Thermoproteus neutrophilus, a hyperthermophilic, chemolithoautotrophic, anaerobic crenarchaeon, uses a novel autotrophic CO(2) fixation pathway, the dicarboxylate/hydroxybutyrate cycle. The regulation of the central carbon metabolism was studied on the level of whole cells, enzyme activity, the proteome, transcription, and gene organization. The organism proved to be a facultative autotroph, which prefers organic acids as carbon sources that can easily feed into the metabolite pools of this cycle. Addition of the preferred carbon sources acetate, pyruvate, succinate, and 4-hydroxybutyrate to cultures resulted in stimulation of the growth rate and a diauxic growth response. The characteristic enzyme activities of the carbon fixation cycle, fumarate hydratase, fumarate reductase, succinyl coenzyme A (CoA) synthetase, and enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA, were differentially downregulated in the presence of acetate and, to a lesser extent, in the presence of other organic substrates. This regulation pattern correlated well with the differential expression profile of the proteome as well as with the transcription of the encoding genes. The genes encoding phosphoenolpyruvate (PEP) carboxylase, fumarate reductase, and four enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA are clustered. Two putative operons, one comprising succinyl-CoA reductase plus 4-hydroxybutyrate-CoA ligase genes and the other comprising 4-hydroxybutyryl-CoA dehydratase plus fumarate reductase genes, were divergently transcribed into leaderless mRNAs. The promoter regions were characterized and used for isolating DNA binding proteins. Besides an Alba protein, a 18-kDa protein characteristic for autotrophic Thermoproteales that bound specifically to the promoter region was identified. This system may be suitable for molecular analysis of the transcriptional regulation of autotrophy-related genes.

Central role of a bacterial two-component gene regulatory system of previously unknown function in pathogen persistence in human saliva.

PubMed

Shelburne, Samuel A; Sumby, Paul; Sitkiewicz, Izabela; Granville, Chanel; DeLeo, Frank R; Musser, James M

2005-11-01

The molecular genetic mechanisms used by bacteria to persist in humans are poorly understood. Group A Streptococcus (GAS) causes the majority of bacterial pharyngitis cases in humans and is prone to persistently inhabit the upper respiratory tract. To gain information about how GAS survives in and infects the oropharynx, we analyzed the transcriptome of a serotype M1 strain grown in saliva. The dynamic pattern of changes in transcripts of genes [spy0874/0875, herein named sptR and sptS (sptR/S), for saliva persistence] encoding a two-component gene regulatory system of unknown function suggested that SptR/S contributed to persistence of GAS in saliva. Consistent with this idea, an isogenic nonpolar mutant strain (DeltasptR) was dramatically less able to survive in saliva compared with the parental strain. Iterative expression microarray analysis of bacteria grown in saliva revealed that transcripts of several known and putative GAS virulence factor genes were decreased significantly in the DeltasptR mutant strain. Compared with the parental strain, the isogenic mutant strain also had altered transcripts of multiple genes encoding proteins involved in complex carbohydrate acquisition and utilization pathways. Western immunoblot analysis and real-time PCR analysis of GAS in throat swabs taken from humans with pharyngitis confirmed the findings. We conclude that SptR/S optimizes persistence of GAS in human saliva, apparently by strategically influencing metabolic pathways and virulence factor production. The discovery of a genetic program that significantly increased persistence of a major human pathogen in saliva enhances understanding of how bacteria survive in the host and suggests new therapeutic strategies.

Expression of Shigella flexneri gluQ-rs gene is linked to dksA and controlled by a transcriptional terminator

PubMed Central

2012-01-01

Background Glutamyl queuosine-tRNAAsp synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNAAsp. Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses. Results The genomic organization, dksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes. Conclusions The results presented here, show that the expression of gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri. PMID:23035718

Expression of Shigella flexneri gluQ-rs gene is linked to dksA and controlled by a transcriptional terminator.

PubMed

Caballero, Valeria C; Toledo, Viviana P; Maturana, Cristian; Fisher, Carolyn R; Payne, Shelley M; Salazar, Juan Carlos

2012-10-05

Glutamyl queuosine-tRNA(Asp) synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNA(Asp). Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses. The genomic organization, dksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes. The results presented here, show that the expression of gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri.

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

PubMed Central

Dziewit, Lukasz; Oscik, Karolina; Bartosik, Dariusz

2014-01-01

ABSTRACT ΦLM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that ΦLM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of ΦLM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage ΦLM21 shows very limited similarity to other known phage genome sequences and is thus considered unique. IMPORTANCE Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, ΦLM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of mimicking the host regulatory mechanisms by viruses is quite common in bacteriophages. PMID:25187538

The Varicella-zoster virus DNA encapsidation genes: Identification and characterization of the putative terminase subunits

PubMed Central

Visalli, Robert J.; Nicolosi, Denise M.; Irven, Karen L.; Goshorn, Bradley; Khan, Tamseel; Visalli, Melissa A.

2007-01-01

The putative DNA encapsidation genes encoded by open reading frames (ORFs) 25, 26, 30, 34, 43, 45/42 and 54 were cloned from Varicella-zoster virus (VZV) strain Ellen. Sequencing revealed that the Ellen ORFs were highly conserved at the amino acid level when compared to those of nineteen previously published VZV isolates. Additionally, RT-PCR provided the first evidence that ORF45/42 was expressed as a spliced transcript in VZV-infected cells. All seven ORFs were expressed in vitro and full length products were identified using a C-terminal V5 epitope tag. The in vitro products of the putative VZV terminase subunits encoded by ORFs 30 and 45/42 proved useful in protein-protein interaction assays. Previous studies have reported the formation of a heterodimeric terminase complex involved in DNA encapsidation for both herpes simplex virus-type 1 (HSV-1) and human cytomegalovirus (HCMV). Here we report that the C-terminal portion of exon II of ORF45/42 (ORF42-C269) interacted in GST-pull down experiments with in vitro synthesized ORF30 and ORF45/42. The interactions were maintained in the presence of anionic detergents and in buffers of increasing ionic strength. Cells transiently transfected with epitope tagged ORF45/42 or ORF30 showed primarily cytoplasmic staining. In contrast, an antiserum directed to the N-terminal portion of ORF45 showed nearly exclusive nuclear localization of the ORF45/42 gene product in infected cells. An ORF30 specific antiserum detected an 87 kDa protein in both the cytoplasmic and nuclear fractions of VZV infected cells. The results were consistent with the localization and function of herpesviral terminase subunits. This is the first study aimed at the identification and characterization of the VZV DNA encapsidation gene products. PMID:17868947

Streptococcus suis serotype 9 strain GZ0565 contains a type VII secretion system putative substrate EsxA that contributes to bacterial virulence and a vanZ-like gene that confers resistance to teicoplanin and dalbavancin in Streptococcus agalactiae.

PubMed

Lai, Liying; Dai, Jiao; Tang, Huanyu; Zhang, Shouming; Wu, Chunyan; Qiu, Wancen; Lu, Chengping; Yao, Huochun; Fan, Hongjie; Wu, Zongfu

2017-06-01

Streptococcus suis (SS), an important pathogen for pigs, is not only considered as a zoonotic agent for humans, but is also recognized as a major reservoir of antimicrobial resistance contributing to the spread of resistance genes to other pathogenic Streptococcus species. In addition to serotype 2 (SS2), serotype 9 (SS9) is another prevalent serotype isolated from diseased pigs. Although many SS strains have been sequenced, the complete genome of a non-SS2 virulent strain has been unavailable to date. Here, we report the complete genome of GZ0565, a virulent strain of SS9, isolated from a pig with meningitis. Comparative genomic analysis revealed five new putative virulence or antimicrobial resistance-associated genes in strain GZ0565 but not in SS2 virulent strains. These five genes encode a putative triacylglycerol lipase, a TipAS antibiotic-recognition domain protein, a putative TetR family transcriptional repressor, a protein containing a LPXTG domain and a G5 domain, and a type VII secretion system (T7SS) putative substrate (EsxA), respectively. Western blot analysis showed that strain GZ0565 can secrete EsxA. We generated an esxA deletion mutant and showed that EsxA contributes to SS virulence in a mouse infection model. Additionally, the antibiotic resistance gene vanZ SS was identified and expression of vanZ SS conferred resistance to teicoplanin and dalbavancin in Streptococcus agalactiae. We believe this is the first experimental demonstration of the existence of the T7SS putative substrate EsxA and its contribution to bacterial virulence in SS. Together, our results contribute to further understanding of the virulence and antimicrobial resistance characteristics of SS. Copyright © 2017 Elsevier B.V. All rights reserved.

A putative positive feedback regulation mechanism in CsACS2 expression suggests a modified model for sex determination in cucumber (Cucumis sativus L.)

PubMed Central

Wang, Shu; Tao, Qianyi; Pan, Junsong; Si, Longting; Gong, Zhenhui; Cai, Run

2012-01-01

It is well established that the plant hormone ethylene plays a key role in cucumber sex determination. Since the unisexual control gene M was cloned and shown to encode an ethylene synthase, instead of an ethylene receptor, the ‘one-hormone hypothesis’, which was used to explain the cucumber sex phenotype, has been challenged. Here, the physiological function of CsACS2 (the gene encoded by the M locus) was studied using the transgenic tobacco system. The results indicated that overexpression of CsACS2 increased ethylene production in the tobacco plant, and the native cucumber promoter had no activity in transgenic tobacco (PM). However, when PM plants were treated with exogenous ethylene, CsACS2 expression could be detected. In cucumber, ethylene treatment could also induce transcription of CsACS2, while inhibition of ethylene action reduced the expression level. These findings suggest a positive feedback regulation mechanism for CsACS2, and a modified ‘one-hormone hypothesis’ for sex determination in cucumber is proposed. PMID:22577183

A putative positive feedback regulation mechanism in CsACS2 expression suggests a modified model for sex determination in cucumber (Cucumis sativus L.).

PubMed

Li, Zheng; Wang, Shu; Tao, Qianyi; Pan, Junsong; Si, Longting; Gong, Zhenhui; Cai, Run

2012-07-01

It is well established that the plant hormone ethylene plays a key role in cucumber sex determination. Since the unisexual control gene M was cloned and shown to encode an ethylene synthase, instead of an ethylene receptor, the 'one-hormone hypothesis', which was used to explain the cucumber sex phenotype, has been challenged. Here, the physiological function of CsACS2 (the gene encoded by the M locus) was studied using the transgenic tobacco system. The results indicated that overexpression of CsACS2 increased ethylene production in the tobacco plant, and the native cucumber promoter had no activity in transgenic tobacco (PM). However, when PM plants were treated with exogenous ethylene, CsACS2 expression could be detected. In cucumber, ethylene treatment could also induce transcription of CsACS2, while inhibition of ethylene action reduced the expression level. These findings suggest a positive feedback regulation mechanism for CsACS2, and a modified 'one-hormone hypothesis' for sex determination in cucumber is proposed.

BPF-1, a pathogen-induced DNA-binding protein involved in the plant defense response.

PubMed

da Costa e Silva, O; Klein, L; Schmelzer, E; Trezzini, G F; Hahlbrock, K

1993-07-01

The mechanisms by which plants restrict the growth of pathogens include transient activation of numerous defense-related genes. Box P is a putative cis-acting element of a distinct group of such genes, including those encoding the enzyme phenylalanine ammonialyase (PAL). A DNA-binding activity to Box P was identified in nuclear extracts from cultured parsley cells and a cDNA encoding the protein BPF-1 (Box P-binding Factor) partially characterized. BPF-1 binds to this element with specificity similar to that of the binding activity in nuclear extracts. BPF-1 mRNA accumulates rapidly in elicitor-treated parsley cells and around fungal infection sites on parsley leaves. This accumulation is, at least partly, due to a rapid and transient increase in the transcription rate of BPF-1. Moreover, tight correlation between the relative amounts of BPF-1 and PAL mRNAs was observed in different organs of a parsley plant. These results are consistent with the hypothesis that BPF-1 is involved in disease resistance by modulating plant defense gene expression.

Actin dynamics involved in gravity perception in Arabidopsis inflorescense stem

NASA Astrophysics Data System (ADS)

Tasaka, Masao; Nakamura, Moritaka; Morita, Miyo T.

The amyloplasts sedimentation in the endodermal cells is important for gravity perception in Arabidopsis shoot. Our previous study suggests that SGR5(SHOOT GRAVITROPISM 5) and SGR9 are synergistically involved in regulation of amyloplast movement in these cells, and shows that sgr5 sgr9 double mutant completely loses gravitropic response. SGR5 encodes putative transcription factor and SGR9 encodes a ring finger containing protein, which surrounds amyloplasts. It has been reported that amyloplasts are surrounded by actin microfilaments (MFs), and that treatment with actin polymerization inhibitor enhances gravitropic organ curvature. However, not only the molecular link between amyolplasts and MFs, but also regulatory role of MFs in gravitropic response is still unclear. Here, we found that treatment with actin polymerization inhibitor restored gravitropic response of sgr5 sgr9 double mutant stems. The result suggests that abnormal amyloplasts movement in the double mutant could result from inhibition of MFs depolymerization, leading to abnormal gravitropism. We are investigating whether SGR5 and SGR9 are involved in amyloplasts movement by regulating actin remodeling in gravity perceptive cells.

A kinase-dependent feedforward loop affects CREBB stability and long term memory formation.

PubMed

Lee, Pei-Tseng; Lin, Guang; Lin, Wen-Wen; Diao, Fengqiu; White, Benjamin H; Bellen, Hugo J

2018-02-23

In Drosophila , long-term memory (LTM) requires the cAMP-dependent transcription factor CREBB, expressed in the mushroom bodies (MB) and phosphorylated by PKA. To identify other kinases required for memory formation, we integrated Trojan exons encoding T2A-GAL4 into genes encoding putative kinases and selected for genes expressed in MB. These lines were screened for learning/memory deficits using UAS-RNAi knockdown based on an olfactory aversive conditioning assay. We identified a novel, conserved kinase, Meng-Po ( MP , CG11221 , SBK1 in human), the loss of which severely affects 3 hr memory and 24 hr LTM, but not learning. Remarkably, memory is lost upon removal of the MP protein in adult MB but restored upon its reintroduction. Overexpression of MP in MB significantly increases LTM in wild-type flies showing that MP is a limiting factor for LTM. We show that PKA phosphorylates MP and that both proteins synergize in a feedforward loop to control CREBB levels and LTM. key words: Drosophila, Mushroom bodies, SBK1, deGradFP, T2A-GAL4, MiMIC.

Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse

PubMed Central

2011-01-01

Background Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. Aspergillus niger has been shown to produce a wide spectrum of polysaccharide hydrolytic enzymes. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse (SEB). Results Herein we report the main cellulase- and hemicellulase-encoding genes with increased expression during growth on SEB. We also sought to determine whether the mRNA accumulation of several SEB-induced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 (58% of A. niger predicted cellulases) and 21 (58% of A. niger predicted hemicellulases) cellulase- and hemicellulase-encoding genes, respectively, that were highly expressed during growth on SEB. Conclusions Degradation of sugarcane bagasse requires production of many different enzymes which are regulated by the type and complexity of the available substrate. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol. PMID:22008461

Characterization of the relationship between integrase, excisionase and antirepressor activities associated with a superinfecting Shiga toxin encoding bacteriophage

PubMed Central

Fogg, P. C. M.; Rigden, D. J.; Saunders, J. R.; McCarthy, A. J.; Allison, H. E.

2011-01-01

Shigatoxigenic Escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of Shiga toxin-encoding lambdoid bacteriophages. At least some of these Stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host's pathogenic profile. Here, increasing levels of Stx phage Φ24B integrase expression in multiple lysogen cultures are demonstrated along with apparently negligible repression of integrase expression by the cognate CI repressor. The Φ24B int transcription start site and promoter region were identified and found to differ from in silico predictions. The unidirectional activity of this integrase was determined in an in situ, inducible tri-partite reaction. This indicated that Φ24B must encode a novel directionality factor that is controlling excision events during prophage induction. This excisionase was subsequently identified and characterized through complementation experiments. In addition, the previous proposal that a putative antirepressor was responsible for the lack of immunity to superinfection through inactivation of CI has been revisited and a new hypothesis involving the role of this protein in promoting efficient induction of the Φ24B prophage is proposed. PMID:21062824

Molecular cloning of the transcription factor TFIIB homolog from Sulfolobus shibatae.

PubMed Central

Qureshi, S A; Khoo, B; Baumann, P; Jackson, S P

1995-01-01

The Archaea (archaebacteria) constitute a group of prokaryotes that are phylogenetically distinct from Eucarya (eukaryotes) and Bacteria (eubacteria). Although Archaea possess only one RNA polymerase, evidence suggests that their transcriptional apparatus is similar to that of Eucarya. For example, Archaea contain a homolog of the TATA-binding protein which interacts with the TATA-box like A-box sequence upstream of many archaeal genes. Here, we report the cloning of a Sulfolobus shibatae gene that encodes a protein (transcription factor TFB) with striking homology to the eukaryotic basal transcription factor TFIIB. We show by primer extension analysis that transcription of the S. shibatae TFB gene initiates 27 bp downstream from a consensus A-box element. Significantly, S. shibatae TFB contains an N-terminal putative metal-binding region and two imperfect direct repeats--structural features that are well conserved in eukaryotic TFIIBs. This suggests that TFB may perform analogous functions in Archaea and Eucarya. Consistent with this, we demonstrate that S. shibatae TFB promotes the binding of S. shibatae TBP to the A-box element of the Sulfolobus 16S/23S rRNA gene. Finally, we show that S. shibatae TFB is significantly more related to TFB of the archaeon Pyrococcus woesei than it is to eukaryotic TFIIBs. These data suggest that TFB arose in the common archaeal/eukaryotic ancestor and that the lineages leading to P. woesei and S. shibatae separated after the divergence of the archaeal and eukaryotic lines of descent. Images Fig. 2 Fig. 3 PMID:7597084

Expression of multidrug resistance efflux pump gene norA is iron responsive in Staphylococcus aureus.

PubMed

Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu; He, Chuan

2012-04-01

Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl(3) repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus.

Expression of Multidrug Resistance Efflux Pump Gene norA Is Iron Responsive in Staphylococcus aureus

PubMed Central

Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu

2012-01-01

Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl3 repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus. PMID:22267518

Divergent homologs of the predicted small RNA BpCand697 in Burkholderia spp.

NASA Astrophysics Data System (ADS)

Damiri, Nadzirah; Mohd-Padil, Hirzahida; Firdaus-Raih, Mohd

2015-09-01

The small RNA (sRNA) gene candidate, BpCand697 was previously reported to be unique to Burkholderia spp. and is encoded at 3' non-coding region of a putative AraC family transcription regulator gene. This study demonstrates the conservation of BpCand697 sequence across 32 Burkholderia spp. including B. pseudomallei, B. mallei, B. thailandensis and Burkholderia sp. by integrating both sequence homology and secondary structural analyses of BpCand697 within the dataset. The divergent sequence of BpCand697 was also used as a discriminatory power in clustering the dataset according to the potential virulence of Burkholderia spp., showing that B. thailandensis was clearly secluded from the virulent cluster of B. pseudomallei and B. mallei. Finally, the differential co-transcript expression of BpCand697 and its flanking gene, bpsl2391 was detected in Burkholderia pseudomallei D286 after grown under two different culture conditions using nutrient-rich and minimal media. It is hypothesized that the differential expression of BpCand697-bpsl2391 co-transcript between the two standard prepared media might correlate with nutrient availability in the culture media, suggesting that the physical co-localization of BpCand697 in B. pseudomallei D286 might be directly or indirectly involved with the transcript regulation of bpsl2391 under the selected in vitro culture conditions.

Characterization of a prototype strain of hepatitis E virus.

PubMed

Tsarev, S A; Emerson, S U; Reyes, G R; Tsareva, T S; Legters, L J; Malik, I A; Iqbal, M; Purcell, R H

1992-01-15

A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was characterized extensively. Six cynomolgus monkeys (Macaca fascicularis) were infected with a strain of hepatitis E virus from Pakistan. Reverse transcription-polymerase chain reaction was used to determine the pattern of virus shedding in feces, bile, and serum relative to hepatitis and induction of specific antibodies. Virtually the entire genome of SAR-55 (7195 nucleotides) was sequenced. Comparison of the sequence of SAR-55 with that of a Burmese strain revealed a high level of homology except for one region encoding 100 amino acids of a putative nonstructural polyprotein. Identification of this region as hypervariable was obtained by partial sequencing of a third isolate of hepatitis E virus from Kirgizia.

Unusual varieties and duplication of Rig-I like receptors encoded in the marine mollusk, Crassostrea gigas

NASA Astrophysics Data System (ADS)

Tian, Z. H.; Jiao, C. Z.

2017-07-01

RIG-I like receptors (RLRs) play key roles in sensing non-self nucleic acids in cytoplasm and trigger antiviral innate immune response in vertebrates and human body. Here we carried out in silico analysis to identify and investigate the putative RLRs encoded in the genome of marine mollusk, Crassostrea gigas (cgRLRs), an invertebrate species. We found the unusual duplication and varieties on domain architecture of putative cgRLRs encoded in the genome of C. gigas. Three putative cgRLRs (accessions numbers are EKC24603, EKC31344.1 and EKC38304.1 on GenBank), have the similar domain architecture with that of human RIG-I or MDA5, and one protein (EKC34573.1) with that of human LGP2; The fifth putative cgRLRs (EKC38303.1) is somewhat similar with human RIG-I/MDA5 except that it has only one caspase activation and recruitment domain (CARD) in its N-terminal. Other nine proteins were identified to be partialy similar with RLRs while with the incomplete sequences, which maybe reflect the events of partial duplication of cgRLRs genes occurred in the oyster genome.

Network analysis of transcriptomics expands regulatory landscapes in Synechococcus sp. PCC 7002

DOE Office of Scientific and Technical Information (OSTI.GOV)

McClure, Ryan S.; Overall, Christopher C.; McDermott, Jason E.

Cyanobacterial regulation of gene expression must contend with a genome organization that lacks apparent functional context, as the majority of cellular processes and metabolic pathways are encoded by genes found at disparate locations across the genome. In addition, the fact that coordinated regulation of cyanobacterial cellular machinery takes place with significantly fewer transcription factors, compared to other Eubacteria, suggests the involvement of post-transcriptional mechanisms and regulatory adaptations which are not fully understood. Global transcript abundance from model cyanobacterium Synechococcus sp. PCC 7002 grown under 42 different conditions was analyzed using context-likelihood of relatedness. The resulting 903-gene network, which was organizedmore » into 11 modules, not only allowed classification of cyanobacterial responses to specific environmental variables but provided insight into the transcriptional network topology and led to the expansion of predicted regulons. When used in conjunction with genome sequence, the global transcript abundance allowed identification of putative post-transcriptional changes in expression as well as novel potential targets of both DNA binding proteins and asRNA regulators. The results offer a new perspective into the multi-level regulation that governs cellular adaptations of fast-growing physiologically robust cyanobacterium Synechococcus sp. PCC 7002 to changing environmental variables. It also extends a methodological knowledge-based framework for studying multi-scale regulatory mechanisms that operate in cyanobacteria. Finally, it provides valuable context for integrating systems-level data to enhance evidence-driven genomic annotation, especially in organisms where traditional context analyses cannot be implemented due to lack of operon-based functional organization.« less

Transcriptome Sequencing and Developmental Regulation of Gene Expression in Anopheles aquasalis

PubMed Central

Silva, Maria C. P.; Lopes, Adriana R.; Barros, Michele S.; Sá-Nunes, Anderson; Kojin, Bianca B.; Carvalho, Eneas; Suesdek, Lincoln; Silva-Neto, Mário Alberto C.; James, Anthony A.; Capurro, Margareth L.

2014-01-01

Background Anopheles aquasalis is a major malaria vector in coastal areas of South and Central America where it breeds preferentially in brackish water. This species is very susceptible to Plasmodium vivax and it has been already incriminated as responsible vector in malaria outbreaks. There has been no high-throughput investigation into the sequencing of An. aquasalis genes, transcripts and proteins despite its epidemiological relevance. Here we describe the sequencing, assembly and annotation of the An. aquasalis transcriptome. Methodology/Principal Findings A total of 419 thousand cDNA sequence reads, encompassing 164 million nucleotides, were assembled in 7544 contigs of ≥2 sequences, and 1999 singletons. The majority of the An. aquasalis transcripts encode proteins with their closest counterparts in another neotropical malaria vector, An. darlingi. Several analyses in different protein databases were used to annotate and predict the putative functions of the deduced An. aquasalis proteins. Larval and adult-specific transcripts were represented by 121 and 424 contig sequences, respectively. Fifty-one transcripts were only detected in blood-fed females. The data also reveal a list of transcripts up- or down-regulated in adult females after a blood meal. Transcripts associated with immunity, signaling networks and blood feeding and digestion are discussed. Conclusions/Significance This study represents the first large-scale effort to sequence the transcriptome of An. aquasalis. It provides valuable information that will facilitate studies on the biology of this species and may lead to novel strategies to reduce malaria transmission on the South American continent. The An. aquasalis transcriptome is accessible at http://exon.niaid.nih.gov/transcriptome/An_aquasalis/Anaquexcel.xlsx. PMID:25033462

Early Cone Setting in Picea abies acrocona Is Associated with Increased Transcriptional Activity of a MADS Box Transcription Factor1[W][OA

PubMed Central

Uddenberg, Daniel; Reimegård, Johan; Clapham, David; Almqvist, Curt; von Arnold, Sara; Emanuelsson, Olof; Sundström, Jens F.

2013-01-01

Conifers normally go through a long juvenile period, for Norway spruce (Picea abies) around 20 to 25 years, before developing male and female cones. We have grown plants from inbred crosses of a naturally occurring spruce mutant (acrocona). One-fourth of the segregating acrocona plants initiate cones already in their second growth cycle, suggesting control by a single locus. The early cone-setting properties of the acrocona mutant were utilized to identify candidate genes involved in vegetative-to-reproductive phase change in Norway spruce. Poly(A+) RNA samples from apical and basal shoots of cone-setting and non-cone-setting plants were subjected to high-throughput sequencing (RNA-seq). We assembled and investigated 33,383 expressed putative protein-coding acrocona transcripts. Eight transcripts were differentially expressed between selected sample pairs. One of these (Acr42124_1) was significantly up-regulated in apical shoot samples from cone-setting acrocona plants, and the encoded protein belongs to the MADS box gene family of transcription factors. Using quantitative real-time polymerase chain reaction with independently derived plant material, we confirmed that the MADS box gene is up-regulated in both needles and buds of cone-inducing shoots when reproductive identity is determined. Our results constitute important steps for the development of a rapid cycling model system that can be used to study gene function in conifers. In addition, our data suggest the involvement of a MADS box transcription factor in the vegetative-to-reproductive phase change in Norway spruce. PMID:23221834

Systematic asymmetric nucleotide exchanges produce human mitochondrial RNAs cryptically encoding for overlapping protein coding genes.

PubMed

Seligmann, Hervé

2013-05-07

GenBank's EST database includes RNAs matching exactly human mitochondrial sequences assuming systematic asymmetric nucleotide exchange-transcription along exchange rules: A→G→C→U/T→A (12 ESTs), A→U/T→C→G→A (4 ESTs), C→G→U/T→C (3 ESTs), and A→C→G→U/T→A (1 EST), no RNAs correspond to other potential asymmetric exchange rules. Hypothetical polypeptides translated from nucleotide-exchanged human mitochondrial protein coding genes align with numerous GenBank proteins, predicted secondary structures resemble their putative GenBank homologue's. Two independent methods designed to detect overlapping genes (one based on nucleotide contents analyses in relation to replicative deamination gradients at third codon positions, and circular code analyses of codon contents based on frame redundancy), confirm nucleotide-exchange-encrypted overlapping genes. Methods converge on which genes are most probably active, and which not, and this for the various exchange rules. Mean EST lengths produced by different nucleotide exchanges are proportional to (a) extents that various bioinformatics analyses confirm the protein coding status of putative overlapping genes; (b) known kinetic chemistry parameters of the corresponding nucleotide substitutions by the human mitochondrial DNA polymerase gamma (nucleotide DNA misinsertion rates); (c) stop codon densities in predicted overlapping genes (stop codon readthrough and exchanging polymerization regulate gene expression by counterbalancing each other). Numerous rarely expressed proteins seem encoded within regular mitochondrial genes through asymmetric nucleotide exchange, avoiding lengthening genomes. Intersecting evidence between several independent approaches confirms the working hypothesis status of gene encryption by systematic nucleotide exchanges. Copyright © 2013 Elsevier Ltd. All rights reserved.

Complete nucleotide sequences and genome characterization of a novel double-stranded RNA virus infecting Rosa multiflora.

PubMed

Salem, Nidá M; Golino, Deborah A; Falk, Bryce W; Rowhani, Adib

2008-01-01

The three double-stranded (ds) RNAs were detected in Rosa multiflora plants showing rose spring dwarf (RSD) symptoms. Northern blot analysis revealed three dsRNAs in preparations of both dsRNA and total RNA from R. multiflora plants. The complete sequences of the dsRNAs (referred to as dsRNA 1, dsRNA 2 and dsRNA 3) were determined based on a combination of shotgun cloning of dsRNA cDNAs and reverse transcription-polymerase chain reaction (RT-PCR). The largest dsRNA (dsRNA 1) was 1,762 bp long with a single open reading frame (ORF) that encoded a putative polypeptide containing 479 amino acid residues with a molecular mass of 55.9 kDa. This polypeptide contains amino acid sequence motifs conserved in the RNA-dependent RNA polymerases (RdRp) of members of the family Partitiviridae. Both dsRNA 2 (1,475 bp) and dsRNA 3 (1,384 bp) contained single ORFs, encoding putative proteins of unknown function. The 5' untranslated regions (UTR) of all three segments shared regions of high sequence homology. Phylogenetic analysis using the RdRp sequences of the various partitiviruses revealed that the new sequences would constitute the genome of a virus in family Partitiviridae. This virus would cluster with Fragaria chiloensis cryptic virus and Raphanus sativus cryptic virus 2. We suggest that the three dsRNA segments constitute the genome of a novel cryptic virus infecting roses; we propose the name Rosa multiflora cryptic virus (RMCV). Detection primers were developed and used for RT-PCR detection of RMCV in rose plants.

Characterization of the response to zinc deficiency in the cyanobacterium Anabaena sp. strain PCC 7120.

PubMed

Napolitano, Mauro; Rubio, Miguel Ángel; Santamaría-Gómez, Javier; Olmedo-Verd, Elvira; Robinson, Nigel J; Luque, Ignacio

2012-05-01

Zur regulators control zinc homeostasis by repressing target genes under zinc-sufficient conditions in a wide variety of bacteria. This paper describes how part of a survey of duplicated genes led to the identification of the open reading frame all2473 as the gene encoding the Zur regulator of the cyanobacterium Anabaena sp. strain PCC 7120. All2473 binds to DNA in a zinc-dependent manner, and its DNA-binding sequence was characterized, which allowed us to determine the relative contribution of particular nucleotides to Zur binding. A zur mutant was found to be impaired in the regulation of zinc homeostasis, showing sensitivity to elevated concentrations of zinc but not other metals. In an effort to characterize the Zur regulon in Anabaena, 23 genes containing upstream putative Zur-binding sequences were identified and found to be regulated by Zur. These genes are organized in six single transcriptional units and six operons, some of them containing multiple Zur-regulated promoters. The identities of genes of the Zur regulon indicate that Anabaena adapts to conditions of zinc deficiency by replacing zinc metalloproteins with paralogues that fulfill the same function but presumably with a lower zinc demand, and with inducing putative metallochaperones and membrane transport systems likely being involved in the scavenging of extracellular zinc, including plasma membrane ABC transport systems and outer membrane TonB-dependent receptors. Among the Zur-regulated genes, the ones showing the highest induction level encode proteins of the outer membrane, suggesting a primary role for components of this cell compartment in the capture of zinc cations from the extracellular medium.

ROOT HAIR DEFECTIVE SIX-LIKE4 (RSL4) promotes root hair elongation by transcriptionally regulating the expression of genes required for cell growth.

PubMed

Vijayakumar, Priya; Datta, Sourav; Dolan, Liam

2016-12-01

ROOT HAIR DEFECTIVE SIX-LIKE4 (RSL4) is necessary and sufficient for root hair elongation in Arabidopsis thaliana. Root hair length is determined by the duration for which RSL4 protein is present in the developing root hair. The aim of this research was to identify genes regulated by RSL4 that affect root hair growth. To identify genes regulated by RSL4, we identified genes whose expression was elevated by induction of RSL4 activity in the presence of an inhibitor of translation. Thirty-four genes were identified as putative targets of RSL transcriptional regulation, and the results suggest that the activities of SUPPRESSOR OF ACTIN (SAC1), EXOCSYT SUBUNIT 70A1 (EXO70A1), PEROXIDASE7 (PRX7) and CALCIUM-DEPENDENT PROTEIN KINASE11 (CPK11) are required for root hair elongation. These data indicate that RSL4 controls cell growth by controlling the expression of genes encoding proteins involved in cell signalling, cell wall modification and secretion. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Transcription analysis of pilS and xpsEL genes from Xylella fastidiosa.

PubMed

Coltri, Patricia P; Rosato, Yoko B

2005-04-01

Xylella fastidiosa is a xylem-limited phytopathogen responsible for diseases in several plants such as citrus and coffee. Analysis of the bacterial genome revealed some putative pathogenicity-related genes that could help to elucidate the molecular mechanisms of plant-pathogen interactions. In the present work, the transcription of three genes of the bacterium, grown in defined and rich media and also in media containing host plant extracts (sweet orange, 'ponkan' and coffee) was analyzed by RT-PCR. The pilS gene, which encodes a sensor histidine kinase responsible for the biosynthesis of fimbriae, was transcribed when the bacterium was grown in more complex media such as PW and in medium containing plant extracts. The xps genes (xpsL and xpsE) which are related to the type II secretion system were also detected when the bacterium was grown in rich media and media with 'ponkan' and coffee extracts. It was thus observed that pilS and xpsEL genes of X. fastidiosa can be modulated by environmental factors and their expression is dependent on the nutritional status of the growth medium.

A core subunit of the RNA-processing/degrading exosome specifically influences cuticular wax biosynthesis in Arabidopsis.

PubMed

Hooker, Tanya S; Lam, Patricia; Zheng, Huanquan; Kunst, Ljerka

2007-03-01

The cuticle is an extracellular matrix composed of cutin polyester and waxes that covers aerial organs of land plants and protects them from environmental stresses. The Arabidopsis thaliana cer7 mutant exhibits reduced cuticular wax accumulation and contains considerably lower transcript levels of ECERIFERUM3/WAX2/YORE-YORE (CER3/WAX2/YRE), a key wax biosynthetic gene. We show here that CER7 protein is a putative 3'-5' exoribonuclease homologous to yeast Ribonuclease PH45 (RRP45p), a core subunit of the RNA processing and degrading exosome that controls the expression of CER3/WAX2/YRE. We propose that CER7 acts by degrading a specific mRNA species encoding a negative regulator of CER3/WAX2/YRE transcription. A second RRP45p homolog found in Arabidopsis, designated At RRP45a, is partially functionally redundant with CER7, and complete loss of RRP45 function in Arabidopsis is lethal. To our knowledge, CER7 is currently the only example of a core exosomal subunit specifically influencing a cellular process.

Molecular characterization of genes encoding inward rectifier potassium (Kir) channels in the bed bug (Cimex lectularius).

PubMed

Mamidala, Praveen; Mittapelly, Priyanka; Jones, Susan C; Piermarini, Peter M; Mittapalli, Omprakash

2013-04-01

The molecular genetics of inward-rectifier potassium (Kir) channels in insects is poorly understood. To date, Kir channel genes have been characterized only from a few representative dipterans (i.e., fruit flies and mosquitoes). The goal of the present study was to characterize Kir channel cDNAs in a hemipteran, the bed bug (Cimex lectularius). Using our previously reported bed bug transcriptome (RNA-seq), we identified two cDNAs that encode putative Kir channels. One was a full-length cDNA that encodes a protein belonging to the insect 'Kir3' clade, which we designate as 'ClKir3'. The other was a partial cDNA that encodes a protein with similarity to both the insect 'Kir1' and 'Kir2' clades, which we designate as 'ClKir1/2'. Quantitative real-time PCR analysis revealed that ClKir1/2 and ClKir3 exhibited peak expression levels in late-instar nymphs and early-instar nymphs, respectively. Furthermore, ClKir3, but not ClKir1/2, showed tissue-specific expression in Malpighian tubules of adult bed bugs. Lastly, using an improved procedure for delivering double-stranded RNA (dsRNA) to male and female bed bugs (via the cervical membrane) we demonstrate rapid and systemic knockdown of ClKir3 transcripts. In conclusion, we demonstrate that the bed bug possesses at least two genes encoding Kir channels, and that RNAi is possible for at least Kir3, thereby offering a potential approach for elucidating the roles of Kir channel genes in bed bug physiology. Copyright © 2013 Elsevier Inc. All rights reserved.

Ovule development: identification of stage-specific and tissue-specific cDNAs.

PubMed Central

Nadeau, J A; Zhang, X S; Li, J; O'Neill, S D

1996-01-01

A differential screening approach was used to identify seven ovule-specific cDNAs representing genes that are expressed in a stage-specific manner during ovule development. The Phalaenopsis orchid takes 80 days to complete the sequence of ovule developmental events, making it a good system to isolate stage-specific ovule genes. We constructed cDNA libraries from orchid ovule tissue during archesporial cell differentiation, megasporocyte formation, and the transition to meiosis, as well as during the final mitotic divisions of female gametophyte development. RNA gel blot hybridization analysis revealed that four clones were stage specific and expressed solely in ovule tissue, whereas one clone was specific to pollen tubes. Two other clones were not ovule specific. Sequence analysis and in situ hybridization revealed the identities and domain of expression of several of the cDNAs. O39 encodes a putative homeobox transcription factor that is expressed early in the differentiation of the ovule primordium; O40 encodes a cytochrome P450 monooxygenase (CYP78A2) that is pollen tube specific. O108 encodes a protein of unknown function that is expressed exclusively in the outer layer of the outer integument and in the female gametophyte of mature ovules. O126 encodes a glycine-rich protein that is expressed in mature ovules, and O141 encodes a cysteine proteinase that is expressed in the outer integument of ovules during seed formation. Sequences homologous to these ovule clones can now be isolated from other organisms, and this should facilitate their functional characterization. PMID:8742709

Multiple genes for functional 6 fatty acyl desaturases (Fad) in Atlantic salmon (Salmo salar L.): gene and cDNA characterization, functional expression, tissue distribution and nutritional regulation.

PubMed

Monroig, Oscar; Zheng, Xiaozhong; Morais, Sofia; Leaver, Michael J; Taggart, John B; Tocher, Douglas R

2010-09-01

Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct Delta5 and Delta6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, Delta6fad_a and Delta5fad, corresponded to the previously cloned Delta6 and Delta5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Delta6fad_b and Delta6fad_c, had only Delta6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for 6Fad_b and Delta6fad_c, respectively. Both 6fad_a and 6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with 6fad_b also highly expressed in gill, whereas 6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the 6fad_a gene in liver and the 6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for 6fad_a in intestine, 6fad_b in liver, or 6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are discussed in relation to gene structures and fish physiology. 2010 Elsevier B.V. All rights reserved.

Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare)WRKY transcription factor family reveals putatively retained functions betweenmonocots and dicots

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mangelsen, Elke; Kilian, Joachim; Berendzen, Kenneth W.

2008-02-01

WRKY proteins belong to the WRKY-GCM1 superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. For the cereal crop barley (Hordeum vulgare), three different WRKY proteins have been characterized so far, as regulators in sucrose signaling, in pathogen defense, and in response to cold and drought, respectively. However, their phylogenetic relationship remained unresolved. In this study, we used the available sequence information to identify a minimum number of 45 barley WRKY transcription factor (HvWRKY) genes. According to their structural features the HvWRKY factors were classified into the previously defined polyphyletic WRKY subgroups 1 tomore » 3. Furthermore, we could assign putative orthologs of the HvWRKY proteins in Arabidopsis and rice. While in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and Arabidopsis only, which is indicative of specific gene radiation events. To gain insight into their putative functions, we examined expression profiles of WRKY genes from publicly available microarray data resources and found group specific expression patterns. While putative orthologs of the HvWRKY transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of WRKY genes in Arabidopsis and barley. Indeed, highly correlative expression profiles were found between some of the putative orthologs. HvWRKY genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. HvWRKY proteins exhibited not only sequence similarities between orthologs with Arabidopsis, but also relatedness in their expression patterns. This correlative expression is indicative for a putative conserved function of related WRKY proteins in mono- and dicot species.« less

Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

PubMed Central

Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

2013-01-01

The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

Medically important carotenoids from Momordica charantia and their gene expressions in different organs.

PubMed

Cuong, Do Manh; Arasu, Mariadhas Valan; Jeon, Jin; Park, Yun Ji; Kwon, Soon-Jae; Al-Dhabi, Naif Abdullah; Park, Sang Un

2017-12-01

Carotenoids, found in the fruit and different organs of bitter melon ( Momordica charantia ), have attracted great attention for their potential health benefits in treating several major chronic diseases. Therefore, study related to the identification and quantification of the medically important carotenoid metabolites is highly important for the treatment of various disorderes. The present study involved in the identification and quantification of the various carotenoids present in the different organs of M. charantia and the identification of the genes responsible for the accumulation of the carotenoids with respect to the transcriptome levels were investigated. In this study, using the transcriptome database of bitter melon, a partial-length cDNA clone encoding geranylgeranyl pyrophosphate synthase ( McGGPPS2 ), and several full-length cDNA clones encoding geranylgeranyl pyrophosphate synthase ( McGGPPS1 ), zeta-carotene desaturase ( McZDS ), lycopene beta-cyclase ( McLCYB ), lycopene epsilon cyclases ( McLCYE1 and McLCYE2 ), beta-carotene hydroxylase ( McCHXB ), and zeaxanthin epoxidase ( McZEP ) were identified in bitter melon . The expression levels of the mRNAs encoding these eight putative biosynthetic enzymes, as well as the accumulation of lycopene, α-carotene, lutein, 13Z-β-carotene, E-β-carotene, 9Z-β-carotene, β-cryptoxanthin, zeaxanthin, antheraxanthin, and violaxanthin were investigated in different organs from M. charantia as well as in the four different stages of its fruit maturation. Transcripts were found to be constitutively expressed at high levels in the leaves where carotenoids were also found at the highest levels . Collectively, these results indicate that the putative McGGPPS2, McZDS, McLCYB, McLCYE1, McLCYE2, and McCHXB enzymes might be key factors in controlling carotenoid content in bitter melon . In conclusion, the over expression of the carotenoid biosynthetic genes from M. charantia crops to increase the yield of these medically important carotenoids.

6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle.

PubMed

Le, Simone Balzer; Heggeset, Tonje Marita Bjerkan; Haugen, Tone; Nærdal, Ingemar; Brautaset, Trygve

2017-05-01

D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The B. methanolicus wild-type strain MGA3 possesses two putative rpe and pfk genes encoded on plasmid pBM19 (rpe1-MGA3 and pfk1-MGA3) and on the chromosome (rpe2-MGA3 and pfk2-MGA3). The wild-type strain PB1 also encodes putative rpe and pfk genes on plasmid pBM20 (rpe1-PB1 and pfk1-PB1*); however, it only harbours a chromosomal pfk gene (pfk2-PB1). Transcription of the plasmid-encoded genes was 10-fold to 15-fold upregulated in cells growing on methanol compared to mannitol, while the chromosomal genes were transcribed at similar levels under both conditions in both strains. All seven gene products were recombinantly produced in Escherichia coli, purified and biochemically characterized. All three RPEs were active as hexamers, catalytically stimulated by Mg 2+ and Mn 2+ and displayed similar K' values (56-75 μM) for ribulose 5-phosphate. Rpe2-MGA3 showed displayed 2-fold lower V max (49 U/mg) and a significantly reduced thermostability compared to the two Rpe1 proteins. Pfk1-PB1* was shown to be non-functional. The PFKs were active both as octamers and as tetramers, were catalytically stimulated by Mg 2+ and Mn 2+ , and displayed similar thermostabilities. The PFKs have similar K m values for fructose 6-phosphate (0.61-0.94 μM) and for ATP (0.38-0.82 μM), while Pfk1-MGA3 had a 2-fold lower V max (6.3 U/mg) compared to the two Pfk2 proteins. Our results demonstrate that MGA3 and PB1 exert alternative solutions to plasmid-dependent methylotrophy, including genetic organization, regulation, and biochemistry of RuMP cycle enzymes.

Microbial responses to xenobiotic compounds. Identification of genes that allow Pseudomonas putida KT2440 to cope with 2,4,6‐trinitrotoluene

PubMed Central

Fernández, Matilde; Duque, Estrella; Pizarro‐Tobías, Paloma; Van Dillewijn, Pieter; Wittich, Rolf‐Michael; Ramos, Juan L.

2009-01-01

Summary Pseudomonas putida KT2440 grows in M9 minimal medium with glucose in the presence of 2,4,6‐trinitrotoluene (TNT) at a similar rate than in the absence of TNT, although global transcriptional analysis using DNA microarrays revealed that TNT exerts some stress. Response to TNT stress is regulated at the transcriptional level, as significant changes in the level of expression of 65 genes were observed. Of these genes, 39 appeared upregulated, and 26 were downregulated. The identity of upregulated genes suggests that P. putida uses two kinds of strategies to overcome TNT toxicity: (i) induction of genes encoding nitroreductases and detoxification‐related enzymes (pnrA, xenD, acpD) and (ii) induction of multidrug efflux pump genes (mexEF/oprN) to reduce intracellular TNT concentrations. Mutants of 13 up‐ and 7 downregulated genes were analysed with regards to TNT toxicity revealing the role of the MexE/MexF/OprN pump and a putative isoquinoline 1‐oxidoreductase in tolerance to TNT. The ORF PP1232 whose transcriptional level did not change in response to TNT affected growth in the presence of nitroaromatic compounds and it was found in a screening of 4000 randomly generated mutants. PMID:21261922

Over-expression of TaMYB33 encoding a novel wheat MYB transcription factor increases salt and drought tolerance in Arabidopsis.

PubMed

Qin, Yuxiang; Wang, Mengcheng; Tian, Yanchen; He, Wenxing; Han, Lu; Xia, Guangmin

2012-06-01

Salt and drought stresses often adversely affect plant growth and productivity, MYB transcription factors have been shown to participate in the response to these stresses. Here we identified a new R2R3-type MYB transcription factor gene TaMYB33 from wheat (Triticum aestivum). TaMYB33 was induced by NaCl, PEG and ABA treatments, and its promoter sequence contains putative ABRE, MYB and other abiotic stress related cis-elements. Ectopic over-expression of TaMYB33 in Arabidopsis thaliana remarkably enhanced its tolerance to drought and NaCl stresses, but not to LiCl and KCl treatments. The expressions of AtP5CS and AtZAT12 which mirror the activities of proline and ascorbate peroxidase synthesis respectively were induced in TaMYB33 over-expression lines, indicating TaMYB33 promotes the ability for osmotic pressure balance-reconstruction and reactive oxidative species (ROS) scavenging. The up-regulation of AtAAO3 along with down-regulation of AtABF3, AtABI1 in TaMYB33 over-expression lines indicated that ABA synthesis was elevated while its signaling was restricted. These results suggest that TaMYB33 enhances salt and drought tolerance partially through superior ability for osmotic balance reconstruction and ROS detoxification.

γ-Lindane Increases Microcystin Synthesis in Microcystis aeruginosa PCC7806

PubMed Central

Ceballos-Laita, Laura; Calvo-Begueria, Laura; Lahoz, Jessica; Bes, María-Teresa; Fillat, María F.; Peleato, María-Luisa

2015-01-01

HCH factories, and the waste dumpsites associated to its production, have become a global environmental concern, and their runoff could pollute ground and surface waters with high levels of the pollutant. In this study, the influence of lindane (γ-HCH) on microcystin production has been investigated in Microcystis aeruginosa PCC7806. This toxic cyanobacterium is highly tolerant to γ-lindane (20 mg/L), and produces more toxin (microcystin) in the presence of the pollutant. Microcystis degrades γ-lindane and presence of γ-lindane induces genes involved in its own degradation (nirA). RT-PCRsq has been used to monitor changes in levels of transcripts encoded by the mcy operon (mcyD, mcyH and mcyJ), responsible for the microcystin synthesis machinery, as well as other genes involved in its transcriptional regulation, such as ntcA and fur family members. The presence of lindane in the culture media induces mcyD expression, as well as ntcA gene transcription, while other genes, such as mcyH, (putative ABC transporter), are downregulated. The amount of microcystin found in the cells and the culture media is higher when M. aeruginosa is treated with γ-lindane than in control cells. The results suggest that in a lindane polluted environment, Microcystis toxic strains may enhance their microcystin synthesis. PMID:26404326

Transcriptional activation of a 37 kDa ethylene responsive cysteine protease gene, RbCP1, is associated with protein degradation during petal abscission in rose

PubMed Central

Tripathi, Siddharth Kaushal; Singh, Amar Pal; Sane, Aniruddha P.; Nath, Pravendra

2009-01-01

Cysteine proteases play an important role in several developmental processes in plants, particularly those related to senescence and cell death. A cysteine protease gene, RbCP1, has been identified that encodes a putative protein of 357 amino acids and is expressed in the abscission zone (AZ) of petals in rose. The gene was responsive to ethylene in petals, petal abscission zones, leaves, and thalamus. The expression of RbCP1 increased during both ethylene-induced as well as natural abscission and was inhibited by 1-MCP. Transcript accumulation of RbCP1 was accompanied by the appearance of a 37 kDa cysteine protease, a concomitant increase in protease activity and a substantial decrease in total protein content in the AZ of petals. Agro-injection of rose petals with a 2.0 kb region upstream of the RbCP1 gene could drive GUS expression in an abscission zone-specific manner and was blocked by 1-MCP. It is concluded that petal abscission is associated with a decrease in total protein content resulting from rapid transcription of RbCP1 and the expression of a 37 kDa protease. PMID:19346241

Large-scale analysis of antisense transcription in wheat using the Affymetrix GeneChip Wheat Genome Array

USDA-ARS?s Scientific Manuscript database

Natural antisense transcripts (NATs) are transcripts of the opposite DNA strand to the sense-strand either at the same locus (cis-encoded) or a different locus (trans-encoded). They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation....

Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.).

PubMed

Yamaguchi, S; Saito, T; Abe, H; Yamane, H; Murofushi, N; Kamiya, Y

1996-08-01

The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region.

Annotation of differentially expressed genes in the somatic embryogenesis of musa and their location in the banana genome.

PubMed

Maldonado-Borges, Josefina Ines; Ku-Cauich, José Roberto; Escobedo-Graciamedrano, Rosa Maria

2013-01-01

Analysis of cDNA-AFLP was used to study the genes expressed in zygotic and somatic embryogenesis of Musa acuminata Colla ssp. malaccensis, and a comparison was made between their differential transcribed fragments (TDFs) and the sequenced genome of the double haploid- (DH-) Pahang of the malaccensis subspecies that is available in the network. A total of 253 transcript-derived fragments (TDFs) were detected with apparent size of 100-4000 bp using 5 pairs of AFLP primers, of which 21 were differentially expressed during the different stages of banana embryogenesis; 15 of the sequences have matched DH-Pahang chromosomes, with 7 of them being homologous to gene sequences encoding either known or putative protein domains of higher plants. Four TDF sequences were located in all Musa chromosomes, while the rest were located in one or two chromosomes. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed. Thus the availability of the genome of Musa and the information of TDFs sequences presented here opens new possibilities for an in-depth study of the molecular and biochemical research of zygotic and somatic embryogenesis of Musa.

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress.

PubMed

Niederwanger, Michael; Dvorak, Martin; Schnegg, Raimund; Pedrini-Martha, Veronika; Bacher, Katharina; Bidoli, Massimo; Dallinger, Reinhard

2017-08-11

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata , one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails.

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress

PubMed Central

Dvorak, Martin; Schnegg, Raimund; Pedrini-Martha, Veronika; Bacher, Katharina; Bidoli, Massimo; Dallinger, Reinhard

2017-01-01

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata, one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails. PMID:28800079

Unsupervised Network Analysis of the Plastic Supraoptic Nucleus Transcriptome Predicts Caprin2 Regulatory Interactions.

PubMed

Loh, Su-Yi; Jahans-Price, Thomas; Greenwood, Michael P; Greenwood, Mingkwan; Hoe, See-Ziau; Konopacka, Agnieszka; Campbell, Colin; Murphy, David; Hindmarch, Charles C T

2017-01-01

The supraoptic nucleus (SON) is a group of neurons in the hypothalamus responsible for the synthesis and secretion of the peptide hormones vasopressin and oxytocin. Following physiological cues, such as dehydration, salt-loading and lactation, the SON undergoes a function related plasticity that we have previously described in the rat at the transcriptome level. Using the unsupervised graphical lasso (Glasso) algorithm, we reconstructed a putative network from 500 plastic SON genes in which genes are the nodes and the edges are the inferred interactions. The most active nodal gene identified within the network was Caprin2 . Caprin2 encodes an RNA-binding protein that we have previously shown to be vital for the functioning of osmoregulatory neuroendocrine neurons in the SON of the rat hypothalamus. To test the validity of the Glasso network, we either overexpressed or knocked down Caprin2 transcripts in differentiated rat pheochromocytoma PC12 cells and showed that these manipulations had significant opposite effects on the levels of putative target mRNAs. These studies suggest that the predicative power of the Glasso algorithm within an in vivo system is accurate, and identifies biological targets that may be important to the functional plasticity of the SON.

Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties.

PubMed

Rama Reddy, Nagaraja Reddy; Mehta, Rucha Harishbhai; Soni, Palak Harendrabhai; Makasana, Jayanti; Gajbhiye, Narendra Athamaram; Ponnuchamy, Manivel; Kumar, Jitendra

2015-01-01

Senna (Cassia angustifolia Vahl.) is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090)', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.

Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties

PubMed Central

Rama Reddy, Nagaraja Reddy; Mehta, Rucha Harishbhai; Soni, Palak Harendrabhai; Makasana, Jayanti; Gajbhiye, Narendra Athamaram; Ponnuchamy, Manivel; Kumar, Jitendra

2015-01-01

Senna (Cassia angustifolia Vahl.) is a world’s natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with ‘green plant database (txid 33090)’, Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna. PMID:26098898

Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization

PubMed Central

Gouzil, Julie; Fablet, Aurore; Lara, Estelle; Caignard, Grégory; Cochet, Marielle; Kundlacz, Cindy; Palmarini, Massimo; Varela, Mariana; Breard, Emmanuel; Sailleau, Corinne; Viarouge, Cyril; Coulpier, Muriel; Zientara, Stéphan

2016-01-01

ABSTRACT Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. IMPORTANCE Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family. PMID:27795408

Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization.

PubMed

Gouzil, Julie; Fablet, Aurore; Lara, Estelle; Caignard, Grégory; Cochet, Marielle; Kundlacz, Cindy; Palmarini, Massimo; Varela, Mariana; Breard, Emmanuel; Sailleau, Corinne; Viarouge, Cyril; Coulpier, Muriel; Zientara, Stéphan; Vitour, Damien

2017-01-01

Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family. Copyright © 2016 Gouzil et al.

Comparative Genomics Reveals the Regulatory Complexity of Bifidobacterial Arabinose and Arabino-Oligosaccharide Utilization.

PubMed

Arzamasov, Aleksandr A; van Sinderen, Douwe; Rodionov, Dmitry A

2018-01-01

Members of the genus Bifidobacterium are common inhabitants of the human gastrointestinal tract. Previously it was shown that arabino-oligosaccharides (AOS) might act as prebiotics and stimulate the bifidobacterial growth in the gut. However, despite the rapid accumulation of genomic data, the precise mechanisms by which these sugars are utilized and associated transcription control still remain unclear. In the current study, we used a comparative genomic approach to reconstruct arabinose and AOS utilization pathways in over 40 bacterial species belonging to the Bifidobacteriaceae family. The results indicate that the gene repertoire involved in the catabolism of these sugars is highly diverse, and even phylogenetically close species may differ in their utilization capabilities. Using bioinformatics analysis we identified potential DNA-binding motifs and reconstructed putative regulons for the arabinose and AOS utilization genes in the Bifidobacteriaceae genomes. Six LacI-family transcriptional factors (named AbfR, AauR, AauU1, AauU2, BauR1 and BauR2) and a TetR-family regulator (XsaR) presumably act as local repressors for AOS utilization genes encoding various α- or β-L-arabinofuranosidases and predicted AOS transporters. The ROK-family regulator AraU and the LacI-family regulator AraQ control adjacent operons encoding putative arabinose transporters and catabolic enzymes, respectively. However, the AraQ regulator is universally present in all Bifidobacterium species including those lacking the arabinose catabolic genes araBDA , suggesting its control of other genes. Comparative genomic analyses of prospective AraQ-binding sites allowed the reconstruction of AraQ regulons and a proposed binary repression/activation mechanism. The conserved core of reconstructed AraQ regulons in bifidobacteria includes araBDA , as well as genes from the central glycolytic and fermentation pathways ( pyk, eno, gap, tkt, tal, galM, ldh ). The current study expands the range of genes involved in bifidobacterial arabinose/AOS utilization and demonstrates considerable variations in associated metabolic pathways and regulons. Detailed comparative and phylogenetic analyses allowed us to hypothesize how the identified reconstructed regulons evolved in bifidobacteria. Our findings may help to improve carbohydrate catabolic phenotype prediction and metabolic modeling, while it may also facilitate rational development of novel prebiotics.

[Cloning and expression analysis of two pro-inflammatory cytokines, IL-1β and its receptor, IL-1R2, in the Asian swamp eel Monopterus albus].

PubMed

Xu, Q Q; Xu, P; Zhou, J W; Pan, T S; Tuo, R; Ai, K; Yang, D Q

2016-01-01

Interleukin-1β (IL-1β) is the prototypic pro-inflammatory cytokine, whose functions are mediated through interaction with its receptors (IL-1R1 and IL-1R2). Herein, we cloned the full-length cDNA and genomic DNA of IL-1β and IL-1R2 in the Asian swamp eel (Monopterus albus). The eel IL-1β cDNA encodes a putative polypeptide of 246 amino acids. The protein sequence includes a typical IL-1 family signature, but lacked an interleukin-converting enzyme cleavage site. The genomic DNA of eel IL-1β was 2520 bp and comprised five exons and four introns. The eel IL-1R2 cDNA encoded a putative propeptide of 423 amino acid residues, comprising a signal peptide, a transmembrane region and two Ig-like domains in the extracellular region. Similar to other vertebrates, the genomic DNA of the eel IL-1R2 has nine exons and eight introns. Real-time PCR analysis indicated that IL-1β and IL-1R2 were constitutively expressed in all tissues, especially in the liver and immune-related organs. After infection with Aeromonas hydrophila, the transcript levels of IL-1β and IL-1R2 were induced in the head kidney and spleen, reaching their highest levels at 6 h post injection. In vitro, IL-1β and IL-1R2 mRNA levels were also upregulated rapidly at 1h post infection with A. hydrophila. Furthermore, acanthocephalan Pallisentis (Neosentis) celatus could induce the expression of both genes in the head kidney and intestine. In infected intestines, the transcript levels of IL-1β and IL-1R2 were increased by 21.4-fold and 20.8-fold, respectively, relative to the control. The present study indicated that IL-1β and IL-1R2 play an important role in inflammation and host defense, especially in the antiacanthocephalan response.

Comparative Genomics Reveals the Regulatory Complexity of Bifidobacterial Arabinose and Arabino-Oligosaccharide Utilization

PubMed Central

Arzamasov, Aleksandr A.; van Sinderen, Douwe; Rodionov, Dmitry A.

2018-01-01

Members of the genus Bifidobacterium are common inhabitants of the human gastrointestinal tract. Previously it was shown that arabino-oligosaccharides (AOS) might act as prebiotics and stimulate the bifidobacterial growth in the gut. However, despite the rapid accumulation of genomic data, the precise mechanisms by which these sugars are utilized and associated transcription control still remain unclear. In the current study, we used a comparative genomic approach to reconstruct arabinose and AOS utilization pathways in over 40 bacterial species belonging to the Bifidobacteriaceae family. The results indicate that the gene repertoire involved in the catabolism of these sugars is highly diverse, and even phylogenetically close species may differ in their utilization capabilities. Using bioinformatics analysis we identified potential DNA-binding motifs and reconstructed putative regulons for the arabinose and AOS utilization genes in the Bifidobacteriaceae genomes. Six LacI-family transcriptional factors (named AbfR, AauR, AauU1, AauU2, BauR1 and BauR2) and a TetR-family regulator (XsaR) presumably act as local repressors for AOS utilization genes encoding various α- or β-L-arabinofuranosidases and predicted AOS transporters. The ROK-family regulator AraU and the LacI-family regulator AraQ control adjacent operons encoding putative arabinose transporters and catabolic enzymes, respectively. However, the AraQ regulator is universally present in all Bifidobacterium species including those lacking the arabinose catabolic genes araBDA, suggesting its control of other genes. Comparative genomic analyses of prospective AraQ-binding sites allowed the reconstruction of AraQ regulons and a proposed binary repression/activation mechanism. The conserved core of reconstructed AraQ regulons in bifidobacteria includes araBDA, as well as genes from the central glycolytic and fermentation pathways (pyk, eno, gap, tkt, tal, galM, ldh). The current study expands the range of genes involved in bifidobacterial arabinose/AOS utilization and demonstrates considerable variations in associated metabolic pathways and regulons. Detailed comparative and phylogenetic analyses allowed us to hypothesize how the identified reconstructed regulons evolved in bifidobacteria. Our findings may help to improve carbohydrate catabolic phenotype prediction and metabolic modeling, while it may also facilitate rational development of novel prebiotics. PMID:29740413

Association of an SNP in a novel DREB2-like gene SiDREB2 with stress tolerance in foxtail millet [Setaria italica (L.)].

PubMed

Lata, Charu; Bhutty, Sarita; Bahadur, Ranjit Prasad; Majee, Manoj; Prasad, Manoj

2011-06-01

The DREB genes code for important plant transcription factors involved in the abiotic stress response and signal transduction. Characterization of DREB genes and development of functional markers for effective alleles is important for marker-assisted selection in foxtail millet. Here the characterization of a cDNA (SiDREB2) encoding a putative dehydration-responsive element-binding protein 2 from foxtail millet and the development of an allele-specific marker (ASM) for dehydration tolerance is reported. A cDNA clone (GenBank accession no. GT090998) coding for a putative DREB2 protein was isolated as a differentially expressed gene from a 6 h dehydration stress SSH library. A 5' RACE (rapid amplification of cDNA ends) was carried out to obtain the full-length cDNA, and sequence analysis showed that SiDREB2 encoded a polypeptide of 234 amino acids with a predicted mol. wt of 25.72 kDa and a theoretical pI of 5.14. A theoretical model of the tertiary structure shows that it has a highly conserved GCC-box-binding N-terminal domain, and an acidic C-terminus that acts as an activation domain for transcription. Based on its similarity to AP2 domains, SiDREB2 was classified into the A-2 subgroup of the DREB subfamily. Quantitative real-time PCR analysis showed significant up-regulation of SiDREB2 by dehydration (polyethylene glycol) and salinity (NaCl), while its expression was less affected by other stresses. A synonymous single nucleotide polymorphism (SNP) associated with dehydration tolerance was detected at the 558th base pair (an A/G transition) in the SiDREB2 gene in a core set of 45 foxtail millet accessions used. Based on the identified SNP, three primers were designed to develop an ASM for dehydration tolerance. The ASM produced a 261 bp fragment in all the tolerant accessions and produced no amplification in the sensitive accessions. The use of this ASM might be faster, cheaper, and more reproducible than other SNP genotyping methods, and thus will enable marker-aided breeding of foxtail millet for dehydration tolerance.

Genomic sequences of murine gamma B- and gamma C-crystallin-encoding genes: promoter analysis and complete evolutionary pattern of mouse, rat and human gamma-crystallins.

PubMed

Graw, J; Liebstein, A; Pietrowski, D; Schmitt-John, T; Werner, T

1993-12-22

The murine genes, gamma B-cry and gamma C-cry, encoding the gamma B- and gamma C-crystallins, were isolated from a genomic DNA library. The complete nucleotide (nt) sequences of both genes were determined from 661 and 711 bp, respectively, upstream from the first exon to the corresponding polyadenylation sites, comprising more than 2650 and 2890 bp, respectively. The new sequences were compared to the partial cDNA sequences available for the murine gamma B-cry and gamma C-cry, as well as to the corresponding genomic sequences from rat and man, at both the nt and predicted amino acid (aa) sequence levels. In the gamma B-cry promoter region, a canonical CCAAT-box, a TATA-box, putative NF-I and C/EBP sites were detected. An R-repeat is inserted 366 bp upstream from the transcription start point. In contrast, the gamma C-cry promoter does not contain a CCAAT-box, but some other putative binding sites for transcription factors (AP-2, UBP-1, LBP-1) were located by computer analysis. The promoter regions of all six gamma-cry from mouse, rat and human, except human psi gamma F-cry, were analyzed for common sequence elements. A complex sequence element of about 70-80 bp was found in the proximal promoter, which contains a gamma-cry-specific and almost invariant sequence (crygpel) of 14 nt, and ends with the also invariant TATA-box. Within the complex sequence element, a minimum of three further features specific for the gamma A-, gamma B- and gamma D/E/F-cry genes can be defined, at least two of which were recently shown to be functional. In addition to these four sequence elements, a subtype-specific structure of inverted repeats with different-sized spacers can be deduced from the multiple sequence alignment. A phylogenetic analysis based on the promoter region, as well as the complete exon 3 of all gamma-cry from mouse, rat and man, suggests separation of only five gamma-cry subtypes (gamma A-, gamma B-, gamma C-, gamma D- and gamma E/F-cry) prior to species separation.

Phosphatidate Phosphatase Plays Role in Zinc-mediated Regulation of Phospholipid Synthesis in Yeast*

PubMed Central

Soto-Cardalda, Aníbal; Fakas, Stylianos; Pascual, Florencia; Choi, Hyeon-Son; Carman, George M.

2012-01-01

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated by mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G. S. (2007) Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion. Biochim. Biophys. Acta 1771, 322–330; Eide, D. J. (2009) Homeostatic and adaptive responses to zinc deficiency in Saccharomyces cerevisiae. J. Biol. Chem. 284, 18565–18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative upstream activating sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme) and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme) but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway. PMID:22128164

The Nostoc punctiforme Genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

John C. Meeks

2001-12-31

Nostoc punctiforme is a filamentous cyanobacterium with extensive phenotypic characteristics and a relatively large genome, approaching 10 Mb. The phenotypic characteristics include a photoautotrophic, diazotrophic mode of growth, but N. punctiforme is also facultatively heterotrophic; its vegetative cells have multiple development alternatives, including terminal differentiation into nitrogen-fixing heterocysts and transient differentiation into spore-like akinetes or motile filaments called hormogonia; and N. punctiforme has broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms and an angiosperm. The shotgun-sequencing phase of the N. punctiforme strain ATCC 29133 genome has been completed by the Joint Genome Institute. Annotation of an 8.9more » Mb database yielded 7432 open reading frames, 45% of which encode proteins with known or probable known function and 29% of which are unique to N. punctiforme. Comparative analysis of the sequence indicates a genome that is highly plastic and in a state of flux, with numerous insertion sequences and multilocus repeats, as well as genes encoding transposases and DNA modification enzymes. The sequence also reveals the presence of genes encoding putative proteins that collectively define almost all characteristics of cyanobacteria as a group. N. punctiforme has an extensive potential to sense and respond to environmental signals as reflected by the presence of more than 400 genes encoding sensor protein kinases, response regulators and other transcriptional factors. The signal transduction systems and any of the large number of unique genes may play essential roles in the cell differentiation and symbiotic interaction properties of N. punctiforme.« less

Identification and characterization of a putative lipopolysaccharide-induced TNF-α factor (LITAF) gene from Amphioxus (Branchiostoma belcheri): an insight into the innate immunity of Amphioxus and the evolution of LITAF.

PubMed

Jin, Ping; Hu, Jing; Qian, Jinjun; Chen, Liming; Xu, Xiaofeng; Ma, Fei

2012-06-01

Innate immunity defenses against infectious agent in all multicultural organisms. TNF-α is an important cytokine that can be stimulated by Lipopolysaccharide (LPS) to regulate the innate immunity. The lipopolysaccharide-induced TNF-α factor (LITAF) functions as a transcription factor for regulating the expression of TNF-α as well as various inflammatory cytokines in response to LPS stimulation. The physiological significance of LITAF gene in the innate immunity of various animals has recently been reported. However, no LITAF gene has yet been identified in amphioxus, which is the best available stand-in for the proximate invertebrate ancestor of the vertebrates. In this study, we identified and characterized an amphioxus LITAF gene (designated as AmphiLITAF). First, we identified the AmphiLITAF from the amphioxus and found that AmphiLITAF gene with ~1.6 kb in length has a 827bp cDNA transcription product which encodes a putative protein with 127 amino acids containing conserved LITAF-domain, and the deduced amino acid of AmphiLITAF shared 37-60% similarity with the LITAFs from other species; second, we uncovered the spatial distribution of the LITAF in different tissues, the expression level of AmphiLITAF mRNA was the highest in hepatic cecum and intestine, moderate in muscles, gills and gonad, and the lowest in notochord. Our findings provide an insight into the innate immune response in the amphioxus and the evolution of the LITAF family. Copyright © 2012 Elsevier Ltd. All rights reserved.

Expression of Olfactory Signaling Genes in the Eye

PubMed Central

Velmeshev, Dmitry; Faghihi, Mohammad; Shestopalov, Valery I.; Slepak, Vladlen Z.

2014-01-01

Purpose To advance our understanding how the outer eye interacts with its environment, we asked which cellular receptors are expressed in the cornea, focusing on G protein-coupled receptors. Methods Total RNA from the mouse cornea was subjected to next-generation sequencing using the Illumina platform. The data was analyzed with TopHat and CuffLinks software packages. Expression of a representative group of genes detected by RNA-seq was further analyzed by RT-PCR and in situ hybridization using RNAscope technology and fluorescent microscopy. Results We generated more than 46 million pair-end reads from mouse corneal RNA. Bioinformatics analysis revealed that the mouse corneal transcriptome reconstructed from these reads represents over 10,000 gene transcripts. We identified 194 GPCR transcripts, of which 96 were putative olfactory receptors. RT-PCR analysis confirmed the presence of several olfactory receptors and related genes, including olfactory marker protein and the G protein associated with olfaction, Gαolf. In situ hybridization showed that mRNA for olfactory marker protein, Gαolf and possibly some olfactory receptors were found in the corneal epithelial cells. In addition to the corneal epithelium, Gαolf was present in the ganglionic and inner nuclear layers of the retina. One of the olfactory receptors, Olfr558, was present primarily in vessels of the eye co-stained with antibodies against alpha-smooth muscle actin, indicating expression in arterioles. Conclusions Several species of mRNA encoding putative olfactory receptors and related genes are expressed in the mouse cornea and other parts of the eye indicating they may play a role in sensing chemicals in the ocular environment. PMID:24789354

ACR4, a putative receptor kinase gene of Arabidopsis thaliana, that is expressed in the outer cell layers of embryos and plants, is involved in proper embryogenesis.

PubMed

Tanaka, Hirokazu; Watanabe, Masaru; Watanabe, Daisuke; Tanaka, Toshihiro; Machida, Chiyoko; Machida, Yasunori

2002-04-01

The surfaces of higher plants are characterized by epidermis, which usually consists of a single layer of cells. The epidermis is derived from the outer cell layer of the embryo or protoderm, which arises as a result of periclinal cell division. After seed germination, most of the epidermal cells of the aerial parts of plants are derived from the outer cell layer of the shoot apical meristem (the L1 layer). Thus, knowledge of how the protoderm and/or L1 layer is established is fundamental to understanding the morphogenesis of higher plants. Here, we report the isolation of a gene encoding an Arabidopsis homologue (ACR4) of the maize putative receptor kinase CRINKLY4 (CR4), which is involved in epidermal differentiation. The domain organization of the predicted amino acid sequence of ACR4 is essentially identical to that of CR4. ACR4-GFP fusion protein localized to the cell surface when expressed in tobacco cell (BY-2) culture. ACR4 transcripts were detected in all the organs of the Arabidopsis plant. In developing embryos and shoot apices, ACR4 transcripts accumulated in protoderm and epidermis at relatively higher levels than in the inner tissues. Over-expression of antisense ACR4 in Arabidopsis plants resulted in malformation of embryos to varying degrees. These results suggest that ACR4 is, at a minimum, involved in the normal morphogenesis of embryos, most likely through properly differentiating protoderm cells.

Transcriptome analysis of the couch potato (CPO) protein reveals an expression pattern associated with early development in the salmon louse Caligus rogercresseyi.

PubMed

Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Nuñez-Acuña, Gustavo; Chávez-Mardones, Jacqueline; Maldonado-Aguayo, Waleska

2014-02-15

The couch potato (CPO) protein is a key biomolecule involved in regulating diapause through the RNA-binding process of the peripheral and central nervous systems in insects and also recently discovered in a few crustacean species. As such, ectoparasitic copepods are interesting model species that have no evidence of developmental arrest. The present study is the first to report on the cloning of a putative CPO gene from the salmon louse Caligus rogercresseyi (CrCPO), as identified by high-throughput transcriptome sequencing. In addition, the transcription expression in larvae and adults was evaluated using quantitative real-time PCR. The CrCPO cDNA sequence showed 3261 base pairs (bp), consisting of 713bp of 5' UTR, 1741bp of 3' UTR, and an open reading frame of 807bp encoding for 268 amino acids. The highly conserved RNA binding regions RNP2 (LFVSGL) and RNP1 (SPVGFVTF), as well the dimerization site (LEF), were also found. Furthermore, eight single nucleotide polymorphisms located in the untranslated regions and one located in the coding region were detected. Gene transcription analysis revealed that CrCPO has ubiquitous expression across larval stages and in adult individuals, with the highest expression from nauplius to copepodid stages. The present study suggests a putative biological function of CrCPO associated with the development of the nervous system in salmon lice and contributes molecular evidence for candidate genes related to host-parasite interactions. Copyright © 2013 Elsevier B.V. All rights reserved.

Abscisic acid regulates pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol accumulation in developing flax (Linum usitatissimum L.) seeds.

PubMed

Renouard, Sullivan; Corbin, Cyrielle; Lopez, Tatiana; Montguillon, Josiane; Gutierrez, Laurent; Lamblin, Frédéric; Lainé, Eric; Hano, Christophe

2012-01-01

Secoisolariciresinol diglucoside (SDG), the main phytoestrogenic lignan of Linum usitatissimum, is accumulated in the seed coat of flax during its development and pinoresinol-lariciresinol reductase (PLR) is a key enzyme in flax for its synthesis. The promoter of LuPLR1, a flax gene encoding a pinoresinol lariciresinol reductase, contains putative regulatory boxes related to transcription activation by abscisic acid (ABA). Gel mobility shift experiments evidenced an interaction of nuclear proteins extracted from immature flax seed coat with a putative cis-acting element involved in ABA response. As ABA regulates a number of physiological events during seed development and maturation we have investigated its involvement in the regulation of this lignan synthesis by different means. ABA and SDG accumulation time courses in the seed as well as LuPLR1 expression were first determined in natural conditions. These results showed that ABA timing and localization of accumulation in the flax seed coat could be correlated with the LuPLR1 gene expression and SDG biosynthesis. Experimental modulations of ABA levels were performed by exogenous application of ABA or fluridone, an inhibitor of ABA synthesis. When submitted to exogenous ABA, immature seeds synthesized 3-times more SDG, whereas synthesis of SDG was reduced in immature seeds treated with fluridone. Similarly, the expression of LuPLR1 gene in the seed coat was up-regulated by exogenous ABA and down-regulated when fluridone was applied. These results demonstrate that SDG biosynthesis in the flax seed coat is positively controlled by ABA through the transcriptional regulation of LuPLR1 gene.

Glucocorticoid Regulation of the Vitamin D Receptor

PubMed Central

Hidalgo, Alejandro A.; Trump, Donald L.; Johnson, Candace S.

2010-01-01

Many studies indicate calcitriol has potent anti-tumor activity in different types of cancers. However, high levels of vitamin D can produce hypercalcemia in some patients. Glucocorticoids are used to ameliorate hypercalcemia and to enhance calcitriol anti-tumor activity. Calcitriol in combination with the glucocorticoid dexamethasone (Dex) increased vitamin D receptor (VDR) protein levels and ligand binding in squamous cell carcinoma VII (SCC). In this study we found that both calcitriol and Dex induce VDR- and glucocorticoid receptor (GR)-mediated transcription respectively, indicating both hormone receptors are active in SCC. Pre-treatment with Dex increases VDR-mediated transcription at the human CYP24A1 promoter. Whereas, pre-treatment with other steroid hormones, including dihydrotestosterone and R1881, has no effect on VDR-mediated transcription. Real-time PCR indicates treatment with Dex increases Vdr transcripts in a time-dependent manner, suggesting Dex may directly regulate expression of Vdr. Numerous putative glucocorticoid response elements (GREs) were found in the Vdr gene. Chromatin immunoprecipitation (ChIP) assay demonstrated GR binding at several putative GREs located within the mouse Vdr gene. However, none of the putative GREs studied increase GR-mediated transcription in luciferase reporter assays. In an attempt to identify the response element responsible for Vdr transcript regulation, future studies will continue to analyze newly identified GREs more distal from the Vdr gene promoter. PMID:20398752

An intact SAM-dependent methyltransferase fold is encoded by the human endothelin-converting enzyme-2 gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tempel, W.; Wu, H.; Dombrovsky, L.

2010-08-17

A recent survey of protein expression patterns in patients with Alzheimer's disease (AD) has identified ece2 (chromosome: 3; Locations: 3q27.1) as the most significantly downregulated gene within the tested group. ece2 encodes endothelin-converting enzyme ECE2, a metalloprotease with a role in neuropeptide processing. Deficiency in the highly homologous ECE1 has earlier been linked to increased levels of AD-related {beta}-amyloid peptide in mice, consistent with a role for ECE in the degradation of that peptide. Initially, ECE2 was presumed to resemble ECE1, in that it comprises a single transmembrane region of {approx}20 residues flanked by a small amino-terminal cytosolic segment andmore » a carboxy-terminal lumenar peptidase domain. The carboxy-terminal domain has significant sequence similarity to both neutral endopeptidase, for which an X-ray structure has been determined, and Kell blood group protein. After their initial discovery, multiple isoforms of ECE1 and ECE2 were discovered, generated by alternative splicing of multiple exons. The originally described ece2 transcript, RefSeq NM{_}174046, contains the amino-terminal cytosolic portion followed by the transmembrane region and peptidase domain (Fig. 1, isoform B). Another ece2 transcript, available from the Mammalian Gene Collection under MGC2408 (Fig. 1, isoform C), RefSeq accession NM{_}032331, is predicted to be translated into a 255 residue peptide with low but detectable sequence similarity to known S-adenosyl-L-methionine (SAM)-dependent methyltransferases (SAM-MTs), such as the hypothetical protein TT1324 from Thermus thermophilis, PDB code 2GS9, which shares 30% amino acid sequence identity with ECE2 over 138 residues of the sequence. Intriguingly, another 'elongated' ece2 transcript (Fig. 1, isoform A) (RefSeq NM{_}014693) contains an amino-terminal portion of the putative SAM-MT domain, the transmembrane domain, and the protease domain. This suggests the possibility for coexistence of the putative SAM-MT and protease domains in a single polypeptide and their transmembrane interplay. Although sequence conservation across the SAM-MT family is weak, the structural fold is highly conserved. The most conserved part of this fold is the SAM-binding subdomain, which is shared between MGC2408 and hypothetical protein TT1324. Typically, the SAM-binding subdomain is flanked by a variable Nterminal extension and, at the C-terminus, by a substrate- binding subdomain, which varies enormously in size but preserves a conserved topology with three antiparallel b-strands. The 'elongated' transcript of ece2 lacks this substrate-binding subdomain. To test the hypothesis that the 255 residue ece2 gene product MGC2408 represents a complete SAM-MT fold, we have determined a crystal structure of this protein in the presence of SAH.« less

Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei

PubMed Central

Derntl, Christian; Rassinger, Alice; Srebotnik, Ewald; Mach, Robert L.

2016-01-01

ABSTRACT The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum. In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1. IMPORTANCE Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum. The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism. PMID:27520818

Localization and expression of putative circadian clock transcripts in the brain of the nudibranch Melibe leonina.

PubMed

Duback, Victoria E; Sabrina Pankey, M; Thomas, Rachel I; Huyck, Taylor L; Mbarani, Izhar M; Bernier, Kyle R; Cook, Geoffrey M; O'Dowd, Colleen A; Newcomb, James M; Watson, Winsor H

2018-09-01

The nudibranch, Melibe leonina, expresses a circadian rhythm of locomotion, and we recently determined the sequences of multiple circadian clock transcripts that may play a role in controlling these daily patterns of behavior. In this study, we used these genomic data to help us: 1) identify putative clock neurons using fluorescent in situ hybridization (FISH); and 2) determine if there is a daily rhythm of expression of clock transcripts in the M. leonina brain, using quantitative PCR. FISH indicated the presence of the clock-related transcripts clock, period, and photoreceptive and non-photoreceptive cryptochrome (pcry and npcry, respectively) in two bilateral neurons in each cerebropleural ganglion and a group of <10 neurons in the anterolateral region of each pedal ganglion. Double-label experiments confirmed colocalization of all four clock transcripts with each other. Quantitative PCR demonstrated that the genes clock, period, pcry and npcry exhibited significant differences in expression levels over 24 h. These data suggest that the putative circadian clock network in M. leonina consists of a small number of identifiable neurons that express circadian genes with a daily rhythm. Copyright © 2018 Elsevier Inc. All rights reserved.

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection

PubMed Central

De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain

2016-01-01

ABSTRACT Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae. Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum. IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete’s foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae. Comparing gene expression during infection on guinea pigs with keratin degradation in vitro, which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo, encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates. PMID:27822542

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection.

PubMed

Tran, Van Du T; De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain; Pagni, Marco; Monod, Michel

2016-01-01

Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae . Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum . IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete's foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae . Comparing gene expression during infection on guinea pigs with keratin degradation in vitro , which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo , encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates.

Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers

PubMed Central

2011-01-01

Background Panax notoginseng (Burk) F.H. Chen is important medicinal plant of the Araliacease family. Triterpene saponins are the bioactive constituents in P. notoginseng. However, available genomic information regarding this plant is limited. Moreover, details of triterpene saponin biosynthesis in the Panax species are largely unknown. Results Using the 454 pyrosequencing technology, a one-quarter GS FLX titanium run resulted in 188,185 reads with an average length of 410 bases for P. notoginseng root. These reads were processed and assembled by 454 GS De Novo Assembler software into 30,852 unique sequences. A total of 70.2% of unique sequences were annotated by Basic Local Alignment Search Tool (BLAST) similarity searches against public sequence databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) assignment discovered 41 unique sequences representing 11 genes involved in triterpene saponin backbone biosynthesis in the 454-EST dataset. In particular, the transcript encoding dammarenediol synthase (DS), which is the first committed enzyme in the biosynthetic pathway of major triterpene saponins, is highly expressed in the root of four-year-old P. notoginseng. It is worth emphasizing that the candidate cytochrome P450 (Pn02132 and Pn00158) and UDP-glycosyltransferase (Pn00082) gene most likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis were discovered from 174 cytochrome P450s and 242 glycosyltransferases by phylogenetic analysis, respectively. Putative transcription factors were detected in 906 unique sequences, including Myb, homeobox, WRKY, basic helix-loop-helix (bHLH), and other family proteins. Additionally, a total of 2,772 simple sequence repeat (SSR) were identified from 2,361 unique sequences, of which, di-nucleotide motifs were the most abundant motif. Conclusion This study is the first to present a large-scale EST dataset for P. notoginseng root acquired by next-generation sequencing (NGS) technology. The candidate genes involved in triterpene saponin biosynthesis, including the putative CYP450s and UGTs, were obtained in this study. Additionally, the identification of SSRs provided plenty of genetic makers for molecular breeding and genetics applications in this species. These data will provide information on gene discovery, transcriptional regulation and marker-assisted selection for P. notoginseng. The dataset establishes an important foundation for the study with the purpose of ensuring adequate drug resources for this species. PMID:22369100

Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

PubMed

Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

2016-01-01

Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides.

SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses

PubMed Central

2010-01-01

Background Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown. Results We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction. Conclusions The analysis of the early changes in the transcriptome of N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process. PMID:20398280

Comparative analysis of long non-coding RNAs in Atlantic and Coho salmon reveals divergent transcriptome responses associated with immunity and tissue repair during sea lice infestation.

PubMed

Valenzuela-Muñoz, Valentina; Valenzuela-Miranda, Diego; Gallardo-Escárate, Cristian

2018-05-24

The increasing capacity of transcriptomic analysis by high throughput sequencing has highlighted the presence of a large proportion of transcripts that do not encode proteins. In particular, long non-coding RNAs (lncRNAs) are sequences with low coding potential and conservation among species. Moreover, cumulative evidence has revealed important roles in post-transcriptional gene modulation in several taxa. In fish, the role of lncRNAs has been scarcely studied and even less so during the immune response against sea lice. In the present study we mined for lncRNAs in Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynkus kisutch), which are affected by the sea louse Caligus rogercresseyi, evaluating the degree of sequence conservation between these two fish species and their putative roles during the infection process. Herein, Atlantic and Coho salmon were infected with 35 lice/fish and evaluated after 7 and 14 days post-infestation (dpi). For RNA sequencing, samples from skin and head kidney were collected. A total of 5658/4140 and 3678/2123 lncRNAs were identified in uninfected/infected Atlantic and Coho salmon transcriptomes, respectively. Species-specific transcription patterns were observed in exclusive lncRNAs according to the tissue analyzed. Furthermore, neighbor gene GO enrichment analysis of the top 100 highly regulated lncRNAs in Atlantic salmon showed that lncRNAs were localized near genes related to the immune response. On the other hand, in Coho salmon the highly regulated lncRNAs were localized near genes involved in tissue repair processes. This study revealed high regulation of lncRNAs closely localized to immune and tissue repair-related genes in Atlantic and Coho salmon, respectively, suggesting putative roles for lncRNAs in salmon against sea lice infestation. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

PubMed Central

Shulami, Smadar; Gat, Orit; Sonenshein, Abraham L.; Shoham, Yuval

1999-01-01

A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-d-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer. PMID:10368143

SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses.

PubMed

Gilardoni, Paola A; Schuck, Stefan; Jüngling, Ruth; Rotter, Björn; Baldwin, Ian T; Bonaventure, Gustavo

2010-04-14

Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown. We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated >or= 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction. The analysis of the early changes in the transcriptome of N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.

Cloning and characterization of two duplicated interleukin-17A/F2 genes in common carp (Cyprinus carpio L.): Transcripts expression and bioactivity of recombinant IL-17A/F2.

PubMed

Li, Hongxia; Yu, Juhua; Li, Jianlin; Tang, Yongkai; Yu, Fan; Zhou, Jie; Yu, Wenjuan

2016-04-01

Interleukin-17 (IL-17) plays an important role in inflammation and host defense in mammals. In this study, we identified two duplicated IL-17A/F2 genes in the common carp (Cyprinus carpio) (ccIL-17A/F2a and ccIL-17A/F2b), putative encoded proteins contain 140 amino acids (aa) with conserved IL-17 family motifs. Expression analysis revealed high constitutive expression of ccIL-17A/F2s in mucosal tissues, including gill, skin and intestine, their expression could be induced by Aeromonas hydrophila, suggesting a potential role in mucosal immunity. Recombinant ccIL-17A/F2a protein (rccIL-17A/F2a) produced in Escherichia coli could induce the expression of proinflammatory cytokines (IL-1β) and the antimicrobial peptides S100A1, S100A10a and S100A10b in the primary kidney in a dose- and time-dependent manner. Above findings suggest that ccIL-17A/F2 plays an important role in both proinflammatory and innate immunity. Two duplicated ccIL-17A/F2s showed different expression level with ccIL-17A/F2a higher than b, comparison of two 5' regulatory regions indicated the length from anticipated promoter to transcriptional start site (TSS) and putative transcription factor binding site (TFBS) were different. Promoter activity of ccIL-17A/F2a was 2.5 times of ccIL-17A/F2b which consistent with expression results of two genes. These suggest mutations in 5'regulatory region contributed to the differentiation of duplicated genes. To our knowledge, this is the first report to analyze 5'regulatory region of piscine IL-17 family genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tuning of RNA editing by ADAR is required in Drosophila

PubMed Central

Keegan, Liam P; Brindle, James; Gallo, Angela; Leroy, Anne; Reenan, Robert A; O'Connell, Mary A

2005-01-01

RNA editing increases during development in more than 20 transcripts encoding proteins involved in rapid synaptic neurotransmission in Drosophila central nervous system and muscle. Adar (adenosine deaminase acting on RNA) mutant flies expressing only genome-encoded, unedited isoforms of ion-channel subunits are viable but show severe locomotion defects. The Adar transcript itself is edited in adult wild-type flies to generate an isoform with a serine to glycine substitution close to the ADAR active site. We show that editing restricts ADAR function since the edited isoform of ADAR is less active in vitro and in vivo than the genome-encoded, unedited isoform. Ubiquitous expression in embryos and larvae of an Adar transcript that is resistant to editing is lethal. Expression of this transcript in embryonic muscle is also lethal, with above-normal, adult-like levels of editing at sites in a transcript encoding a muscle voltage-gated calcium channel. PMID:15920480

Characterization of a prototype strain of hepatitis E virus.

PubMed Central

Tsarev, S A; Emerson, S U; Reyes, G R; Tsareva, T S; Legters, L J; Malik, I A; Iqbal, M; Purcell, R H

1992-01-01

A strain of hepatitis E virus (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, was characterized extensively. Six cynomolgus monkeys (Macaca fascicularis) were infected with a strain of hepatitis E virus from Pakistan. Reverse transcription-polymerase chain reaction was used to determine the pattern of virus shedding in feces, bile, and serum relative to hepatitis and induction of specific antibodies. Virtually the entire genome of SAR-55 (7195 nucleotides) was sequenced. Comparison of the sequence of SAR-55 with that of a Burmese strain revealed a high level of homology except for one region encoding 100 amino acids of a putative nonstructural polyprotein. Identification of this region as hypervariable was obtained by partial sequencing of a third isolate of hepatitis E virus from Kirgizia. Images PMID:1731327

Tetrahymena thermophila acidic ribosomal protein L37 contains an archaebacterial type of C-terminus.

PubMed

Hansen, T S; Andreasen, P H; Dreisig, H; Højrup, P; Nielsen, H; Engberg, J; Kristiansen, K

1991-09-15

We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63 nucleotides upstream from the translational start codon. The uppermost tsp mapped to the first T in a putative T. thermophila RNA polymerase II initiator element, TATAA. The coding region of L37 predicts a protein of 109 amino acid (aa) residues. A substantial part of the deduced aa sequence was verified by protein sequencing. The T. thermophila L37 clearly belongs to the P1-type family of eukaryotic A-proteins, but the C-terminal region has the hallmarks of archaebacterial A-proteins.

AmrZ Beta-Sheet Residues Are Essential for DNA Binding and Transcriptional Control of Pseudomonas aeruginosa Virulence Genes ▿ †

PubMed Central

Waligora, Elizabeth A.; Ramsey, Deborah M.; Pryor, Edward E.; Lu, Haiping; Hollis, Thomas; Sloan, Gina P.; Deora, Rajendar; Wozniak, Daniel J.

2010-01-01

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection. PMID:20709902

A single-repeat R3-MYB transcription factor MYBC1 negatively regulates freezing tolerance in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhai, Hong, E-mail: Zhai.h@hotmail.com; Bai, Xi, E-mail: baixi@neau.edu.cn; Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn

2010-04-16

We had previously identified the MYBC1 gene, which encodes a single-repeat R3-MYB protein, as a putative osmotic responding gene; however, no R3-MYB transcription factor has been reported to regulate osmotic stress tolerance. Thus, we sought to elucidate the function of MYBC1 in response to osmotic stresses. Real-time RT-PCR analysis indicated that MYBC1 expression responded to cold, dehydration, salinity and exogenous ABA at the transcript level. mybc1 mutants exhibited an increased tolerance to freezing stress, whereas 35S::MYBC1 transgenic plants exhibited decreased cold tolerance. Transcript levels of some cold-responsive genes, including CBF/DREB genes, KIN1, ADC1, ADC2 and ZAT12, though, were not alteredmore » in the mybc1 mutants or the 35S::MYBC1 transgenic plants in response to cold stress, as compared to the wild type. Microarray analysis results that are publically available were investigated and found transcript level of MYBC1 was not altered by overexpression of CBF1, CBF2, and CBF3, suggesting that MYBC1 is not down regulated by these CBF family members. Together, these results suggested that MYBC1is capable of negatively regulating the freezing tolerance of Arabidopsis in the CBF-independent pathway. In transgenic Arabidopsis carrying an MYBC1 promoter driven {beta}-glucuronidase (GUS) construct, GUS activity was observed in all tissues and was relatively stronger in the vascular tissues. Fused MYBC1 and GFP protein revealed that MYBC1 was localized exclusively in the nuclear compartment.« less

A Novel C2H2 Transcription Factor that Regulates gliA Expression Interdependently with GliZ in Aspergillus fumigatus

PubMed Central

Schoberle, Taylor J.; Nguyen-Coleman, C. Kim; Herold, Jennifer; Yang, Ally; Weirauch, Matt; Hughes, Timothy R.; McMurray, John S.; May, Gregory S.

2014-01-01

Secondary metabolites are produced by numerous organisms and can either be beneficial, benign, or harmful to humans. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Gliotoxin, which is produced by a variety of Aspergillus species, Trichoderma species, and Penicillium species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn2Cys6 binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal species. Using a high-copy inducer screen in A. fumigatus, our lab has identified a novel C2H2 transcription factor, which plays an important role in regulating the gliotoxin biosynthetic cluster. This transcription factor, named GipA, induces gliotoxin production when present in extra copies. Furthermore, loss of gipA reduces gliotoxin production significantly. Through protein binding microarray and mutagenesis, we have identified a DNA binding site recognized by GipA that is in extremely close proximity to a potential GliZ DNA binding site in the 5′ untranslated region of gliA, which encodes an efflux pump within the gliotoxin cluster. Not surprisingly, GliZ and GipA appear to work in an interdependent fashion to positively control gliA expression. PMID:24784729

Transcriptional Profiling of Human Endogenous Retrovirus Group HERV-K(HML-2) Loci in Melanoma

PubMed Central

Schmitt, Katja; Reichrath, Jörg; Roesch, Alexander; Meese, Eckart; Mayer, Jens

2013-01-01

Recent studies suggested a role for the human endogenous retrovirus (HERV) group HERV-K(HML-2) in melanoma because of upregulated transcription and expression of HERV-K(HML-2)-encoded proteins. Very little is known about which HML-2 loci are transcribed in melanoma. We assigned >1,400 HML-2 cDNA sequences generated from various melanoma and related samples to genomic HML-2 loci, identifying a total of 23 loci as transcribed. Transcription profiles of loci differed significantly between samples. One locus was found transcribed only in melanoma-derived samples but not in melanocytes and might represent a marker for melanoma. Several of the transcribed loci harbor ORFs for retroviral Gag and/or Env proteins. Env-encoding loci were transcribed only in melanoma. Specific investigation of rec and np9 transcripts indicated transcription of protein encoding loci in melanoma and melanocytes hinting at the relevance of Rec and Np9 in melanoma. UVB irradiation changed transcription profiles of loci and overall transcript levels decreased in melanoma and melanocytes. We further identified transcribed HML-2 loci formed by reverse transcription of spliced HML-2 transcripts by L1 machinery or in a retroviral fashion, with loci potentially encoding HML-2-like proteins. We reveal complex, sample-specific transcription of HML-2 loci in melanoma and related samples. Identified HML-2 loci and proteins encoded by those loci are particularly relevant for further studying the role of HML-2 in melanoma. Transcription of HERVs appears as a complex mechanism requiring specific studies to elucidate which HERV loci are transcribed and how transcribed HERVs may be involved in disease. PMID:23338945

Transcriptome profiling of Nasonia vitripennis testis reveals novel transcripts expressed from the selfish B chromosome, paternal sex ratio.

PubMed

Akbari, Omar S; Antoshechkin, Igor; Hay, Bruce A; Ferree, Patrick M

2013-09-04

A widespread phenomenon in nature is sex ratio distortion of arthropod populations caused by microbial and genetic parasites. Currently little is known about how these agents alter host developmental processes to favor one sex or the other. The paternal sex ratio (PSR) chromosome is a nonessential, paternally transmitted centric fragment that segregates in natural populations of the jewel wasp, Nasonia vitripennis. To persist, PSR is thought to modify the hereditary material of the developing sperm, with the result that all nuclear DNA other than the PSR chromosome is destroyed shortly after fertilization. This results in the conversion of a fertilized embryo--normally a female--into a male, thereby insuring transmission of the "selfish" PSR chromosome, and simultaneously leading to wasp populations that are male-biased. To begin to understand this system at the mechanistic level, we carried out transcriptional profiling of testis from WT and PSR-carrying males. We identified a number of transcripts that are differentially expressed between these conditions. We also discovered nine transcripts that are uniquely expressed from the PSR chromosome. Four of these PSR-specific transcripts encode putative proteins, whereas the others have very short open reading frames and no homology to known proteins, suggesting that they are long noncoding RNAs. We propose several different models for how these transcripts could facilitate PSR-dependent effects. Our analyses also revealed 15.71 MB of novel transcribed regions in the N. vitripennis genome, thus increasing the current annotation of total transcribed regions by 53.4%. Finally, we detected expression of multiple meiosis-related genes in the wasp testis, despite the lack of conventional meiosis in the male sex.

Transcriptome Profiling of Nasonia vitripennis Testis Reveals Novel Transcripts Expressed from the Selfish B Chromosome, Paternal Sex Ratio

PubMed Central

Akbari, Omar S.; Antoshechkin, Igor; Hay, Bruce A.; Ferree, Patrick M.

2013-01-01

A widespread phenomenon in nature is sex ratio distortion of arthropod populations caused by microbial and genetic parasites. Currently little is known about how these agents alter host developmental processes to favor one sex or the other. The paternal sex ratio (PSR) chromosome is a nonessential, paternally transmitted centric fragment that segregates in natural populations of the jewel wasp, Nasonia vitripennis. To persist, PSR is thought to modify the hereditary material of the developing sperm, with the result that all nuclear DNA other than the PSR chromosome is destroyed shortly after fertilization. This results in the conversion of a fertilized embryo—normally a female—into a male, thereby insuring transmission of the “selfish” PSR chromosome, and simultaneously leading to wasp populations that are male-biased. To begin to understand this system at the mechanistic level, we carried out transcriptional profiling of testis from WT and PSR-carrying males. We identified a number of transcripts that are differentially expressed between these conditions. We also discovered nine transcripts that are uniquely expressed from the PSR chromosome. Four of these PSR-specific transcripts encode putative proteins, whereas the others have very short open reading frames and no homology to known proteins, suggesting that they are long noncoding RNAs. We propose several different models for how these transcripts could facilitate PSR-dependent effects. Our analyses also revealed 15.71 MB of novel transcribed regions in the N. vitripennis genome, thus increasing the current annotation of total transcribed regions by 53.4%. Finally, we detected expression of multiple meiosis-related genes in the wasp testis, despite the lack of conventional meiosis in the male sex. PMID:23893741

The Oncoprotein Tax Binds the SRC-1-Interacting Domain of CBP/p300 To Mediate Transcriptional Activation

PubMed Central

Scoggin, Kirsten E. S.; Ulloa, Aida; Nyborg, Jennifer K.

2001-01-01

Oncogenesis associated with human T-cell leukemia virus (HTLV) infection is directly linked to the virally encoded transcription factor Tax. To activate HTLV-1 transcription Tax interacts with the cellular protein CREB and the pleiotropic coactivators CBP and p300. While extensively studied, the molecular mechanisms of Tax transcription function and coactivator utilization are not fully understood. Previous studies have focused on Tax binding to the KIX domain of CBP, as this was believed to be the key step in recruiting the coactivator to the HTLV-1 promoter. In this study, we identify a carboxy-terminal region of CBP (and p300) that strongly interacts with Tax and mediates Tax transcription function. Through deletion mutagenesis, we identify amino acids 2003 to 2212 of CBP, which we call carboxy-terminal region 2 (CR2), as the minimal region for Tax interaction. Interestingly, this domain corresponds to the steroid receptor coactivator 1 (SRC-1)-interacting domain of CBP. We show that a double point mutant targeted to one of the putative α-helical motifs in this domain significantly compromises the interaction with Tax. We also characterize the region of Tax responsible for interaction with CR2 and show that the previously identified transactivation domain of Tax (amino acids 312 to 319) participates in CR2 binding. This region of Tax corresponds to a consensus amphipathic helix, and single point mutations targeted to amino acids on the face of this helix abolish interaction with CR2 and dramatically reduce Tax transcription function. Finally, we demonstrate that Tax and SRC-1 bind to CR2 in a mutually exclusive fashion. Together, these studies identify a novel Tax-interacting site on CBP/p300 and extend our understanding of the molecular mechanism of Tax transactivation. PMID:11463834

Transcription factor FOXA2-centered transcriptional regulation network in non-small cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Sang-Min; An, Joo-Hee; Kim, Chul-Hong

2015-08-07

Lung cancer is the leading cause of cancer-mediated death. Although various therapeutic approaches are used for lung cancer treatment, these mainly target the tumor suppressor p53 transcription factor, which is involved in apoptosis and cell cycle arrest. However, p53-targeted therapies have limited application in lung cancer, since p53 is found to be mutated in more than half of lung cancers. In this study, we propose tumor suppressor FOXA2 as an alternative target protein for therapies against lung cancer and reveal a possible FOXA2-centered transcriptional regulation network by identifying new target genes and binding partners of FOXA2 by using various screeningmore » techniques. The genes encoding Glu/Asp-rich carboxy-terminal domain 2 (CITED2), nuclear receptor subfamily 0, group B, member 2 (NR0B2), cell adhesion molecule 1 (CADM1) and BCL2-associated X protein (BAX) were identified as putative target genes of FOXA2. Additionally, the proteins including highly similar to heat shock protein HSP 90-beta (HSP90A), heat shock 70 kDa protein 1A variant (HSPA1A), histone deacetylase 1 (HDAC1) and HDAC3 were identified as novel interacting partners of FOXA2. Moreover, we showed that FOXA2-dependent promoter activation of BAX and p21 genes is significantly reduced via physical interactions between the identified binding partners and FOXA2. These results provide opportunities to understand the FOXA2-centered transcriptional regulation network and novel therapeutic targets to modulate this network in p53-deficient lung cancer. - Highlights: • Identification of new target genes of FOXA2. • Identifications of novel interaction proteins of FOXA2. • Construction of FOXA2-centered transcriptional regulatory network in non-small cell lung cancer.« less

Next generation sequencing and de novo transcriptome analysis of Costus pictus D. Don, a non-model plant with potent anti-diabetic properties

PubMed Central

2012-01-01

Background Phyto-remedies for diabetic control are popular among patients with Type II Diabetes mellitus (DM), in addition to other diabetic control measures. A number of plant species are known to possess diabetic control properties. Costus pictus D. Don is popularly known as “Insulin Plant” in Southern India whose leaves have been reported to increase insulin pools in blood plasma. Next Generation Sequencing is employed as a powerful tool for identifying molecular signatures in the transcriptome related to physiological functions of plant tissues. We sequenced the leaf transcriptome of C. pictus using Illumina reversible dye terminator sequencing technology and used combination of bioinformatics tools for identifying transcripts related to anti-diabetic properties of C. pictus. Results A total of 55,006 transcripts were identified, of which 69.15% transcripts could be annotated. We identified transcripts related to pathways of bixin biosynthesis and geraniol and geranial biosynthesis as major transcripts from the class of isoprenoid secondary metabolites and validated the presence of putative norbixin methyltransferase, a precursor of Bixin. The transcripts encoding these terpenoids are known to be Peroxisome Proliferator-Activated Receptor (PPAR) agonists and anti-glycation agents. Sequential extraction and High Performance Liquid Chromatography (HPLC) confirmed the presence of bixin in C. pictus methanolic extracts. Another significant transcript identified in relation to anti-diabetic, anti-obesity and immuno-modulation is of Abscisic Acid biosynthetic pathway. We also report many other transcripts for the biosynthesis of antitumor, anti-oxidant and antimicrobial metabolites of C. pictus leaves. Conclusion Solid molecular signatures (transcripts related to bixin, abscisic acid, and geranial and geraniol biosynthesis) for the anti-diabetic properties of C. pictus leaves and vital clues related to the other phytochemical functions like antitumor, anti-oxidant, immuno-modulatory, anti-microbial and anti-malarial properties through the secondary metabolite pathway annotations are reported. The data provided will be of immense help to researchers working in the treatment of DM using herbal therapies. PMID:23176672

Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning.

PubMed

Albert, Nick W; Lewis, David H; Zhang, Huaibi; Schwinn, Kathy E; Jameson, Paula E; Davies, Kevin M

2011-03-01

We present an investigation of anthocyanin regulation over the entire petunia plant, determining the mechanisms governing complex floral pigmentation patterning and environmentally induced vegetative anthocyanin synthesis. DEEP PURPLE (DPL) and PURPLE HAZE (PHZ) encode members of the R2R3-MYB transcription factor family that regulate anthocyanin synthesis in petunia, and control anthocyanin production in vegetative tissues and contribute to floral pigmentation. In addition to these two MYB factors, the basic helix-loop-helix (bHLH) factor ANTHOCYANIN1 (AN1) and WD-repeat protein AN11, are also essential for vegetative pigmentation. The induction of anthocyanins in vegetative tissues by high light was tightly correlated to the induction of transcripts for PHZ and AN1. Interestingly, transcripts for PhMYB27, a putative R2R3-MYB active repressor, were highly expressed during non-inductive shade conditions and repressed during high light. The competitive inhibitor PhMYBx (R3-MYB) was expressed under high light, which may provide feedback repression. In floral tissues DPL regulates vein-associated anthocyanin pigmentation in the flower tube, while PHZ determines light-induced anthocyanin accumulation on exposed petal surfaces (bud-blush). A model is presented suggesting how complex floral and vegetative pigmentation patterns are derived in petunia in terms of MYB, bHLH and WDR co-regulators. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Identification, cloning, and sequencing of a fragment of Amsacta moorei entomopoxvirus DNA containing the spheroidin gene and three vaccinia virus-related open reading frames.

PubMed Central

Hall, R L; Moyer, R W

1991-01-01

Entomopoxvirus virions are frequently contained within crystalline occlusion bodies, which are composed of primarily a single protein, spheroidin, which is analogous to the polyhedrin protein of baculovirus. The spheroidin gene of Amsacta moorei entomopoxvirus was identified following the microsequencing of polypeptides generated from cyanogen bromide treatment of spheroidin and the subsequent synthesis of oligonucleotide hybridization probes. DNA sequencing of a 6.8-kb region of DNA containing the spheroidin gene showed that the spheroidin protein is derived from a 3.0-kb open reading frame potentially encoding a protein of 115 kDa. Three copies of the heptanucleotide, TTTTTNT, a sequence associated with early gene transcription in the vertebrate poxviruses, and four in-frame translational termination signals were found within 60 bp upstream of the putative spheroidin gene promoter (TAAATG). The spheroidin gene promoter region contains the sequence TAAATG, which is found in many late promoters of the vertebrate poxviruses and which serves as the site of transcriptional initiation, as shown by primer extension. Primer extension experiments also showed that spheroidin gene transcripts contain 5' poly(A) sequences typical of vertebrate poxvirus late transcripts. The 92 bases upstream of the initiating TAAATG are unusually A + T rich and contain only 7 G or C residues. An analysis of open reading frames around the spheroidin gene suggests that the colinear core of "essential genes" typical of the vertebrate poxviruses is absent in A. moorei entomopoxvirus. Images PMID:1942245

Transcriptional Analysis of the vanC Cluster from Enterococcus gallinarum Strains with Constitutive and Inducible Vancomycin Resistance

PubMed Central

Panesso, Diana; Abadía-Patiño, Lorena; Vanegas, Natasha; Reynolds, Peter E.; Courvalin, Patrice; Arias, Cesar A.

2005-01-01

The vanC glycopeptide resistance gene cluster encodes enzymes required for synthesis of peptidoglycan precursors ending in d-Ala-d-Ser. Enterococcus gallinarum BM4174 and SC1 are constitutively and inducibly resistant to vancomycin, respectively. Analysis of peptidoglycan precursors in both strains indicated that UDP-MurNAc-tetrapeptide and UDP-MurNAc-pentapeptide[d-Ser] were synthesized in E. gallinarum SC1 only in the presence of vancomycin (4 μg/ml), whereas the “resistance” precursors accumulated in the cytoplasm of BM4174 cells under both inducing and noninducing conditions. Northern hybridization and reverse transcription-PCR experiments revealed that all the genes from the cluster, vanC-1, vanXYC, vanT, vanRC, and vanSC, were transcribed from a single promoter. In the inducible SC1 isolate, transcriptional regulation appeared to be responsible for inducible expression of resistance. Promoter mapping in E. gallinarum BM4174 revealed that the transcriptional start site was located 30 nucleotides upstream from vanC-1 and that the −10 promoter consensus sequence had high identity with that of the vanA cluster. Comparison of the deduced sequence of the vanSC genes from isolates with constitutive and inducible resistance revealed several amino acid substitutions located in the X box (R200L) and in the region between the F and G2 boxes (D312N, D312A, and G320S) of the putative sensor kinase proteins from isolates with constitutive resistance. PMID:15728903

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom[OPEN

PubMed Central

Smith, Sarah R.; McCrow, John P.; Tan, Maxine; Lichtle, Christian; Goodenough, Ursula; Bowler, Chris P.; Dupont, Christopher L.

2017-01-01

The ecological prominence of diatoms in the ocean environment largely results from their superior competitive ability for dissolved nitrate (NO3−). To investigate the cellular and genetic basis of diatom NO3− assimilation, we generated a knockout in the nitrate reductase gene (NR-KO) of the model pennate diatom Phaeodactylum tricornutum. In NR-KO cells, N-assimilation was abolished although NO3− transport remained intact. Unassimilated NO3− accumulated in NR-KO cells, resulting in swelling and associated changes in biochemical composition and physiology. Elevated expression of genes encoding putative vacuolar NO3− chloride channel transporters plus electron micrographs indicating enlarged vacuoles suggested vacuolar storage of NO3−. Triacylglycerol concentrations in the NR-KO cells increased immediately following the addition of NO3−, and these increases coincided with elevated gene expression of key triacylglycerol biosynthesis components. Simultaneously, induction of transcripts encoding proteins involved in thylakoid membrane lipid recycling suggested more abrupt repartitioning of carbon resources in NR-KO cells compared with the wild type. Conversely, ribosomal structure and photosystem genes were immediately deactivated in NR-KO cells following NO3− addition, followed within hours by deactivation of genes encoding enzymes for chlorophyll biosynthesis and carbon fixation and metabolism. N-assimilation pathway genes respond uniquely, apparently induced simultaneously by both NO3− replete and deplete conditions. PMID:28765511

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

DOE PAGES

McCarthy, James K.; Smith, Sarah R.; McCrow, John P.; ...

2017-09-07

The ecological prominence of diatoms in the ocean environment largely results from their superior competitive ability for dissolved nitrate (NO 3 -). To investigate the cellular and genetic basis of diatom NO 3 - assimilation, in this paper we generated a knockout in the nitrate reductase gene (NR-KO) of the model pennate diatom Phaeodactylum tricornutum. In NR-KO cells, N-assimilation was abolished although NO 3 - transport remained intact. Unassimilated NO 3 - accumulated in NR-KO cells, resulting in swelling and associated changes in biochemical composition and physiology. Elevated expression of genes encoding putative vacuolar NO 3 - chloride channel transportersmore » plus electron micrographs indicating enlarged vacuoles suggested vacuolar storage of NO 3 -. Triacylglycerol concentrations in the NR-KO cells increased immediately following the addition of NO 3 -, and these increases coincided with elevated gene expression of key triacylglycerol biosynthesis components. Simultaneously, induction of transcripts encoding proteins involved in thylakoid membrane lipid recycling suggested more abrupt repartitioning of carbon resources in NR-KO cells compared with the wild type. Conversely, ribosomal structure and photosystem genes were immediately deactivated in NR-KO cells following NO 3 - addition, followed within hours by deactivation of genes encoding enzymes for chlorophyll biosynthesis and carbon fixation and metabolism. Finally, N-assimilation pathway genes respond uniquely, apparently induced simultaneously by both NO 3 - replete and deplete conditions.« less

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCarthy, James K.; Smith, Sarah R.; McCrow, John P.

The ecological prominence of diatoms in the ocean environment largely results from their superior competitive ability for dissolved nitrate (NO 3 -). To investigate the cellular and genetic basis of diatom NO 3 - assimilation, in this paper we generated a knockout in the nitrate reductase gene (NR-KO) of the model pennate diatom Phaeodactylum tricornutum. In NR-KO cells, N-assimilation was abolished although NO 3 - transport remained intact. Unassimilated NO 3 - accumulated in NR-KO cells, resulting in swelling and associated changes in biochemical composition and physiology. Elevated expression of genes encoding putative vacuolar NO 3 - chloride channel transportersmore » plus electron micrographs indicating enlarged vacuoles suggested vacuolar storage of NO 3 -. Triacylglycerol concentrations in the NR-KO cells increased immediately following the addition of NO 3 -, and these increases coincided with elevated gene expression of key triacylglycerol biosynthesis components. Simultaneously, induction of transcripts encoding proteins involved in thylakoid membrane lipid recycling suggested more abrupt repartitioning of carbon resources in NR-KO cells compared with the wild type. Conversely, ribosomal structure and photosystem genes were immediately deactivated in NR-KO cells following NO 3 - addition, followed within hours by deactivation of genes encoding enzymes for chlorophyll biosynthesis and carbon fixation and metabolism. Finally, N-assimilation pathway genes respond uniquely, apparently induced simultaneously by both NO 3 - replete and deplete conditions.« less

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

PubMed

Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

2008-12-01

Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

Avirulence gene mapping in the Hessian fly (Mayetiola destructor) reveals a protein phosphatase 2C effector gene family.

PubMed

Zhao, Chaoyang; Shukle, Richard; Navarro-Escalante, Lucio; Chen, Mingshun; Richards, Stephen; Stuart, Jeffrey J

2016-01-01

The genetic tractability of the Hessian fly (HF, Mayetiola destructor) provides an opportunity to investigate the mechanisms insects use to induce plant gall formation. Here we demonstrate that capacity using the newly sequenced HF genome by identifying the gene (vH24) that elicits effector-triggered immunity in wheat (Triticum spp.) seedlings carrying HF resistance gene H24. vH24 was mapped within a 230-kb genomic fragment near the telomere of HF chromosome X1. That fragment contains only 21 putative genes. The best candidate vH24 gene in this region encodes a protein containing a secretion signal and a type-2 serine/threonine protein phosphatase (PP2C) domain. This gene has an H24-virulence associated insertion in its promoter that appears to silence transcription of the gene in H24-virulent larvae. Candidate vH24 is a member of a small family of genes that encode secretion signals and PP2C domains. It belongs to the fraction of genes in the HF genome previously predicted to encode effector proteins. Because PP2C proteins are not normally secreted, our results suggest that these are PP2C effectors that HF larvae inject into wheat cells to redirect, or interfere, with wheat signal transduction pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243.

PubMed

Onaka, Hiroyasu; Taniguchi, Shin-ichi; Igarashi, Yasuhiro; Furumai, Tamotsu

2003-01-01

The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

PubMed Central

2011-01-01

Background Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation. Results We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere. Conclusions We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation. PMID:21689454

Integration of light and circadian signals that regulate chloroplast transcription by a nuclear-encoded sigma factor.

PubMed

Belbin, Fiona E; Noordally, Zeenat B; Wetherill, Sarah J; Atkins, Kelly A; Franklin, Keara A; Dodd, Antony N

2017-01-01

We investigated the signalling pathways that regulate chloroplast transcription in response to environmental signals. One mechanism controlling plastid transcription involves nuclear-encoded sigma subunits of plastid-encoded plastid RNA polymerase. Transcripts encoding the sigma factor SIG5 are regulated by light and the circadian clock. However, the extent to which a chloroplast target of SIG5 is regulated by light-induced changes in SIG5 expression is unknown. Moreover, the photoreceptor signalling pathways underlying the circadian regulation of chloroplast transcription by SIG5 are unidentified. We monitored the regulation of chloroplast transcription in photoreceptor and sigma factor mutants under controlled light regimes in Arabidopsis thaliana. We established that a chloroplast transcriptional response to light intensity was mediated by SIG5; a chloroplast transcriptional response to the relative proportions of red and far red light was regulated by SIG5 through phytochrome and photosynthetic signals; and the circadian regulation of chloroplast transcription by SIG5 was predominantly dependent on blue light and cryptochrome. Our experiments reveal the extensive integration of signals concerning the light environment by a single sigma factor to regulate chloroplast transcription. This may originate from an evolutionarily ancient mechanism that protects photosynthetic bacteria from high light stress, which subsequently became integrated with higher plant phototransduction networks. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Mutually Exclusive Alterations in Secondary Metabolism Are Critical for the Uptake of Insoluble Iron Compounds by Arabidopsis and Medicago truncatula1[C][W

PubMed Central

Rodríguez-Celma, Jorge; Lin, Wen-Dar; Fu, Guin-Mau; Abadía, Javier; López-Millán, Ana-Flor; Schmidt, Wolfgang

2013-01-01

The generally low bioavailability of iron in aerobic soil systems forced plants to evolve sophisticated genetic strategies to improve the acquisition of iron from sparingly soluble and immobile iron pools. To distinguish between conserved and species-dependent components of such strategies, we analyzed iron deficiency-induced changes in the transcriptome of two model species, Arabidopsis (Arabidopsis thaliana) and Medicago truncatula. Transcriptional profiling by RNA sequencing revealed a massive up-regulation of genes coding for enzymes involved in riboflavin biosynthesis in M. truncatula and phenylpropanoid synthesis in Arabidopsis upon iron deficiency. Coexpression and promoter analysis indicated that the synthesis of flavins and phenylpropanoids is tightly linked to and putatively coregulated with other genes encoding proteins involved in iron uptake. We further provide evidence that the production and secretion of phenolic compounds is critical for the uptake of iron from sources with low bioavailability but dispensable under conditions where iron is readily available. In Arabidopsis, homozygous mutations in the Fe(II)- and 2-oxoglutarate-dependent dioxygenase family gene F6′H1 and defects in the expression of PLEIOTROPIC DRUG RESISTANCE9, encoding a putative efflux transporter for products from the phenylpropanoid pathway, compromised iron uptake from an iron source of low bioavailability. Both mutants were partially rescued when grown alongside wild-type Arabidopsis or M. truncatula seedlings, presumably by secreted phenolics and flavins. We concluded that production and secretion of compounds that facilitate the uptake of iron is an essential but poorly understood aspect of the reduction-based iron acquisition strategy, which is likely to contribute substantially to the efficiency of iron uptake in natural conditions. PMID:23735511

Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum

PubMed Central

2012-01-01

Background The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. Results By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. Conclusions The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation. PMID:22243621

Molecular characterization of a KIF3B-like kinesin gene in the testis of Octopus tankahkeei (Cephalopoda, Octopus).

PubMed

Dang, Ran; Zhu, Jun-Quan; Tan, Fu-Qing; Wang, Wei; Zhou, Hong; Yang, Wan-Xi

2012-05-01

KIF3B is known for maintaining and assembling cilia and flagellum. To date, the function of KIF3B and its relationship with KIF3A during spermiogenesis in the cephalopod Octopus tankahkeei remains unknown. In the present study, we characterized a gene encoding a homologue of rat KIF3B in the O. tankahkeei testis and examined its temporal and spatial expression pattern during spermiogenesis. The cDNA of KIF3B was obtained with degenerate and RACE PCR and the distribution pattern of ot-kif3b were observed with RT-PCR. The morphological development during spermiogenesis was illustrated by histological and transmission electron microscopy and mRNA expression of ot-kif3b was observed by in situ hybridization. The 2,365 nucleotides cDNA consisted of a 102 bp 5' untranslated region (UTR), a 2,208 bp open reading frame (ORF) encoding a protein of 736 amino acids, and a 55 bp 3' UTR. Multiple alignments revealed that the putative Ot-KIF3B shared 68, 68, 69, 68, and 67% identity with that of Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis, respectively, along with high identities with Ot-KIF3A in fundamental structures. Ot-kif3b transcripts appeared gradually in early spermatids, increased in intermediate spermatids and maximized in drastically remodeled and final spermatids. The kif3b gene is identified and its expression pattern is demonstrated for the first time in O. tankahkeei. Compared to ot-kif3a reported by our laboratory before, our data suggested that the putative heterodimeric motor proteins Ot-KIF3A/B may be involved in intraspermatic transport and might contribute to structural changes during spermiogenesis.

Blunt Snout Bream (Megalobrama amblycephala) MyD88 and TRAF6: Characterisation, Comparative Homology Modelling and Expression

PubMed Central

Tran, Ngoc Tuan; Liu, Han; Jakovlić, Ivan; Wang, Wei-Min

2015-01-01

MyD88 and TRAF6 play an essential role in the innate immune response in most animals. This study reports the full-length MaMyD88 and MaTRAF6 genes identified from the blunt snout bream (Megalobrama amblycephala) transcriptome profile. MaMyD88 is 2501 base pairs (bp) long, encoding a putative protein of 284 amino acids (aa), including the N-terminal DEATH domain of 78 aa and the C-terminal TIR domain of 138 aa. MaTRAF6 is 2252 bp long, encoding a putative protein of 542 aa, including the N-terminal low-complexity region, RING domain (40 aa), a coiled-coil region (64 aa) and C-terminal MATH domain (147 aa). Coding regions of MaMyD88 and MaTRAF6 genomic sequences consisted of five and six exons, respectively. Physicochemical and functional characteristics of the proteins were analysed. Alpha helices were dominant in the secondary structure of the proteins. Homology models of the MaMyD88 and MaTRAF6 domains were constructed applying the comparative modelling method. RT-qPCR was used to analyse the expression of MaMyD88 and MaTRAF6 mRNA transcripts in response to Aeromonas hydrophila challenge. Both genes were highly upregulated in the liver, spleen and kidney during the first 24 h after the challenge. While MyD88 and TRAF6 have been reported in various aquatic species, this is the first report and characterisation of these genes in blunt snout bream. This research also provides evidence of the important roles of these two genes in the blunt snout bream innate immune system. PMID:25830478

Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

PubMed

Reddy, M K; Nair, S; Singh, B N; Mudgil, Y; Tewari, K K; Sopory, S K

2001-01-24

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum.

PubMed

Schneider, Jens; Peters-Wendisch, Petra; Stansen, K Corinna; Götker, Susanne; Maximow, Stanislav; Krämer, Reinhard; Wendisch, Volker F

2012-01-13

The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation.

Molecular cloning and characterization of an acetylcholinesterase cDNA in the brown planthopper, Nilaparvata lugens.

PubMed

Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

2010-01-01

A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain.

cDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

1999-05-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

cDNA encoding a polypeptide including a hev ein sequence

DOEpatents

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

2000-07-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

cDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

1999-05-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

CDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

1995-03-21

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

Leukocyte common antigen-related phosphatase (LRP) gene structure: Conservation of the genomic organization of transmembrane protein tyrosine phosphatases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wong, E.C.C.; Mullersman, J.E.; Thomas, M.L.

1993-07-01

The leukocyte common antigen-related protein tyrosine phosphatase (LRP) is a widely expressed transmembrane glycoprotein thought to be involved in cell growth and differentiation. Similar to most other transmembrane protein tyrosine phosphatases, LRP contains two tandem cytoplasmic phosphatase domains. To understand further the regulation and evolution of LRP, the authors have isolated and characterized mouse [lambda] genomic clones. Thirteen genomic clones could be divided into two non-overlapping clusters. The first cluster contained the transcription initiation site and the exon encoding most of the 5[prime] untranslated region. The second cluster contained the remaining exons encoding the protein and the 3[prime] untranslated region.more » The gene consists of 22 exons spanning over 75 kb. The distance between exon 1 and exon 2 is at least 25 kb. Characterization of the 5[prime] ends of LRP mRNA by S1 nuclease protection identifies putative initiation start sites within a G/C-rich region. The upstream region does not contain a TATA box. Comparison of the LRP gene structure to the mammalian protein tyrosine phosphatase gene, CD45, shows striking similarities in size and genomic organization. 29 refs., 5 figs., 1 tab.« less

cDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

1995-03-21

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

Identification of avian wax synthases

PubMed Central

2012-01-01

Background Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS) which have been identified in pro- and eukaryotic organisms. Results Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands. Conclusions We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities. PMID:22305293

The major facilitator superfamily transporter Knq1p modulates boron homeostasis in Kluyveromyces lactis.

PubMed

Svrbicka, Alexandra; Toth Hervay, Nora; Gbelska, Yvetta

2016-03-01

Boron is an essential micronutrient for living cells, yet its excess causes toxicity. To date, the mechanisms of boron toxicity are poorly understood. Recently, the ScATR1 gene has been identified encoding the main boron efflux pump in Saccharomyces cerevisiae. In this study, we analyzed the ScATR1 ortholog in Kluyveromyces lactis--the KNQ1 gene, to understand whether it participates in boron stress tolerance. We found that the KNQ1 gene, encoding a permease belonging to the major facilitator superfamily, is required for K. lactis boron tolerance. Deletion of the KNQ1 gene led to boron sensitivity and its overexpression increased K. lactis boron tolerance. The KNQ1 expression was induced by boron and the intracellular boron concentration was controlled by Knq1p. The KNQ1 promoter contains two putative binding motifs for the AP-1-like transcription factor KlYap1p playing a central role in oxidative stress defense. Our results indicate that the induction of the KNQ1 expression requires the presence of KlYap1p and that Knq1p like its ortholog ScAtr1p in S. cerevisiae functions as a boron efflux pump providing boron resistance in K. lactis.

A Zn(II)2Cys6 DNA binding protein regulates the sirodesmin PL biosynthetic gene cluster in Leptosphaeria maculans

PubMed Central

Fox, Ellen M.; Gardiner, Donald M.; Keller, Nancy P.; Howlett, Barbara J.

2008-01-01

A gene, sirZ, encoding a Zn(II)2Cys6 DNA binding protein is present in a cluster of genes responsible for the biosynthesis of the epipolythiodioxopiperazine (ETP) toxin, sirodesmin PL in the ascomycete plant pathogen, Leptosphaeria maculans. RNA-mediated silencing of sirZ gives rise to transformants that produce only residual amounts of sirodesmin PL and display a decrease in the transcription of several sirodesmin PL biosynthetic genes. This indicates that SirZ is a major regulator of this gene cluster. Proteins similar to SirZ are encoded in the gliotoxin biosynthetic gene cluster of Aspergillus fumigatus (gliZ) and in an ETP-like cluster in Penicillium lilacinoechinulatum (PlgliZ). Despite its high level of sequence similarity to gliZ, PlgliZ is unable to complement the gliotoxin-deficiency of a mutant of gliZ in A. fumigatus. Putative binding sites for these regulatory proteins in the promoters of genes in these clusters were predicted using bioinformatic analysis. These sites are similar to those commonly bound by other proteins with Zn(II)2Cys6 DNA binding domains. PMID:18023597

The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation.

PubMed

Henry, Romain; Bruneau, Emmanuelle; Gardan, Rozenn; Bertin, Stéphane; Fleuchot, Betty; Decaris, Bernard; Leblond-Bourget, Nathalie

2011-10-07

Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

Evolution of the nuclear receptor gene superfamily.

PubMed Central

Laudet, V; Hänni, C; Coll, J; Catzeflis, F; Stéhelin, D

1992-01-01

Nuclear receptor genes represent a large family of genes encoding receptors for various hydrophobic ligands such as steroids, vitamin D, retinoic acid and thyroid hormones. This family also contains genes encoding putative receptors for unknown ligands. Nuclear receptor gene products are composed of several domains important for transcriptional activation, DNA binding (C domain), hormone binding and dimerization (E domain). It is not known whether these genes have evolved through gene duplication from a common ancestor or if their different domains came from different independent sources. To test these possibilities we have constructed and compared the phylogenetic trees derived from two different domains of 30 nuclear receptor genes. The tree built from the DNA binding C domain clearly shows a common progeny of all nuclear receptors, which can be grouped into three subfamilies: (i) thyroid hormone and retinoic acid receptors, (ii) orphan receptors and (iii) steroid hormone receptors. The tree constructed from the central part of the E domain which is implicated in transcriptional regulation and dimerization shows the same distribution in three subfamilies but two groups of receptors are in a different position from that in the C domain tree: (i) the Drosophila knirps family genes have acquired very different E domains during evolution, and (ii) the vitamin D and ecdysone receptors, as well as the FTZ-F1 and the NGF1B genes, seem to have DNA binding and hormone binding domains belonging to different classes. These data suggest a complex evolutionary history for nuclear receptor genes in which gene duplication events and swapping between domains of different origins took place. PMID:1312460

Molecular cloning and transcriptional analysis of a NPY receptor-like in common Chinese cuttlefish Sepiella japonica

NASA Astrophysics Data System (ADS)

Yang, Jingwen; Xu, Yuchao; Xu, Ke; Ping, Hongling; Shi, Huilai; Lü, Zhenming; Wu, Changwen; Wang, Tianming

2017-08-01

Neuropeptide Y (NPY) has a pivotal role in the regulation of many physiological processes. In this study, the gene encoding a NPY receptor-like from the common Chinese cuttlefish Sepiella japonica (SjNPYR-like) was identified and characterized. The full-length SjNPYR-like cDNA was cloned containing a 492-bp of 5' untranslated region (UTR), 1 182 bp open reading frame (ORF) encoding a protein of 393 amino acid residues, and 228 bp of 3' UTR. The putative protein was predicted to have a molecular weight of 45.54 kDa and an isoelectric point (pI) of 8.13. By informatic analyses, SjNPYR-like was identified as belonging to the class A G protein coupled receptor (GPCR) family (the rhodopsin-type). The amino acid sequence contained 12 potential phosphorylation sites and five predicted N-linked glycosylation sites. Multiple sequence alignment and 3D structure modeling were conducted to clarify SjNPYR bioinformatics characteristics. Phylogenetic analysis identifies it as an NPYR with identity of 33% to Lymnaea stagnalis NPFR. Transmembrane properties of SjNPYR-like were demonstrated in vitro using HEK293 cells and the pEGFP-N1 plasmid. Relative quantification of SjNPYR-like mRNA level confirmed a high level expression and broad distribution of SjNPYR - like in various tissues of female S. japonica. In addition, the transcriptional profile of SjNPYR - like in the brain, liver, and ovary during gonadal development was analyzed. The results provide basic understanding on the molecular characteristics of SjNPYR-like and its potentially physical functions.

Evolution of multicomponent pheromone signals in small ermine moths involves a single fatty-acyl reductase gene

PubMed Central

Liénard, Marjorie A.; Hagström, Åsa K.; Lassance, Jean-Marc; Löfstedt, Christer

2010-01-01

Fatty-acyl CoA reductases (FAR) convert fatty acids into fatty alcohols in pro- and eukaryotic organisms. In the Lepidoptera, members of the FAR gene family serve in the biosynthesis of sex pheromones involved in mate communication. We used a group of closely related species, the small ermine moths (Lepidoptera: Yponomeutidae) as a model to investigate the role of FARs in the biosynthesis of complex pheromone blends. Homology-based molecular cloning in three Yponomeuta species led to the identification of multiple putative FAR transcripts homologous to FAR genes from the Bombyx mori genome. The expression of one transcript was restricted to the female pheromone-gland tissue, suggesting a role in pheromone biosynthesis, and the encoded protein belonged to a recently identified Lepidoptera-specific pgFAR gene subfamily. The Yponomeuta evonymellus pgFAR mRNA was up-regulated in sexually mature females and exhibited a 24-h cyclic fluctuation pattern peaking in the pheromone production period. Heterologous expression confirmed that the Yponomeuta pgFAR orthologs in all three species investigated [Y. evonymellus (L.), Yponomeuta padellus (L.), and Yponomeuta rorellus (Hübner)] encode a functional FAR with a broad substrate range that efficiently promoted accumulation of primary alcohols in recombinant yeast supplied with a series of biologically relevant C14- or C16-acyl precursors. Taken together, our data evidence that a single alcohol-producing pgFAR played a critical function in the production of the multicomponent pheromones of yponomeutids and support the hypothesis of moth pheromone-biosynthetic FARs belonging to a FAR gene subfamily unique to Lepidoptera. PMID:20534481

Expression, regulation and functional assessment of the 80 amino acid Small Adipocyte Factor 1 (Smaf1) protein in adipocytes.

PubMed

Ren, Gang; Eskandari, Parisa; Wang, Siqian; Smas, Cynthia M

2016-01-15

The gene for Small Adipocyte Factor 1, Smaf1 (also known as adipogenin, ADIG), encodes a ∼600 base transcript that is highly upregulated during 3T3-L1 in vitro adipogenesis and markedly enriched in adipose tissues. Based on the lack of an obvious open reading frame in the Smaf1 transcript, it is not known if the Smaf1 gene is protein coding or non-coding RNA. Using a peptide from a putative open reading frame of Smaf1 as antigen, we generated antibodies for western analysis. Our studies prove that Smaf1 encodes an adipose-enriched protein which in western blot analysis migrates at ∼10 kDa. Rapid induction of Smaf1 protein occurs during in vitro adipogenesis and its expression in 3T3-L1 adipocytes is positively regulated by insulin and glucose. Moreover, siRNA studies reveal that expression of Smaf1 in adipocytes is wholly dependent on PPARγ. On the other hand, use of siRNA for Smaf1 to nearly abolish its protein expression in adipocytes revealed that Smaf1 does not have a major role in adipocyte triglyceride accumulation, lipolysis or insulin-stimulated pAkt induction. However, immunolocalization studies using HA-tagged Smaf1 reveal enrichment at adipocyte lipid droplets. Together our findings show that Smaf1 is a novel small protein endogenous to adipocytes and that Smaf1 expression is closely tied to PPARγ-mediated signals and the adipocyte phenotype. Copyright © 2015 Elsevier Inc. All rights reserved.

The 20-hydroxyecdysone-induced signalling pathway in G2/M arrest of Plodia interpunctella imaginal wing cells.

PubMed

Siaussat, David; Bozzolan, Françoise; Porcheron, Patrick; Debernard, Stéphane

2008-05-01

The mechanisms involved in the control of cellular proliferation by the steroid hormone 20-hydroxyecdysone (20E) in insects are not known. We dissected the 20E signalling pathway responsible for G2/M arrest of imaginal cells from the IAL-PID2 cells of the Indian meal moth Plodia interpunctella. We first used a 5'-3' RACE-based strategy to clone a 4479bp cDNA encoding a putative P. interpunctella HR3 transcription factor named PiHR3. The deduced amino acid sequence of PiHR3 was highly similar to those of HR3 proteins from other lepidopterans, e.g. Manduca sexta and Bombyx mori. Using double-stranded RNA-mediated interference (dsRNAi), we then succeeded in blocking the ability of 20E to induce the expression of PiEcR-B1, PiUSP-2 and PiHR3 genes that encode the P. interpunctella ecdysone receptor B1-isoform, Ultraspiracle-2 isoform, the insect homologue of the vertebrate retinoid X receptor, and the HR3 transcription factor. We showed that inhibiting the 20E induction of PiEcR-B1, PiUSP-2 and PiHR3 mRNAs prevented the decreased expression of B cyclin and consequently the G2/M arrest of IAL-PID2 cells. Using this functional approach, we revealed the participation of EcR, USP and HR3 in a 20E signalling pathway that controls the proliferation of imaginal cells by regulating the expression of B cyclin.

Cloning and characterization of an alternatively spliced gene in proximal Xq28 deleted in two patients with intersexual genitalia and myotubular myopathy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laporte, J.; Hu, Ling-Jia; Kretz, C.

1997-05-01

We have identified a novel human gene that is entirely deleted in two boys with abnormal genital development and myotubular myopathy (MTM1). The gene, F18, is located in proximal Xq28, approximately 80 kb centromeric to the recently isolated MTM1 gene. Northern analysis of mRNA showed a ubiquitous pattern and suggested high levels of expression in skeletal muscle, brain, and heart. A transcript of 4.6 kb was detected in a range of tissues, and additional alternate forms of 3.8 and 2.6 kb were present in placenta and pancreas, respectively. The gene extends over 100 kb and is composed of at leastmore » seven exons, of which two are non-coding. Sequence analysis of a 4.6-kb cDNA contig revealed two overlapping open reading frames (ORFs) that encode putative proteins of 701 and 424 amino acids, respectively. Two alternative spliced transcripts affecting the large open reading frame were identified that, together with the Northern blot results, suggest that distinct proteins are derived from the gene. No significant homology to other known proteins was detected, but segments of the first ORF encode polyglutamine tracts and proline-rich domains, which are frequently observed in DNA-binding proteins. The F18 gene is a strong candidate for being implicated in the intersexual genitalia present in the two MTM1-deleted patients. The gene also serves as a candidate for other disorders that map to proximal Xq28. 15 refs., 3 figs., 1 tab.« less

The LuWD40-1 gene encoding WD repeat protein regulates growth and pollen viability in flax (Linum Usitatissimum L.).

PubMed

Kumar, Santosh; Jordan, Mark C; Datla, Raju; Cloutier, Sylvie

2013-01-01

As a crop, flax holds significant commercial value for its omega-3 rich oilseeds and stem fibres. Canada is the largest producer of linseed but there exists scope for significant yield improvements. Implementation of mechanisms such as male sterility can permit the development of hybrids to assist in achieving this goal. Temperature sensitive male sterility has been reported in flax but the leakiness of this system in field conditions limits the production of quality hybrid seeds. Here, we characterized a 2,588 bp transcript differentially expressed in male sterile lines of flax. The twelve intron gene predicted to encode a 368 amino acid protein has five WD40 repeats which, in silico, form a propeller structure with putative nucleic acid and histone binding capabilities. The LuWD40-1 protein localized to the nucleus and its expression increased during the transition and continued through the vegetative stages (seed, etiolated seedling, stem) while the transcript levels declined during reproductive development (ovary, anthers) and embryonic morphogenesis of male fertile plants. Knockout lines for LuWD40-1 in flax failed to develop shoots while overexpression lines showed delayed growth phenotype and were male sterile. The non-viable flowers failed to open and the pollen grains from these flowers were empty. Three independent transgenic lines overexpressing the LuWD40-1 gene had ∼80% non-viable pollen, reduced branching, delayed flowering and maturity compared to male fertile genotypes. The present study provides new insights into a male sterility mechanism present in flax.

Gene expansion shapes genome architecture in the human pathogen Lichtheimia corymbifera: an evolutionary genomics analysis in the ancient terrestrial mucorales (Mucoromycotina).

PubMed

Schwartze, Volker U; Winter, Sascha; Shelest, Ekaterina; Marcet-Houben, Marina; Horn, Fabian; Wehner, Stefanie; Linde, Jörg; Valiante, Vito; Sammeth, Michael; Riege, Konstantin; Nowrousian, Minou; Kaerger, Kerstin; Jacobsen, Ilse D; Marz, Manja; Brakhage, Axel A; Gabaldón, Toni; Böcker, Sebastian; Voigt, Kerstin

2014-08-01

Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1-4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparison to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae.

Gene Expansion Shapes Genome Architecture in the Human Pathogen Lichtheimia corymbifera: An Evolutionary Genomics Analysis in the Ancient Terrestrial Mucorales (Mucoromycotina)

PubMed Central

Wehner, Stefanie; Linde, Jörg; Valiante, Vito; Sammeth, Michael; Riege, Konstantin; Nowrousian, Minou; Kaerger, Kerstin; Jacobsen, Ilse D.; Marz, Manja; Brakhage, Axel A.; Gabaldón, Toni; Böcker, Sebastian; Voigt, Kerstin

2014-01-01

Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1–4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparision to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae. PMID:25121733

The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes.

PubMed

Stout, Jake M; Boubakir, Zakia; Ambrose, Stephen J; Purves, Randy W; Page, Jonathan E

2012-08-01

The psychoactive and analgesic cannabinoids (e.g. Δ(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids. © 2012 National Research Council of Canada. The Plant Journal © 2012 Blackwell Publishing Ltd.

A Genomic View of the Sea Urchin Nervous System

PubMed Central

Burke, RD; Angerer, LM; Elphick, MR; Humphrey, GW; Yaguchi, S; Kiyama, T; Liang, S; Mu, X; Agca, C; Klein, WH; Brandhorst, BP; Rowe, M; Wilson, K; Churcher, AM; Taylor, JS; Chen, N; Murray, G; Wang, D; Mellott, D; Olinski, R; Hallböök, F; Thorndyke, MC

2007-01-01

The sequencing of the Strongylocentrotus purpuratus genome provides a unique opportunity to investigate the function and evolution of neural genes. The neurobiology of sea urchins is of particular interest because they have a close phylogenetic relationship with chordates, yet a distinctive pentaradiate body plan and unusual neural organization. Orthologues of transcription factors that regulate neurogenesis in other animals have been identified and several are expressed in neurogenic domains before gastrulation indicating that they may operate near the top of a conserved neural gene regulatory network. A family of genes encoding voltage-gated ion channels is present but, surprisingly, genes encoding gap junction proteins (connexins and pannexins) appear to be absent. Genes required for synapse formation and function have been identified and genes for synthesis and transport of neurotransmitters are present. There is a large family of G-protein-coupled receptors, including 874 rhodopsin-type receptors, 28 metabotropic glutamate-like receptors and a remarkably expanded group of 161 secretin receptor-like proteins. Absence of cannabinoid, lysophospholipid and melanocortin receptors indicates that this group may be unique to chordates. There are at least 37 putative G-protein coupled peptide receptors and precursors for several neuropeptides and peptide hormones have been identified, including SALMFamides, NGFFFamide, a vasotocin-like peptide, glycoprotein hormones, and insulin/insulin-like growth factors. Identification of a neurotrophin-like gene and Trk receptor in sea urchin indicates that this neural signaling system is not unique to chordates. Several hundred chemoreceptor genes have been predicted using several approaches, a number similar to that for other animals. Intriguingly, genes encoding homologues of rhodopsin, Pax6 and several other key mammalian retinal transcription factors are expressed in tube feet, suggesting tube feet function as photosensory organs. Analysis of the sea urchin genome presents a unique perspective on the evolutionary history of deuterostome nervous systems and reveals new approaches to investigate the development and neurobiology of sea urchins. PMID:16965768

Gene encoding prolactin in cinnamon clownfish Amphiprion melanopus and its expression upon acclimation to low salinities

PubMed Central

2013-01-01

Background Prolactin (PRL) is a key hormone for osmoregulation in fish. Levels of PRL in the pituitary gland and plasma ion composition of clownfish seem to change to regulate their hydromineral balance during adaptation to waters of different salinities. In order to understand osmoregulatory mechanism and its association with growth performance and PRL in fish, the gene encoding PRL and its expression level in cinnamon clownfish Amphiprion melanopus upon acclimation to low salinity was analyzed. Results The PRL gene of A. melanopus encoded a protein of 212 amino acid residues comprised of a putative signal peptide of 24 amino acids and a mature protein of 188 amino acids. Analysis of growth performance under different salinities of 34, 25, 15, and 10 ppt indicated that cinnamon clownfish could survive under salinities as low as 10 ppt. A higher rate of growth was observed at the lower salinities as compared to that of 34 ppt. Upon shifting the salinity of the surrounding water from 34 ppt to 15 ppt, the level of the PRL transcripts gradually increased to reach the peak level until 24 h of acclimation at 15 ppt, but decreased back as adaptation continued to 144 h. In contrast, levels of plasma Na+, Cl-, and osmolality decreased at the initial stage (4–8 h) of acclimation at 15 pt but increased back as adaptation continued till 144 h. Conclusion Cinnamon clownfish could survive under salinities as low as 10 ppt. Upon shifting the salinity of the surrounding water from 34 ppt to 15 ppt, the level of the PRL transcripts gradually increased during the initial stage of acclimation but decreased back to the normal level as adaptation continued. An opposite pattern of changes - decrease at the beginning followed by an increase - in the levels of plasma Na+, Cl-, and osmolality was found upon acclimation to low salinity. The results suggest an involvement of PRL in the processes of osmoregulation and homeostasis in A. melanopus. PMID:23276106

Gene encoding prolactin in cinnamon clownfish Amphiprion melanopus and its expression upon acclimation to low salinities.

PubMed

Noh, Gyeong Eon; Rho, Sum; Chang, Yong Jin; Min, Byung Hwa; Kim, Jong-Myoung

2013-01-01

Prolactin (PRL) is a key hormone for osmoregulation in fish. Levels of PRL in the pituitary gland and plasma ion composition of clownfish seem to change to regulate their hydromineral balance during adaptation to waters of different salinities. In order to understand osmoregulatory mechanism and its association with growth performance and PRL in fish, the gene encoding PRL and its expression level in cinnamon clownfish Amphiprion melanopus upon acclimation to low salinity was analyzed. The PRL gene of A. melanopus encoded a protein of 212 amino acid residues comprised of a putative signal peptide of 24 amino acids and a mature protein of 188 amino acids. Analysis of growth performance under different salinities of 34, 25, 15, and 10 ppt indicated that cinnamon clownfish could survive under salinities as low as 10 ppt. A higher rate of growth was observed at the lower salinities as compared to that of 34 ppt. Upon shifting the salinity of the surrounding water from 34 ppt to 15 ppt, the level of the PRL transcripts gradually increased to reach the peak level until 24 h of acclimation at 15 ppt, but decreased back as adaptation continued to 144 h. In contrast, levels of plasma Na+, Cl-, and osmolality decreased at the initial stage (4-8 h) of acclimation at 15 pt but increased back as adaptation continued till 144 h. Cinnamon clownfish could survive under salinities as low as 10 ppt. Upon shifting the salinity of the surrounding water from 34 ppt to 15 ppt, the level of the PRL transcripts gradually increased during the initial stage of acclimation but decreased back to the normal level as adaptation continued. An opposite pattern of changes - decrease at the beginning followed by an increase - in the levels of plasma Na+, Cl-, and osmolality was found upon acclimation to low salinity. The results suggest an involvement of PRL in the processes of osmoregulation and homeostasis in A. melanopus.

Microarray and differential display identify genes involved in jasmonate-dependent anther development.

PubMed

Mandaokar, Ajin; Kumar, V Dinesh; Amway, Matt; Browse, John

2003-07-01

Jasmonate (JA) is a signaling compound essential for anther development and pollen fertility in Arabidopsis. Mutations that block the pathway of JA synthesis result into male sterility. To understand the processes of anther and pollen maturation, we used microarray and differential display approaches to compare gene expression pattern in anthers of wild-type Arabidopsis and the male-sterile mutant, opr3. Microarray experiment revealed 25 genes that were up-regulated more than 1.8-fold in wild-type anthers as compared to mutant anthers. Experiments based on differential display identified 13 additional genes up-regulated in wild-type anthers compared to opr3 for a total of 38 differentially expressed genes. Searches of the Arabidopsis and non-redundant databases disclosed known or likely functions for 28 of the 38 genes identified, while 10 genes encode proteins of unknown function. Northern blot analysis of eight representative clones as probes confirmed low expression in opr3 anthers compared with wild-type anthers. JA responsiveness of these same genes was also investigated by northern blot analysis of anther RNA isolated from wild-type and opr3 plants, In these experiments, four genes were induced in opr3 anthers within 0.5-1 h of JA treatment while the remaining genes were up-regulated only 1-8 h after JA application. None of these genes was induced by JA in anthers of the coil mutant that is deficient in JA responsiveness. The four early-induced genes in opr3 encode lipoxygenase, a putative bHLH transcription factor, epithiospecifier protein and an unknown protein. We propose that these and other early components may be involved in JA signaling and in the initiation of developmental processes. The four late genes encode an extensin-like protein, a peptide transporter and two unknown proteins, which may represent components required later in anther and pollen maturation. Transcript profiling has provided a successful approach to identify genes involved in anther and pollen maturation in Arabidopsis.

A New Set of ESTs from Chickpea (Cicer arietinum L.) Embryo Reveals Two Novel F-Box Genes, CarF-box_PP2 and CarF-box_LysM, with Potential Roles in Seed Development

PubMed Central

Gupta, Shefali; Garg, Vanika; Bhatia, Sabhyata

2015-01-01

Considering the economic importance of chickpea (C. arietinum L.) seeds, it is important to understand the mechanisms underlying seed development for which a cDNA library was constructed from 6 day old chickpea embryos. A total of 8,186 ESTs were obtained from which 4,048 high quality ESTs were assembled into 1,480 unigenes that majorly encoded genes involved in various metabolic and regulatory pathways. Of these, 95 ESTs were found to be involved in ubiquitination related protein degradation pathways and 12 ESTs coded specifically for putative F-box proteins. Differential transcript accumulation of these putative F-box genes was observed in chickpea tissues as evidenced by quantitative real-time PCR. Further, to explore the role of F-box proteins in chickpea seed development, two F-box genes were selected for molecular characterization. These were named as CarF-box_PP2 and CarF-box_LysM depending on their C-terminal domains, PP2 and LysM, respectively. Their highly conserved structures led us to predict their target substrates. Subcellular localization experiment revealed that CarF-box_PP2 was localized in the cytoplasm and CarF-box_LysM was localized in the nucleus. We demonstrated their physical interactions with SKP1 protein, which validated that they function as F-box proteins in the formation of SCF complexes. Sequence analysis of their promoter regions revealed certain seed specific cis-acting elements that may be regulating their preferential transcript accumulation in the seed. Overall, the study helped in expanding the EST database of chickpea, which was further used to identify two novel F-box genes having a potential role in seed development. PMID:25803812

Genomic characterisation of Wongabel virus reveals novel genes within the Rhabdoviridae.

PubMed

Gubala, Aneta J; Proll, David F; Barnard, Ross T; Cowled, Chris J; Crameri, Sandra G; Hyatt, Alex D; Boyle, David B

2008-06-20

Viruses belonging to the family Rhabdoviridae infect a variety of different hosts, including insects, vertebrates and plants. Currently, there are approximately 200 ICTV-recognised rhabdoviruses isolated around the world. However, the majority remain poorly characterised and only a fraction have been definitively assigned to genera. The genomic and transcriptional complexity displayed by several of the characterised rhabdoviruses indicates large diversity and complexity within this family. To enable an improved taxonomic understanding of this family, it is necessary to gain further information about the poorly characterised members of this family. Here we present the complete genome sequence and predicted transcription strategy of Wongabel virus (WONV), a previously uncharacterised rhabdovirus isolated from biting midges (Culicoides austropalpalis) collected in northern Queensland, Australia. The 13,196 nucleotide genome of WONV encodes five typical rhabdovirus genes N, P, M, G and L. In addition, the WONV genome contains three genes located between the P and M genes (U1, U2, U3) and two open reading frames overlapping with the N and G genes (U4, U5). These five additional genes and their putative protein products appear to be novel, and their functions are unknown. Predictive analysis of the U5 gene product revealed characteristics typical of viroporins, and indicated structural similarities with the alpha-1 protein (putative viroporin) of viruses in the genus Ephemerovirus. Phylogenetic analyses of the N and G proteins of WONV indicated closest similarity with the avian-associated Flanders virus; however, the genomes of these two viruses are significantly diverged. WONV displays a novel and unique genome structure that has not previously been described for any animal rhabdovirus.

Ectoparasitic growth of Magnaporthe on barley triggers expression of the putative barley wax biosynthesis gene CYP96B22 which is involved in penetration resistance

PubMed Central

2014-01-01

Background Head blast caused by the fungal plant pathogen Magnaporthe oryzae is an upcoming threat for wheat and barley cultivation. We investigated the nonhost response of barley to an isolate of the Magnaporthe species complex which is pathogenic on Pennisetum spp. as a potential source for novel resistance traits. Results Array experiments identified a barley gene encoding a putative cytochrome P450 monooxygenase whose transcripts accumulate to a higher concentration in the nonhost as compared to the host interaction. The gene clusters within the CYP96 clade of the P450 plant gene family and is designated as CYP96B22. Expression of CYP96B22 was triggered during the ectoparasitic growth of the pathogen on the outside of the leaf. Usage of a fungicidal treatment and a Magnaporthe mutant confirmed that penetration was not necessary for this early activation of CYP96B22. Transcriptional silencing of CYP96B22 using Barley stripe mosaic virus led to a decrease in penetration resistance of barley plants to Magnaporthe host and nonhost isolates. This phenotype seems to be specific for the barley-Magnaporthe interaction, since penetration of the adapted barley powdery mildew fungus was not altered in similarly treated plants. Conclusion Taken together our results suggest a cross-talk between barley and Magnaporthe isolates across the plant surface. Since members of the plant CYP96 family are known to be involved in synthesis of epicuticular waxes, these substances or their derivatives might act as signal components. We propose a functional overlap of CYP96B22 in the execution of penetration resistance during basal and nonhost resistance of barley against different Magnaporthe species. PMID:24423145

Molecular Characterization of the Llamas (Lama glama) Casein Cluster Genes Transcripts (CSN1S1, CSN2, CSN1S2, CSN3) and Regulatory Regions

PubMed Central

Pauciullo, Alfredo; Erhardt, Georg

2015-01-01

In the present paper, we report for the first time the characterization of llama (Lama glama) caseins at transcriptomic and genetic level. A total of 288 casein clones transcripts were analysed from two lactating llamas. The most represented mRNA populations were those correctly assembled (85.07%) and they encoded for mature proteins of 215, 217, 187 and 162 amino acids respectively for the CSN1S1, CSN2, CSN1S2 and CSN3 genes. The exonic subdivision evidenced a structure made of 21, 9, 17 and 6 exons for the αs1-, β-, αs2- and κ-casein genes respectively. Exon skipping and duplication events were evidenced. Two variants A and B were identified in the αs1-casein gene as result of the alternative out-splicing of the exon 18. An additional exon coding for a novel esapeptide was found to be cryptic in the κ-casein gene, whereas one extra exon was found in the αs2-casein gene by the comparison with the Camelus dromedaries sequence. A total of 28 putative phosphorylated motifs highlighted a complex heterogeneity and a potential variable degree of post-translational modifications. Ninety-six polymorphic sites were found through the comparison of the lama casein cDNAs with the homologous camel sequences, whereas the first description and characterization of the 5’- and 3’-regulatory regions allowed to identify the main putative consensus sequences involved in the casein genes expression, thus opening the way to new investigations -so far- never achieved in this species. PMID:25923814

Molecular Characterization of the Llamas (Lama glama) Casein Cluster Genes Transcripts (CSN1S1, CSN2, CSN1S2, CSN3) and Regulatory Regions.

PubMed

Pauciullo, Alfredo; Erhardt, Georg

2015-01-01

In the present paper, we report for the first time the characterization of llama (Lama glama) caseins at transcriptomic and genetic level. A total of 288 casein clones transcripts were analysed from two lactating llamas. The most represented mRNA populations were those correctly assembled (85.07%) and they encoded for mature proteins of 215, 217, 187 and 162 amino acids respectively for the CSN1S1, CSN2, CSN1S2 and CSN3 genes. The exonic subdivision evidenced a structure made of 21, 9, 17 and 6 exons for the αs1-, β-, αs2- and κ-casein genes respectively. Exon skipping and duplication events were evidenced. Two variants A and B were identified in the αs1-casein gene as result of the alternative out-splicing of the exon 18. An additional exon coding for a novel esapeptide was found to be cryptic in the κ-casein gene, whereas one extra exon was found in the αs2-casein gene by the comparison with the Camelus dromedaries sequence. A total of 28 putative phosphorylated motifs highlighted a complex heterogeneity and a potential variable degree of post-translational modifications. Ninety-six polymorphic sites were found through the comparison of the lama casein cDNAs with the homologous camel sequences, whereas the first description and characterization of the 5'- and 3'-regulatory regions allowed to identify the main putative consensus sequences involved in the casein genes expression, thus opening the way to new investigations -so far- never achieved in this species.

Analysis of high iron rice lines reveals new miRNAs that target iron transporters in roots

PubMed Central

Paul, Soumitra; Gayen, Dipak; Datta, Swapan K.; Datta, Karabi

2016-01-01

The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants . Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds. PMID:27729476

Discovery of numerous novel small genes in the intergenic regions of the Escherichia coli O157:H7 Sakai genome

PubMed Central

Hücker, Sarah M.; Ardern, Zachary; Goldberg, Tatyana; Schafferhans, Andrea; Bernhofer, Michael; Vestergaard, Gisle; Nelson, Chase W.; Schloter, Michael; Rost, Burkhard; Scherer, Siegfried

2017-01-01

In the past, short protein-coding genes were often disregarded by genome annotation pipelines. Transcriptome sequencing (RNAseq) signals outside of annotated genes have usually been interpreted to indicate either ncRNA or pervasive transcription. Therefore, in addition to the transcriptome, the translatome (RIBOseq) of the enteric pathogen Escherichia coli O157:H7 strain Sakai was determined at two optimal growth conditions and a severe stress condition combining low temperature and high osmotic pressure. All intergenic open reading frames potentially encoding a protein of ≥ 30 amino acids were investigated with regard to coverage by transcription and translation signals and their translatability expressed by the ribosomal coverage value. This led to discovery of 465 unique, putative novel genes not yet annotated in this E. coli strain, which are evenly distributed over both DNA strands of the genome. For 255 of the novel genes, annotated homologs in other bacteria were found, and a machine-learning algorithm, trained on small protein-coding E. coli genes, predicted that 89% of these translated open reading frames represent bona fide genes. The remaining 210 putative novel genes without annotated homologs were compared to the 255 novel genes with homologs and to 250 short annotated genes of this E. coli strain. All three groups turned out to be similar with respect to their translatability distribution, fractions of differentially regulated genes, secondary structure composition, and the distribution of evolutionary constraint, suggesting that both novel groups represent legitimate genes. However, the machine-learning algorithm only recognized a small fraction of the 210 genes without annotated homologs. It is possible that these genes represent a novel group of genes, which have unusual features dissimilar to the genes of the machine-learning algorithm training set. PMID:28902868

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez, Diego; Challacombe, Jean; Morgenstern, Ingo

2009-02-04

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in media containing cellulose as sole carbon source, transcripts corresponding tomore » many hemicellulases and to a single putative β-1-4 endoglucanase were expressed at high levels relative to glucose grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also upregulated under cellulolytic culture conditions were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. In particular, comparisons between P. placenta and the closely related white-rot fungus, Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which efficient depolymerization of lignin was lost.« less

Genome, transcriptome, and secretome analysis of wood decay fungus postia placenta supports unique mechanisms of lignocellulose conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez, Diego; Challacombe, Jean F; Misra, Monica

2008-01-01

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding tomore » many hemicellulases and to a single putative {beta}-1-4 endoglucanase were expressed at high levels relative to glucose grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC{center_dot}MSIMS). Also upregulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H202. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H202 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons to the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.« less

Isolation and characterisation of a dwarf rice mutant exhibiting defective gibberellins biosynthesis.

PubMed

Ji, S H; Gururani, M A; Lee, J W; Ahn, B-O; Chun, S-C

2014-03-01

We have isolated a severe dwarf mutant derived from a Ds (Dissociation) insertion mutant rice (Oryza sativa var. japonica c.v. Dongjin). This severe dwarf phenotype, has short and dark green leaves, reduced shoot growth early in the seedling stage, and later severe dwarfism with failure to initiate flowering. When treated with bioactive GA3 , mutants are restored to the normal wild-type phenotype. Reverse transcription PCR analyses of 22 candidate genes related to the gibberellin (GA) biosynthesis pathway revealed that among 22 candidate genes tested, a dwarf mutant transcript was not expressed only in one OsKS2 gene. Genetic analysis revealed that the severe dwarf phenotype was controlled by recessive mutation of a single nuclear gene. The putative OsKS2 gene was a chromosome 4-located ent-kaurene synthase (KS), encoding the enzyme that catalyses an early step of the GA biosynthesis pathway. Sequence analysis revealed that osks2 carried a 1-bp deletion in the ORF region of OsKS2, which led to a loss-of-function mutation. The expression pattern of OsKS2 in wild-type cv Dongjin, showed that it is expressed in all organs, most prominently in the stem and floral organs. Morphological characteristics of the dwarf mutant showed dramatic modifications in internal structure and external morphology. We propose that dwarfism in this mutant is caused by a point mutation in OsKS2, which plays a significant role in growth and development of higher plants. Further investigation on OsKS2 and other OsKS-like proteins is underway and may yield better understanding of the putative role of OsKS in severe dwarf mutants. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Structural and functional studies of a family of Dictyostelium discoideum developmentally regulated, prestalk genes coding for small proteins.

PubMed

Vicente, Juan J; Galardi-Castilla, María; Escalante, Ricardo; Sastre, Leandro

2008-01-03

The social amoeba Dictyostelium discoideum executes a multicellular development program upon starvation. This morphogenetic process requires the differential regulation of a large number of genes and is coordinated by extracellular signals. The MADS-box transcription factor SrfA is required for several stages of development, including slug migration and spore terminal differentiation. Subtractive hybridization allowed the isolation of a gene, sigN (SrfA-induced gene N), that was dependent on the transcription factor SrfA for expression at the slug stage of development. Homology searches detected the existence of a large family of sigN-related genes in the Dictyostelium discoideum genome. The 13 most similar genes are grouped in two regions of chromosome 2 and have been named Group1 and Group2 sigN genes. The putative encoded proteins are 87-89 amino acids long. All these genes have a similar structure, composed of a first exon containing a 13 nucleotides long open reading frame and a second exon comprising the remaining of the putative coding region. The expression of these genes is induced at10 hours of development. Analyses of their promoter regions indicate that these genes are expressed in the prestalk region of developing structures. The addition of antibodies raised against SigN Group 2 proteins induced disintegration of multi-cellular structures at the mound stage of development. A large family of genes coding for small proteins has been identified in D. discoideum. Two groups of very similar genes from this family have been shown to be specifically expressed in prestalk cells during development. Functional studies using antibodies raised against Group 2 SigN proteins indicate that these genes could play a role during multicellular development.

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion

PubMed Central

Martinez, Diego; Challacombe, Jean; Morgenstern, Ingo; Hibbett, David; Schmoll, Monika; Kubicek, Christian P.; Ferreira, Patricia; Ruiz-Duenas, Francisco J.; Martinez, Angel T.; Kersten, Phil; Hammel, Kenneth E.; Vanden Wymelenberg, Amber; Gaskell, Jill; Lindquist, Erika; Sabat, Grzegorz; Splinter BonDurant, Sandra; Larrondo, Luis F.; Canessa, Paulo; Vicuna, Rafael; Yadav, Jagjit; Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Pisabarro, Antonio G.; Lavín, José L.; Oguiza, José A.; Master, Emma; Henrissat, Bernard; Coutinho, Pedro M.; Harris, Paul; Magnuson, Jon Karl; Baker, Scott E.; Bruno, Kenneth; Kenealy, William; Hoegger, Patrik J.; Kües, Ursula; Ramaiya, Preethi; Lucas, Susan; Salamov, Asaf; Shapiro, Harris; Tu, Hank; Chee, Christine L.; Misra, Monica; Xie, Gary; Teter, Sarah; Yaver, Debbie; James, Tim; Mokrejs, Martin; Pospisek, Martin; Grigoriev, Igor V.; Brettin, Thomas; Rokhsar, Dan; Berka, Randy; Cullen, Dan

2009-01-01

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative β-1–4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost. PMID:19193860

The Oenococcus oeni clpX Homologue Is a Heat Shock Gene Preferentially Expressed in Exponential Growth Phase

PubMed Central

Jobin, Michel-Philippe; Garmyn, Dominique; Diviès, Charles; Guzzo, Jean

1999-01-01

Using degenerated primers from conserved regions of previously studied clpX gene products, we cloned the clpX gene of the malolactic bacterium Oenococcus oeni. The clpX gene was sequenced, and the deduced protein of 413 amino acids (predicted molecular mass of 45,650 Da) was highly similar to previously analyzed clpX gene products from other organisms. An open reading frame located upstream of the clpX gene was identified as the tig gene by similarity of its predicted product to other bacterial trigger factors. ClpX was purified by using a maltose binding protein fusion system and was shown to possess an ATPase activity. Northern analyses indicated the presence of two independent 1.6-kb monocistronic clpX and tig mRNAs and also showed an increase in clpX mRNA amount after a temperature shift from 30 to 42°C. The clpX transcript is abundant in the early exponential growth phase and progressively declines to undetectable levels in the stationary phase. Thus, unlike hsp18, the gene encoding one of the major small heat shock proteins of Oenococcus oeni, clpX expression is related to the exponential growth phase and requires de novo protein synthesis. Primer extension analysis identified the 5′ end of clpX mRNA which is located 408 nucleotides upstream of a putative AUA start codon. The putative transcription start site allowed identification of a predicted promoter sequence with a high similarity to the consensus sequence found in the housekeeping gene promoter of gram-positive bacteria as well as Escherichia coli. PMID:10542163

Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens.

PubMed

Rodríguez-Moya, Javier; Argandoña, Montserrat; Reina-Bueno, Mercedes; Nieto, Joaquín J; Iglesias-Guerra, Fernando; Jebbar, Mohamed; Vargas, Carmen

2010-10-13

Osmosensing and associated signal transduction pathways have not yet been described in obligately halophilic bacteria. Chromohalobacter salexigens is a halophilic bacterium with a broad range of salt tolerance. In response to osmotic stress, it synthesizes and accumulates large amounts of the compatible solutes ectoine and hydroxyectoine. In a previous work, we showed that ectoines can be also accumulated upon transport from the external medium, and that they can be used as carbon sources at optimal, but not at low salinity. This was related to an insufficient ectoine(s) transport under these conditions. A C. salexigens Tn1732-induced mutant (CHR95) showed a delayed growth with glucose at low and optimal salinities, could not grow at high salinity, and was able to use ectoines as carbon sources at low salinity. CHR95 was affected in the transport and/or metabolism of glucose, and showed a deregulated ectoine uptake at any salinity, but it was not affected in ectoine metabolism. Transposon insertion in CHR95 caused deletion of three genes, Csal0865-Csal0867: acs, encoding an acetyl-CoA synthase, mntR, encoding a transcriptional regulator of the DtxR/MntR family, and eupR, encoding a putative two-component response regulator with a LuxR_C-like DNA-binding helix-turn-helix domain. A single mntR mutant was sensitive to manganese, suggesting that mntR encodes a manganese-dependent transcriptional regulator. Deletion of eupR led to salt-sensitivity and enabled the mutant strain to use ectoines as carbon source at low salinity. Domain analysis included EupR as a member of the NarL/FixJ family of two component response regulators. Finally, the protein encoded by Csal869, located three genes downstream of eupR was suggested to be the cognate histidine kinase of EupR. This protein was predicted to be a hybrid histidine kinase with one transmembrane and one cytoplasmic sensor domain. This work represents the first example of the involvement of a two-component response regulator in the osmoadaptation of a true halophilic bacterium. Our results pave the way to the elucidation of the signal transduction pathway involved in the control of ectoine transport in C. salexigens.

Regulation of the Salmonella enterica std fimbrial operon by DNA adenine methylation, SeqA, and HdfR.

PubMed

Jakomin, Marcello; Chessa, Daniela; Bäumler, Andreas J; Casadesús, Josep

2008-11-01

DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium grown under laboratory conditions express the std fimbrial operon, which is tightly repressed in the wild type. Here, we show that uncontrolled production of Std fimbriae in S. enterica serovar Typhimurium dam mutants contributes to attenuation in mice, as indicated by the observation that an stdA dam strain is more competitive than a dam strain upon oral infection. Dam methylation appears to regulate std transcription, rather than std mRNA stability or turnover. A genetic screen for std regulators showed that the GATC-binding protein SeqA directly or indirectly represses std expression, while the poorly characterized yifA gene product serves as an std activator. YifA encodes a putative LysR-like protein and has been renamed HdfR, like its Escherichia coli homolog. Activation of std expression by HdfR is observed only in dam and seqA backgrounds. These data suggest that HdfR directly or indirectly activates std transcription. Since SeqA is unable to bind nonmethylated DNA, it is possible that std operon derepression in dam and seqA mutants may result from unconstrained HdfR-mediated activation of std transcription. Derepression of std in dam and seqA mutants of S. enterica occurs in only a fraction of the bacterial population, suggesting the occurrence of either bistable expression or phase variation.

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

PubMed

Håvik, B; Ragnhildstveit, E; Lorens, J B; Saelemyr, K; Fauske, O; Knudsen, L K; Fjose, A

1999-12-20

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences. Copyright 1999 Academic Press.

Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor

PubMed Central

Nair, Ramesh V.; Green, Edward M.; Watson, David E.; Bennett, George N.; Papoutsakis, Eleftherios T.

1999-01-01

A gene (orf1, now designated solR) previously identified upstream of the aldehyde/alcohol dehydrogenase gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 176:871–885, 1994) was found to encode a repressor of the sol locus (aad, ctfA, ctfB and adc) genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824. Primer extension analysis identified a transcriptional start site 35 bp upstream of the solR start codon. Amino acid comparisons of SolR identified a potential helix-turn-helix DNA-binding motif in the C-terminal half towards the center of the protein, suggesting a regulatory role. Overexpression of SolR in strain ATCC 824(pCO1) resulted in a solvent-negative phenotype owing to its deleterious effect on the transcription of the sol locus genes. Inactivation of solR in C. acetobutylicum via homologous recombination yielded mutants B and H (ATCC 824 solR::pO1X) which exhibited deregulated solvent production characterized by increased flux towards butanol and acetone formation, earlier induction of aad, lower overall acid production, markedly improved yields of solvents on glucose, a prolonged solvent production phase, and increased biomass accumulation compared to those of the wild-type strain. PMID:9864345

Transcriptome Analysis of Spartina pectinata in Response to Freezing Stress

PubMed Central

Nah, Gyoungju; Lee, Moonsub; Kim, Do-Soon; Rayburn, A. Lane; Voigt, Thomas; Lee, D. K.

2016-01-01

Prairie cordgrass (Spartina pectinata), a perennial C4 grass native to the North American prairie, has several distinctive characteristics that potentially make it a model crop for production in stressful environments. However, little is known about the transcriptome dynamics of prairie cordgrass despite its unique freezing stress tolerance. Therefore, the purpose of this work was to explore the transcriptome dynamics of prairie cordgrass in response to freezing stress at -5°C for 5 min and 30 min. We used a RNA-sequencing method to assemble the S. pectinata leaf transcriptome and performed gene-expression profiling of the transcripts under freezing treatment. Six differentially expressed gene (DEG) groups were categorized from the profiling. In addition, two major consecutive orders of gene expression were observed in response to freezing; the first being the acute up-regulation of genes involved in plasma membrane modification, calcium-mediated signaling, proteasome-related proteins, and transcription regulators (e.g., MYB and WRKY). The follow-up and second response was of genes involved in encoding the putative anti-freezing protein and the previously known DNA and cell-damage-repair proteins. Moreover, we identified the genes involved in epigenetic regulation and circadian-clock expression. Our results indicate that freezing response in S. pectinata reflects dynamic changes in rapid-time duration, as well as in metabolic, transcriptional, post-translational, and epigenetic regulation. PMID:27032112

Semester-long inquiry-based molecular biology laboratory: Transcriptional regulation in yeast.

PubMed

Oelkers, Peter M

2017-03-04

A single semester molecular biology laboratory has been developed in which students design and execute a project examining transcriptional regulation in Saccharomyces cerevisiae. Three weeks of planning are allocated to developing a hypothesis through literature searches and use of bioinformatics. Common experimental plans address a cell process and how three genes that encode for proteins involved in that process are transcriptionally regulated in response to changing environmental conditions. Planning includes designing oligonucleotides to amplify the putative promoters of the three genes of interest. After the PCR, each product is cloned proximal to β-galactosidase in a yeast reporter plasmid. Techniques used include agarose electrophoresis, extraction of DNA from agarose, plasmid purification from bacteria, restriction digestion, ligation, and bacterial transformation. This promoter/reporter plasmid is then transformed into yeast. Transformed yeast are cultured in conditions prescribed in the experimental design, lysed and β-galactosidase activity is measured. The course provides an independent research experience in a group setting. Notebooks are maintained on-line with regular feedback. Projects culminate with the presentation of a poster worth 60% of the grade. Over the last three years, about 65% of students met expectations for experimental design, data acquisition, and analysis. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):145-151, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

A microRNA regulates the response of corals to thermal stress.

PubMed

Gajigan, Andrian P; Conaco, Cecilia

2017-07-01

Coral reefs are diverse ecosystems of great ecological and economic importance. However, corals are vulnerable to a variety of stressors, including rising seawater temperatures, and yet little is known about the genetic mechanisms underlying their survival and adaptation to stress. Like other animals, corals possess genes for key members of the microRNA (miRNA) machinery. miRNAs are short RNAs that regulate diverse cellular processes, including organismal stress response, through post-transcriptional repression of gene transcripts. Through small RNA sequencing, we identified 26 miRNAs in the coral, Acropora digitifera. Many of the identified miRNAs are novel, while eight are conserved with miRNAs previously identified in other cnidarians. One of the identified miRNAs is differentially expressed in coral tissues exposed to acute thermal stress. This thermally responsive miRNA putatively regulates multiple pathways of the organismal stress response, DNA/RNA expression regulation, repair mechanisms, tissue morphogenesis, and signalling. We propose a model by which miRNA regulation allows the coral to mount a robust stress response through sequestration of a pool of nontranslated transcripts encoding stress response proteins. Release of miRNA-mediated repression under stress conditions may result in rapid and abundant translation of proteins that help the coral maintain cellular homoeostasis. These findings highlight the potential importance of miRNAs in the thermal resilience of corals. © 2017 John Wiley & Sons Ltd.

RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria

PubMed Central

Vogel, Jörg; Bartels, Verena; Tang, Thean Hock; Churakov, Gennady; Slagter-Jäger, Jacoba G.; Hüttenhofer, Alexander; Wagner, E. Gerhart H.

2003-01-01

Recent bioinformatics-aided searches have identified many new small RNAs (sRNAs) in the intergenic regions of the bacterium Escherichia coli. Here, a shot-gun cloning approach (RNomics) was used to generate cDNA libraries of small sized RNAs. Besides many of the known sRNAs, we found new species that were not predicted previously. The present work brings the number of sRNAs in E.coli to 62. Experimental transcription start site mapping showed that some sRNAs were encoded from independent genes, while others were processed from mRNA leaders or trailers, indicative of a parallel transcriptional output generating sRNAs co-expressed with mRNAs. Two of these RNAs (SroA and SroG) consist of known (THI and RFN) riboswitch elements. We also show that two recently identified sRNAs (RyeB and SraC/RyeA) interact, resulting in RNase III-dependent cleavage. To the best of our knowledge, this represents the first case of two non-coding RNAs interacting by a putative antisense mechanism. In addition, intracellular metabolic stabilities of sRNAs were determined, including ones from previous screens. The wide range of half-lives (<2 to >32 min) indicates that sRNAs cannot generally be assumed to be metabolically stable. The experimental characterization of sRNAs analyzed here suggests that the definition of an sRNA is more complex than previously assumed. PMID:14602901

In-vivo-induced antigenic determinants of Fusobacterium nucleatum subsp. nucleatum.

PubMed

Lee, H-R; Rhyu, I-C; Kim, H-D; Jun, H-K; Min, B-M; Lee, S-H; Choi, B-K

2011-04-01

Fusobacterium nucleatum plays a pivotal role in dental plaque biofilm formation and is known to be involved in chronic inflammatory systemic disease. However, limited knowledge of F. nucleatum genes expressed in vivo interferes with our understanding of pathogenesis. In this study, we identified F. nucleatum genes induced in vivo using in-vivo-induced antigen technology (IVIAT). Among 30,000 recombinant clones screened, 87 reacted reproducibly with pooled sera from 10 patients with periodontitis. The clones encoded for 32 different proteins, of which 28 could be assigned to their functions, which were categorized in translation, transcription, transport, energy metabolism, cell envelope, cellular process, fatty acid and phospholipid metabolism, transposition, cofactor biosynthesis, amino acid biosynthesis, and DNA replication. Putative virulence factors detected were ABC transporter, butyrate-acetoacetate CoA-transferase, hemin receptor, hemolysin, hemolysin-related protein, LysR family transcriptional regulator, serine protease, and transposase. Analysis of immune responses to the in-vivo-induced (ivi) antigens in five patients demonstrated that most were reactive to these proteins, confirming results with pooled sera. IVIAT-identified F. nucleatum genes in this study may accelerate the elucidation of F. nucleatum-mediated molecular pathogenesis. © 2011 John Wiley & Sons A/S.

Transcriptional response of the extremophile red alga Cyanidioschyzon merolae to changes in CO2 concentrations.

PubMed

Rademacher, Nadine; Wrobel, Thomas J; Rossoni, Alessandro W; Kurz, Samantha; Bräutigam, Andrea; Weber, Andreas P M; Eisenhut, Marion

2017-10-01

Cyanidioschyzon merolae (C. merolae) is an acidophilic red alga growing in a naturally low carbon dioxide (CO 2 ) environment. Although it uses a ribulose 1,5-bisphosphate carboxylase/oxygenase with high affinity for CO 2 , the survival of C. merolae relies on functional photorespiratory metabolism. In this study, we quantified the transcriptomic response of C. merolae to changes in CO 2 conditions. We found distinct changes upon shifts between CO 2 conditions, such as a concerted up-regulation of photorespiratory genes and responses to carbon starvation. We used the transcriptome data set to explore a hypothetical CO 2 concentrating mechanism in C. merolae, based on the assumption that photorespiratory genes and possible candidate genes involved in a CO 2 concentrating mechanism are co-expressed. A putative bicarbonate transport protein and two α-carbonic anhydrases were identified, which showed enhanced transcript levels under reduced CO 2 conditions. Genes encoding enzymes of a PEPCK-type C 4 pathway were co-regulated with the photorespiratory gene cluster. We propose a model of a hypothetical low CO 2 compensation mechanism in C. merolae integrating these low CO 2 -inducible components. Copyright © 2017 Elsevier GmbH. All rights reserved.

Differential expression of candidate salivary effector proteins in field collections of Hessian fly, Mayetiola destructor

PubMed Central

Johnson, A J; Shukle, R H; Chen, M-S; Srivastava, S; Subramanyam, S; Schemerhorn, B J; Weintraub, P G; Abdel Moniem, H E M; Flanders, K L; Buntin, G D; Williams, C E

2015-01-01

Evidence is emerging that some proteins secreted by gall-forming parasites of plants act as effectors responsible for systemic changes in the host plant, such as galling and nutrient tissue formation. A large number of secreted salivary gland proteins (SSGPs) that are the putative effectors responsible for the physiological changes elicited in susceptible seedling wheat by Hessian fly, Mayetiola destructor (Say), larvae have been documented. However, how the genes encoding these candidate effectors might respond under field conditions is unknown. The goal of this study was to use microarray analysis to investigate variation in SSGP transcript abundance amongst field collections from different geographical regions (southeastern USA, central USA, and the Middle East). Results revealed significant variation in SSGP transcript abundance amongst the field collections studied. The field collections separated into three distinct groups that corresponded to the wheat classes grown in the different geographical regions as well as to recently described Hessian fly populations. These data support previous reports correlating Hessian fly population structure with micropopulation differences owing to agro-ecosystem parameters such as cultivation of regionally adapted wheat varieties, deployment of resistance genes and variation in climatic conditions. PMID:25528896

Genome Sequence and Analysis of the Soil Cellulolytic ActinomyceteThermobifida fusca

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lykidis, Athanasios; Mavromatis, Konstantinos; Ivanova, Natalia

Thermobifida fusca is a moderately thermophilic soilbacterium that belongs to Actinobacteria. 3 It is a major degrader ofplant cell walls and has been used as a model organism for the study of 4secreted, thermostable cellulases. The complete genome sequence showedthat T. fusca has a 5 single circular chromosome of 3642249 bp predictedto encode 3117 proteins and 65 RNA6 species with a coding densityof 85percent. Genome analysis revealed the existence of 29 putative 7glycoside hydrolases in addition to the previously identified cellulasesand xylanases. The 8 glycosyl hydrolases include enzymes predicted toexhibit mainly dextran/starch and xylan 9 degrading functions. T. fuscapossesses twomore » protein secretion systems: the sec general secretion 10system and the twin-arginine translocation system. Several of thesecreted cellulases have 11 sequence signatures indicating theirsecretion may be mediated by the twin-arginine12 translocation system. T.fusca has extensive transport systems for import of carbohydrates 13coupled to transcriptional regulators controlling the expression of thetransporters and14 glycosylhydrolases. In addition to providing anoverview of the physiology of a soil 15 actinomycete, this study presentsinsights on the transcriptional regulation and secretion of16 cellulaseswhich may facilitate the industrial exploitation of thesesystems.« less

Two ABREs, two redundant root-specific and one W-box cis-acting elements are functional in the sunflower HAHB4 promoter.

PubMed

Manavella, Pablo A; Dezar, Carlos A; Ariel, Federico D; Chan, Raquel L

2008-10-01

HAHB4 is a sunflower gene encoding a homeodomain-leucine zipper (HD-Zip) transcription factor. It was previously demonstrated that this gene is regulated at the transcriptional level by several abiotic factors and hormones. A previous analysis in the PLACE database revealed the presence of four putative ABREs. In this work these four elements and also one W-box and two root-specific expression elements were characterized as functional. Site-directed mutagenesis on the promoter, stable transformation of Arabidopis plants as well as transient transformation of sunflower leaves, were performed. The analysis of the transformants was carried out by histochemistry and real time RT-PCR. The results indicate that just one ABRE out of the four is responsible for ABA, NaCl and drought regulation. However, NaCl induction occurs also by an additional ABA-independent way involving another two overlapped ABREs. On the other hand, it was determined that the W-box located 5' upstream is responsive to ethylene and only two root-specific expression elements, among the several detected, are functional but redundant. Conservation of molecular mechanisms between sunflower and Arabidopsis is strongly supported by this experimental work.

TAIL1: an isthmin-like gene, containing type 1 thrombospondin-repeat and AMOP domain, mapped to ARVD1 critical region.

PubMed

Rossi, Valeria; Beffagna, Giorgia; Rampazzo, Alessandra; Bauce, Barbara; Danieli, Gian Antonio

2004-06-23

Isthmins represent a novel family of vertebrate secreted proteins containing one copy of the thrombospondin type 1 repeat (TSR), which in mammals is shared by several proteins with diverse biological functions, including cell adhesion, angiogenesis, and patterning of developing nervous system. We have determined the genomic organization of human TAIL1 (thrombospondin and AMOP containing isthmin-like 1), a novel isthmin-like gene encoding a protein that contains a TSR and a C-terminal AMOP domain (adhesion-associated domain in MUC4 and other proteins), characteristic of extracellular proteins involved in adhesion processes. TAIL1 gene encompasses more than 24.4 kb. Analysis of the DNA sequence surrounding the putative transcriptional start region revealed a TATA-less promoter located in a CpG island. Several consensus binding sites for the transcription factors Sp1 and MZF-1 were identified in this promoter region. In humans, TAIL1 gene is located on chromosome 14q24.3 within ARVD1 (arrhythmogenic right ventricular dysplasia/cardiomyopathy, type 1) critical region; preliminary evidence suggests that it is expressed in several tissues, showing multiple alternative splicing.

Inflexible ethanol intake: A putative link with the Lrrk2 pathway.

PubMed

da Silva E Silva, Daniel Almeida; Frozino Ribeiro, Andrea; Damasceno, Samara; Rocha, Cristiane S; Berenguer de Matos, Alexandre H; Boerngen-Lacerda, Roseli; Correia, Diego; Brunialti Godard, Ana Lúcia

2016-10-15

Alcoholism is a complex multifactorial disorder with a strong genetic influence. Although several studies have shown the impact of high ethanol intake on the striatal gene expression, few have addressed the relationship between the patterns of gene expression underlying the compulsive behaviour associated with the two major concerns in addiction: the excessive drug consumption and relapsing. In this study, we used a chronic three-bottle free-choice murine model to address striatal transcript regulation among animals with different ethanol intakes and preferences: Light Drinkers (preference for water throughout the experiment), Heavy Drinkers (preference for ethanol with a non-compulsive intake) and Inflexible Drinkers (preference for ethanol and simultaneous loss of control over the drug intake). Our aim was to correlate the intake patterns observed in this model with gene expression changes in the striatum, a brain region critical for the development of alcohol addiction. We found that the transcripts of the Lrrk2 gene, which encodes a multifunctional protein with kinase and GTPase activities, is upregulated only in Inflexible Drinkers suggesting, for the first time, that the Lrrk2 pathway plays a major role in the compulsive ethanol intake behaviour of addicted subjects. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome-Wide Identification and Characterization of Novel Laccase Genes in the White-Rot Fungus Flammulina velutipes

PubMed Central

Kim, Hong-Il; Kwon, O-Chul; Kong, Won-Sik; Lee, Chang-Soo

2014-01-01

The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges, fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1 (Asn-454), fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac4 (Asn-402 and Asn-457). In addition, the first 19~20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes. PMID:25606003

Transcriptional Analysis of a Whole-Body Form of Long-Term Habituation in "Aplysia Californica"

ERIC Educational Resources Information Center

Holmes, Geraldine; Herdegen, Samantha; Schuon, Jonathan; Cyriac, Ashly; Lass, Jamie; Conte, Catherine; Calin-Jageman, Irina E.; Calin-Jageman, Robert J.

2015-01-01

Habituation is the simplest form of learning, but we know little about the transcriptional mechanisms that encode long-term habituation memory. A key obstacle is that habituation is relatively stimulus-specific and is thus encoded in small sets of neurons, providing poor signal/noise ratios for transcriptional analysis. To overcome this obstacle,…

A Glycine Riboswitch in Streptococcus pyogenes Controls Expression of a Sodium:Alanine Symporter Family Protein Gene.

PubMed

Khani, Afsaneh; Popp, Nicole; Kreikemeyer, Bernd; Patenge, Nadja

2018-01-01

Regulatory RNAs play important roles in the control of bacterial gene expression. In this study, we investigated gene expression regulation by a putative glycine riboswitch located in the 5'-untranslated region of a sodium:alanine symporter family (SAF) protein gene in the group A Streptococcus pyogenes serotype M49 strain 591. Glycine-dependent gene expression mediated by riboswitch activity was studied using a luciferase reporter gene system. Maximal reporter gene expression was observed in the absence of glycine and in the presence of low glycine concentrations. Differences in glycine-dependent gene expression were not based on differential promoter activity. Expression of the SAF protein gene and the downstream putative cation efflux protein gene was investigated in wild-type bacteria by RT-qPCR transcript analyses. During growth in the presence of glycine (≥1 mM), expression of the genes were downregulated. Northern blot analyses revealed premature transcription termination in the presence of high glycine concentrations. Growth in the presence of 0.1 mM glycine led to the production of a full-length transcript. Furthermore, stability of the SAF protein gene transcript was drastically reduced in the presence of glycine. We conclude that the putative glycine riboswitch in S. pyogenes serotype M49 strain 591 represses expression of the SAF protein gene and the downstream putative cation efflux protein gene in the presence of high glycine concentrations. Sequence and secondary structure comparisons indicated that the streptococcal riboswitch belongs to the class of tandem aptamer glycine riboswitches.

Reactive Oxygen Species-Inducible ECF σ Factors of Bradyrhizobium japonicum

PubMed Central

Masloboeva, Nadezda; Reutimann, Luzia; Stiefel, Philipp; Follador, Rainer; Leimer, Nadja; Hennecke, Hauke; Mesa, Socorro; Fischer, Hans-Martin

2012-01-01

Extracytoplasmic function (ECF) σ factors control the transcription of genes involved in different cellular functions, such as stress responses, metal homeostasis, virulence-related traits, and cell envelope structure. The genome of Bradyrhizobium japonicum, the nitrogen-fixing soybean endosymbiont, encodes 17 putative ECF σ factors belonging to nine different ECF σ factor families. The genes for two of them, ecfQ (bll1028) and ecfF (blr3038), are highly induced in response to the reactive oxygen species hydrogen peroxide (H2O2) and singlet oxygen (1O2). The ecfF gene is followed by the predicted anti-σ factor gene osrA (blr3039). Mutants lacking EcfQ, EcfF plus OsrA, OsrA alone, or both σ factors plus OsrA were phenotypically characterized. While the symbiotic properties of all mutants were indistinguishable from the wild type, they showed increased sensitivity to singlet oxygen under free-living conditions. Possible target genes of EcfQ and EcfF were determined by microarray analyses, and candidate genes were compared with the H2O2-responsive regulon. These experiments disclosed that the two σ factors control rather small and, for the most part, distinct sets of genes, with about half of the genes representing 13% of the members of H2O2-responsive regulon. To get more insight into transcriptional regulation of both σ factors, the 5′ ends of ecfQ and ecfF mRNA were determined. The presence of conserved sequence motifs in the promoter region of ecfQ and genes encoding EcfQ-like σ factors in related α-proteobacteria suggests regulation via a yet unknown transcription factor. By contrast, we have evidence that ecfF is autoregulated by transcription from an EcfF-dependent consensus promoter, and its product is negatively regulated via protein-protein interaction with OsrA. Conserved cysteine residues 129 and 179 of OsrA are required for normal function of OsrA. Cysteine 179 is essential for release of EcfF from an EcfF-OsrA complex upon H2O2 stress while cysteine 129 is possibly needed for EcfF-OsrA interaction. PMID:22916258

A Proteomic Analysis Reveals Differential Regulation of the σS-Dependent yciGFE(katN) Locus by YncC and H-NS in Salmonella and Escherichia coli K-12

PubMed Central

Beraud, Mélanie; Kolb, Annie; Monteil, Véronique; D'Alayer, Jacques; Norel, Françoise

2010-01-01

The stationary phase sigma factor σS (RpoS) controls a regulon required for general stress resistance of the closely related enterobacteria Salmonella and Escherichia coli. The σS-dependent yncC gene encodes a putative DNA binding regulatory protein. Application of the surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) ProteinChip technology for proteome profiling of wild-type and mutant strains of Salmonella enterica serovar Typhimurium revealed potential protein targets for YncC regulation, which were identified by mass spectrometry, and subsequently validated. These proteins are encoded by the σS-dependent operon yciGFEkatN and regulation of their expression by YncC operates at the transcriptional level, as demonstrated by gene fusion analyses and by in vitro transcription and DNase I footprinting experiments with purified YncC. The yciGFE genes are present (without katN) in E. coli K-12 but are poorly expressed, compared with the situation in Salmonella. We report that the yciGFE(katN) locus is silenced by the histone-like protein H-NS in both species, but that σS efficiently relieves silencing in Salmonella but not in E. coli K-12. In Salmonella, YncC acts in concert with σS to activate transcription at the yciG promoter (pyciG). When overproduced, YncC also activated σS-dependent transcription at pyciG in E. coli K-12, but solely by countering the negative effect of H-NS. Our results indicate that differences between Salmonella and E. coli K-12, in the architecture of cis-acting regulatory sequences upstream of pyciG, contribute to the differential regulation of the yciGFE(katN) genes by H-NS and YncC in these two enterobacteria. In E. coli, this locus is subject to gene rearrangements and also likely to horizontal gene transfer, consistent with its repression by the xenogeneic silencer H-NS. PMID:20713450

Unique Transcriptional Profile of Sustained Ligand-Activated Preconditioning in Pre- and Post-Ischemic Myocardium

PubMed Central

Ashton, Kevin J.; Tupicoff, Amanda; Williams-Pritchard, Grant; Kiessling, Can J.; See Hoe, Louise E.; Headrick, John P.; Peart, Jason N.

2013-01-01

Background Opioidergic SLP (sustained ligand-activated preconditioning) induced by 3–5 days of opioid receptor (OR) agonism induces persistent protection against ischemia-reperfusion (I-R) injury in young and aged hearts, and is mechanistically distinct from conventional preconditioning responses. We thus applied unbiased gene-array interrogation to identify molecular effects of SLP in pre- and post-ischemic myocardium. Methodology/Principal Findings Male C57Bl/6 mice were implanted with 75 mg morphine or placebo pellets for 5 days. Resultant SLP did not modify cardiac function, and markedly reduced dysfunction and injury in perfused hearts subjected to 25 min ischemia/45 min reperfusion. Microarray analysis identified 14 up- and 86 down-regulated genes in normoxic hearts from SLP mice (≥1.3-fold change, FDR≤5%). Induced genes encoded sarcomeric/contractile proteins (Myh7, Mybpc3,Myom2,Des), natriuretic peptides (Nppa,Nppb) and stress-signaling elements (Csda,Ptgds). Highly repressed genes primarily encoded chemokines (Ccl2,Ccl4,Ccl7,Ccl9,Ccl13,Ccl3l3,Cxcl3), cytokines (Il1b,Il6,Tnf) and other proteins involved in inflammation/immunity (C3,Cd74,Cd83, Cd86,Hla-dbq1,Hla-drb1,Saa1,Selp,Serpina3), together with endoplasmic stress proteins (known: Dnajb1,Herpud1,Socs3; putative: Il6, Gadd45g,Rcan1) and transcriptional controllers (Egr2,Egr3, Fos,Hmox1,Nfkbid). Biological themes modified thus related to inflammation/immunity, together with cellular/cardiovascular movement and development. SLP also modified the transcriptional response to I-R (46 genes uniquely altered post-ischemia), which may influence later infarction/remodeling. This included up-regulated determinants of cellular resistance to oxidant (Mgst3,Gstm1,Gstm2) and other forms of stress (Xirp1,Ankrd1,Clu), and repression of stress-response genes (Hspa1a,Hspd1,Hsp90aa,Hsph1,Serpinh1) and Txnip. Conclusions Protection via SLP is associated with transcriptional repression of inflammation/immunity, up-regulation of sarcomeric elements and natriuretic peptides, and modulation of cell stress, growth and development, while conventional protective molecules are unaltered. PMID:23991079

Unsupervised Network Analysis of the Plastic Supraoptic Nucleus Transcriptome Predicts Caprin2 Regulatory Interactions

PubMed Central

Jahans-Price, Thomas; Greenwood, Michael P.; Greenwood, Mingkwan; Hoe, See-Ziau; Konopacka, Agnieszka

2017-01-01

Abstract The supraoptic nucleus (SON) is a group of neurons in the hypothalamus responsible for the synthesis and secretion of the peptide hormones vasopressin and oxytocin. Following physiological cues, such as dehydration, salt-loading and lactation, the SON undergoes a function related plasticity that we have previously described in the rat at the transcriptome level. Using the unsupervised graphical lasso (Glasso) algorithm, we reconstructed a putative network from 500 plastic SON genes in which genes are the nodes and the edges are the inferred interactions. The most active nodal gene identified within the network was Caprin2. Caprin2 encodes an RNA-binding protein that we have previously shown to be vital for the functioning of osmoregulatory neuroendocrine neurons in the SON of the rat hypothalamus. To test the validity of the Glasso network, we either overexpressed or knocked down Caprin2 transcripts in differentiated rat pheochromocytoma PC12 cells and showed that these manipulations had significant opposite effects on the levels of putative target mRNAs. These studies suggest that the predicative power of the Glasso algorithm within an in vivo system is accurate, and identifies biological targets that may be important to the functional plasticity of the SON. PMID:29279858

Organization of genes responsible for the stereospecific conversion of hydantoins to alpha-amino acids in Arthrobacter aurescens DSM 3747.

PubMed

Wiese, A; Syldatk, C; Mattes, R; Altenbuchner, J

2001-09-01

Arthrobacter aurescens DSM 3747 hydrolyzes stereospecifically 5'-monosubstituted hydantoins to alpha-amino acids. The genes involved in hydantoin utilization (hyu) were isolated on an 8.7-kb DNA fragment, and by DNA sequence analysis eight ORFs were identified. The hyu gene cluster includes four genes: hyuP encoding a putative transport protein, the hydantoin racemase gene hyuA, the hydantoinase gene hyuH, and the carbamoylase gene hyuC. The four genes are transcribed in the same direction. Upstream of hyuP and in opposite orientation to the hyu genes, three ORFs were found showing similarities to cytochrome P450 monooxygenase (ORF1, incomplete), to membrane proteins (ORF2), and to ferredoxin (ORF3). ORF8 was found downstream of hyuC and again in opposite orientation to the hyu genes. The gene product of ORF8 displayed similarities to the LacI/GalR family of transcriptional regulators. Reverse transcriptase PCR experiments and Northern blot analysis revealed that the genes hyuPAHC are coexpressed in A. aurescens after induction with 3-N-CH3-IMH. The expression of the hyu operon was not regulated by the putative regulator ORF8 as shown by gene disruption and mobility-shift experiments.

Annotation of Differentially Expressed Genes in the Somatic Embryogenesis of Musa and Their Location in the Banana Genome

PubMed Central

Maldonado-Borges, Josefina Ines; Ku-Cauich, José Roberto; Escobedo-GraciaMedrano, Rosa Maria

2013-01-01

Analysis of cDNA-AFLP was used to study the genes expressed in zygotic and somatic embryogenesis of Musa acuminata Colla ssp. malaccensis, and a comparison was made between their differential transcribed fragments (TDFs) and the sequenced genome of the double haploid- (DH-) Pahang of the malaccensis subspecies that is available in the network. A total of 253 transcript-derived fragments (TDFs) were detected with apparent size of 100–4000 bp using 5 pairs of AFLP primers, of which 21 were differentially expressed during the different stages of banana embryogenesis; 15 of the sequences have matched DH-Pahang chromosomes, with 7 of them being homologous to gene sequences encoding either known or putative protein domains of higher plants. Four TDF sequences were located in all Musa chromosomes, while the rest were located in one or two chromosomes. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed. Thus the availability of the genome of Musa and the information of TDFs sequences presented here opens new possibilities for an in-depth study of the molecular and biochemical research of zygotic and somatic embryogenesis of Musa. PMID:24027442

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus

PubMed Central

Selle, Kurt; Goh, Yong J.; Johnson, Brant R.; O’Flaherty, Sarah; Andersen, Joakim M.; Barrangou, Rodolphe; Klaenhammer, Todd R.

2017-01-01

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall. PMID:28443071

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus.

PubMed

Selle, Kurt; Goh, Yong J; Johnson, Brant R; O'Flaherty, Sarah; Andersen, Joakim M; Barrangou, Rodolphe; Klaenhammer, Todd R

2017-01-01

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus . Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus , suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

Identification of Novel Androgen-Regulated Pathways and mRNA Isoforms through Genome-Wide Exon-Specific Profiling of the LNCaP Transcriptome

PubMed Central

Carling, Phillippa J.; Buist, Thomas; Zhang, Chaolin; Grellscheid, Sushma N.; Armstrong, Kelly; Stockley, Jacqueline; Simillion, Cedric; Gaughan, Luke; Kalna, Gabriela; Zhang, Michael Q.; Robson, Craig N.; Leung, Hing Y.; Elliott, David J.

2011-01-01

Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa. PMID:22194994

A deletion mutation at the ep locus causes low seed coat peroxidase activity in soybean.

PubMed

Gijzen, M

1997-11-01

The Ep locus severely affects the amount of peroxidase enzyme in soybean seed coats. Plants containing the dominant Ep allele accumulate large amounts of peroxidase in the hourglass cells of the sub-epidermis. Homozygous recessive epep genotypes do not accumulate peroxidase in the hourglass cells and are much reduced in total seed coat peroxidase activity. To isolate the gene encoding the seed coat peroxidase and to determine whether it corresponds to the Ep locus, a cDNA library was constructed from developing seed coats and an abundant 1.3 kb peroxidase transcript was cloned. The corresponding structural gene was also isolated from a genomic library. Sequence analysis shows that the seed coat peroxidase is translated as a 352 amino acid precursor protein of 38 kDa. Processing of a putative 26 amino acid signal sequence results in a mature protein of 326 residues with a calculated mass of 35 kDa and a pl of 4.4. Using probes derived from the cDNA, genomic DNA blot hybridization and polymerase chain reaction analysis detected polymorphisms that distinguished EpEp and epep genotypes. Co-segregation of the polymorphisms in an F2 population from a cross of EpEp and epep plants shows that the Ep locus encodes the seed coat peroxidase protein. Comparison of Ep and ep alleles indicates that the recessive gene lacks 87 bp of sequence encompassing the translation start codon. Analysis by RNA blot hybridization shows that epep plants have drastically reduced amounts of peroxidase transcript compared with EpEp plants. The peroxidase mRNA is abundant in seed coat tissues of EpEp plants during the late stages of seed maturation, and could also be detected in root tissues, but not in the flower, embryo, pod or leaf. The results indicate that the lack of peroxidase accumulation in seed coats of homozygous recessive epep plants is due to a mutation of the structural gene that reduces transcript abundance.