Activation of Pathogenesis-related Genes by the Rhizobacterium, Bacillus sp. JS, Which Induces Systemic Resistance in Tobacco Plants.

PubMed

Kim, Ji-Seong; Lee, Jeongeun; Lee, Chan-Hui; Woo, Su Young; Kang, Hoduck; Seo, Sang-Gyu; Kim, Sun-Hyung

2015-06-01

Plant growth promoting rhizobacteria (PGPR) are known to confer disease resistance to plants. Bacillus sp. JS demonstrated antifungal activities against five fungal pathogens in in vitro assays. To verify whether the volatiles of Bacillus sp. JS confer disease resistance, tobacco leaves pre-treated with the volatiles were damaged by the fungal pathogen, Rhizoctonia solani and oomycete Phytophthora nicotianae. Pre-treated tobacco leaves had smaller lesion than the control plant leaves. In pathogenesis-related (PR) gene expression analysis, volatiles of Bacillus sp. JS caused the up-regulation of PR-2 encoding β-1,3-glucanase and acidic PR-3 encoding chitinase. Expression of acidic PR-4 encoding chitinase and acidic PR-9 encoding peroxidase increased gradually after exposure of the volatiles to Bacillus sp. JS. Basic PR-14 encoding lipid transfer protein was also increased. However, PR-1 genes, as markers of salicylic acid (SA) induced resistance, were not expressed. These results suggested that the volatiles of Bacillus sp. JS confer disease resistance against fungal and oomycete pathogens through PR genes expression.

Structure, Expression, Chromosomal Location and Product of the Gene Encoding Adh2 in Petunia

PubMed Central

Gregerson, R. G.; Cameron, L.; McLean, M.; Dennis, P.; Strommer, J.

1993-01-01

In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes. PMID:8096485

Dynamics of biomass partitioning, stem gene expression, cell wall biosynthesis, and sucrose accumulation during development of Sorghum bicolor.

PubMed

McKinley, Brian; Rooney, William; Wilkerson, Curtis; Mullet, John

2016-11-01

Biomass accumulated preferentially in leaves of the sweet sorghum Della until floral initiation, then stems until anthesis, followed by panicles until grain maturity, and apical tillers. Sorghum stem RNA-seq transcriptome profiles and composition data were collected for approximately 100 days of development beginning at floral initiation. The analysis identified >200 differentially expressed genes involved in stem growth, cell wall biology, and sucrose accumulation. Genes encoding expansins and xyloglucan endotransglucosylase/hydrolases were differentially expressed in growing stem internodes. Genes encoding enzymes involved in the synthesis of cellulose, lignin, and glucuronoarabinoxylan were expressed at elevated levels in stems until approximately 7 days before anthesis and then down-regulated. CESA genes involved in primary and secondary cell wall synthesis showed different temporal patterns of expression. Following floral initiation, the level of sucrose and other non-structural carbohydrates increased to approximately 50% of the stem's dry weight. Stem sucrose accumulation was inversely correlated with >100-fold down-regulation of SbVIN1, a gene encoding a vacuolar invertase. Accumulation of stem sucrose was also correlated with cessation of leaf and stem growth at anthesis, decreased expression of genes involved in stem cell wall synthesis, and approximately 10-fold lower expression of SbSUS4, a gene encoding sucrose synthase that generates UDP-glucose from sucrose for cell wall biosynthesis. Genes for mixed linkage glucan synthesis (CSLF) and turnover were expressed at high levels in stems throughout development. Overall, the stem transcription profile resource and the genes and regulatory dynamics identified in this study will be useful for engineering sorghum stem composition for improved conversion to biofuels and bio-products. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Functional differentiation and spatial-temporal co-expression networks of the NBS-encoding gene family in Jilin ginseng, Panax ginseng C.A. Meyer.

PubMed

Yin, Rui; Zhao, Mingzhu; Wang, Kangyu; Lin, Yanping; Wang, Yanfang; Sun, Chunyu; Wang, Yi; Zhang, Meiping

2017-01-01

Ginseng, Panax ginseng C.A. Meyer, is one of the most important medicinal plants for human health and medicine. It has been documented that over 80% of genes conferring resistance to bacteria, viruses, fungi and nematodes are contributed by the nucleotide binding site (NBS)-encoding gene family. Therefore, identification and characterization of NBS genes expressed in ginseng are paramount to its genetic improvement and breeding. However, little is known about the NBS-encoding genes in ginseng. Here we report genome-wide identification and systems analysis of the NBS genes actively expressed in ginseng (PgNBS genes). Four hundred twelve PgNBS gene transcripts, derived from 284 gene models, were identified from the transcriptomes of 14 ginseng tissues. These genes were classified into eight types, including TNL, TN, CNL, CN, NL, N, RPW8-NL and RPW8-N. Seven conserved motifs were identified in both the Toll/interleukine-1 receptor (TIR) and coiled-coil (CC) typed genes whereas six were identified in the RPW8 typed genes. Phylogenetic analysis showed that the PgNBS gene family is an ancient family, with a vast majority of its genes originated before ginseng originated. In spite of their belonging to a family, the PgNBS genes have functionally dramatically differentiated and been categorized into numerous functional categories. The expressions of the across tissues, different aged roots and the roots of different genotypes. However, they are coordinating in expression, forming a single co-expression network. These results provide a deeper understanding of the origin, evolution and functional differentiation and expression dynamics of the NBS-encoding gene family in plants in general and in ginseng particularly, and a NBS gene toolkit useful for isolation and characterization of disease resistance genes and for enhanced disease resistance breeding in ginseng and related species.

Functional differentiation and spatial-temporal co-expression networks of the NBS-encoding gene family in Jilin ginseng, Panax ginseng C.A. Meyer

PubMed Central

Wang, Kangyu; Lin, Yanping; Wang, Yanfang; Sun, Chunyu; Wang, Yi

2017-01-01

Ginseng, Panax ginseng C.A. Meyer, is one of the most important medicinal plants for human health and medicine. It has been documented that over 80% of genes conferring resistance to bacteria, viruses, fungi and nematodes are contributed by the nucleotide binding site (NBS)-encoding gene family. Therefore, identification and characterization of NBS genes expressed in ginseng are paramount to its genetic improvement and breeding. However, little is known about the NBS-encoding genes in ginseng. Here we report genome-wide identification and systems analysis of the NBS genes actively expressed in ginseng (PgNBS genes). Four hundred twelve PgNBS gene transcripts, derived from 284 gene models, were identified from the transcriptomes of 14 ginseng tissues. These genes were classified into eight types, including TNL, TN, CNL, CN, NL, N, RPW8-NL and RPW8-N. Seven conserved motifs were identified in both the Toll/interleukine-1 receptor (TIR) and coiled-coil (CC) typed genes whereas six were identified in the RPW8 typed genes. Phylogenetic analysis showed that the PgNBS gene family is an ancient family, with a vast majority of its genes originated before ginseng originated. In spite of their belonging to a family, the PgNBS genes have functionally dramatically differentiated and been categorized into numerous functional categories. The expressions of the across tissues, different aged roots and the roots of different genotypes. However, they are coordinating in expression, forming a single co-expression network. These results provide a deeper understanding of the origin, evolution and functional differentiation and expression dynamics of the NBS-encoding gene family in plants in general and in ginseng particularly, and a NBS gene toolkit useful for isolation and characterization of disease resistance genes and for enhanced disease resistance breeding in ginseng and related species. PMID:28727829

Single cell transcriptome analysis of MCF-7 reveals consistently and inconsistently expressed gene groups each associated with distinct cellular localization and functions

PubMed Central

Chen, Tzu-Han; Shiau, Hsin-Chieh

2018-01-01

Single cell transcriptome (SCT) analysis provides superior resolution to illustrate tumor cell heterogeneity for clinical implications. We characterized four SCTs of MCF-7 using 143 housekeeping genes (HKGs) as control, of which lactate dehydrogenase B (LDHB) expression is silenced. These SCT libraries mapped to 11,423, 11,486, 10,380, and 11,306 RefSeq genes (UCSC), respectively. High consistency in HKG expression levels across all four SCTs, along with transcriptional silencing of LDHB, was observed, suggesting a high sensitivity and reproducibility of the SCT analysis. Cross-library comparison on expression levels by scatter plotting revealed a linear correlation and an 83–94% overlap in transcript isoforms and expressed genes were also observed. To gain insight of transcriptional diversity among the SCTs, expressed genes were split into consistently expressed (CE) (expressed in all SCTs) and inconsistently expressed (IE) (expressed in some but not all SCTs) genes for further characterization, along with the 142 expressed HKGs as a reference. Distinct transcriptional strengths were found among these groups, with averages of 1,612.0, 88.0 and 1.2 FPKM for HKGs, CE and IE, respectively. Comparison between CE and IE groups further indicated that expressions of CE genes vary more significantly than that of IE genes. Gene Ontology analysis indicated that proteins encoded by CE genes are mainly involved in fundamental intracellular activities, while proteins encoded by IE genes are mainly for extracellular activities, especially acting as receptors or ion channels. The diversified gene expressions, especially for those encoded by IE genes, may contribute to cancer drug resistance. PMID:29920548

Genome-wide identification, classification, and expression analysis of the arabinogalactan protein gene family in rice (Oryza sativa L.)

PubMed Central

Zhao, Jie

2010-01-01

Arabinogalactan proteins (AGPs) comprise a family of hydroxyproline-rich glycoproteins that are implicated in plant growth and development. In this study, 69 AGPs are identified from the rice genome, including 13 classical AGPs, 15 arabinogalactan (AG) peptides, three non-classical AGPs, three early nodulin-like AGPs (eNod-like AGPs), eight non-specific lipid transfer protein-like AGPs (nsLTP-like AGPs), and 27 fasciclin-like AGPs (FLAs). The results from expressed sequence tags, microarrays, and massively parallel signature sequencing tags are used to analyse the expression of AGP-encoding genes, which is confirmed by real-time PCR. The results reveal that several rice AGP-encoding genes are predominantly expressed in anthers and display differential expression patterns in response to abscisic acid, gibberellic acid, and abiotic stresses. Based on the results obtained from this analysis, an attempt has been made to link the protein structures and expression patterns of rice AGP-encoding genes to their functions. Taken together, the genome-wide identification and expression analysis of the rice AGP gene family might facilitate further functional studies of rice AGPs. PMID:20423940

Material and methods to increase plant growth and yield

DOEpatents

Kirst, Matias

2015-09-15

The present invention relates to materials and methods for modulating growth rates, yield, and/or resistance to drought conditions in plants. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene or expression or activity of the protein encoded by an hc1 gene results in increased growth rate, yield, and/or drought resistance in the plant.

Materials and methods to increase plant growth and yield

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

Kirst, Matias

The present invention relates to materials and methods for modulating growth rates, yield, and/or resistance to drought conditions in plants. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene or expression or activity of the protein encoded by an hc1 gene results in increased growth rate, yield, and/or drought resistance in the plant.

The prrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

PubMed Central

Reinhart, Alexandria A.; Powell, Daniel A.; Nguyen, Angela T.; O'Neill, Maura; Djapgne, Louise; Wilks, Angela; Ernst, Robert K.

2014-01-01

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence. PMID:25510881

Transcriptional Modulation of Genes Encoding Structural Characteristics of Differentiating Enterocytes During Development of a Polarized Epithelium In Vitro

PubMed Central

Halbleib, Jennifer M.; Sääf, Annika M.

2007-01-01

Although there is considerable evidence implicating posttranslational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell–cell adhesion–initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell–cell contacts between neighboring cells. Expression of genes involved in cell proliferation was down-regulated concomitant with induction of genes necessary for functional specialization of polarized epithelial cells. Transcriptional up-regulation of these latter genes correlated with formation of important structural and functional features in enterocyte differentiation and establishment of structural and functional cell polarity; components of the apical microvilli were induced as the brush border formed during polarization; as barrier function was established, expression of tight junction transmembrane proteins peaked; transcripts encoding components of the apical, but not the basal-lateral trafficking machinery were increased during polarization. Coordinated expression of genes encoding components of functional cell structures were often observed indicating temporal control of expression and assembly of multiprotein complexes. PMID:17699590

Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

PubMed Central

Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

2013-01-01

Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

Insight into the expression variation of metal-responsive genes in the seedling of date palm (Phoenix dactylifera).

PubMed

Chaâbene, Zayneb; Rorat, Agnieszka; Rekik Hakim, Imen; Bernard, Fabien; Douglas, Grubb C; Elleuch, Amine; Vandenbulcke, Franck; Mejdoub, Hafedh

2018-04-01

Phytochelatin synthase and metallothionein gene expressions were monitored via qPCR in order to investigate the molecular mechanisms involved in Cd and Cr detoxification in date palm (Phoenix dactylifera). A specific reference gene validation procedure using BestKeeper, NormFinder and geNorm programs allowed selection of the three most stable reference genes in a context of Cd or Cr contamination among six reference gene candidates, namely elongation factor α1, actin, aldehyde dehydrogenase, SAND family, tubulin 6 and TaTa box binding protein. Phytochelatin synthase (pcs) and metallothionein (mt) encoding gene expression were induced from the first days of exposure. At low Cd stress (0.02 mM), genes were still up-regulated until 60th day of exposure. At the highest metal concentrations, however, pcs and mt gene expressions decreased. pcs encoding gene was significantly up-regulated under Cr exposure, and was more responsive to increasing Cr concentration than mt encoding gene. Moreover, exposure to Cd or Cr influenced clearly seed germination and hypocotyls elongation. Thus, the results have proved that both analyzed genes participate in metal detoxification and their expression is regulated at transcriptional level in date palm subjected to Cr and Cd stress. Consequently, variations of expression of mt and pcs genes may serve as early-warning biomarkers of metal stress in this species. Copyright © 2018 Elsevier Ltd. All rights reserved.

Expression and characterization of hyperthermostable exo-polygalacturonase RmGH28 from Rhodothermus marinus

USDA-ARS?s Scientific Manuscript database

The gene RmGH28 from the organism Rhodothermus marinus putatively encoding a glycosyl hydrolase family 28 polygalacturonase was expressed in E. coli, and the enzyme purified and biochemically characterized. The gene was found to encode an exo- polygalacturonase, with galacturonic acid monomer and th...

Disruption of the psbA gene by the copy correction mechanism reveals that the expression of plastid-encoded genes is regulated by photosynthesis activity.

PubMed

Khan, Muhammad Sarwar; Hameed, Waqar; Nozoe, Mikio; Shiina, Takashi

2007-05-01

The functional analysis of genes encoded by the chloroplast genome of tobacco by reverse genetics is routine. Nevertheless, for a small number of genes their deletion generates heteroplasmic genotypes, complicating their analysis. There is thus the need for additional strategies to develop deletion mutants for these genes. We have developed a homologous copy correction-based strategy for deleting/mutating genes encoded on the chloroplast genome. This system was used to produce psbA knockouts. The resulting plants are homoplasmic and lack photosystem II (PSII) activity. Further, the deletion mutants exhibit a distinct phenotype; young leaves are green, whereas older leaves are bleached, irrespective of light conditions. This suggests that senescence is promoted by the absence of psbA. Analysis of the transcript levels indicates that NEP (nuclear-encoded plastid RNA polymerase)-dependent plastid genes are up regulated in the psbA deletion mutants, whereas the bleached leaves retain plastid-encoded plastid RNA polymerase activity. Hence, the expression of NEP-dependent plastid genes may be regulated by photosynthesis, either directly or indirectly.

Degradation of triglycerides by a pseudomonad isolated from milk: molecular analysis of a lipase-encoding gene and its expression in Escherichia coli.

PubMed Central

Johnson, L A; Beacham, I R; MacRae, I C; Free, M L

1992-01-01

Psychrotrophic lipolytic bacteria represent a significant problem in the storage of refrigerated dairy products. A lipase-encoding gene has been cloned and characterized from a highly lipolytic strain of Pseudomonas. The nucleotide sequence of the gene predicts a polypeptide of M(r) 49,905, which was identified when the gene was expressed in Escherichia coli. Images PMID:1622251

Comparison of gene expression levels of appA, ppsR, and EL368 in Erythrobacter litoralis spheroplasts under aerobic and anaerobic conditions, and under blue light, red light, and dark conditions.

PubMed

Nishino, Koki; Takahashi, Sawako; Nishida, Hiromi

2018-03-31

We compared the gene expression levels of the blue-light-responsive genes, appA (encoding photosynthesis promoting protein AppA), ppsR (encoding photosynthesis suppressing protein PpsR), and EL368 (encoding a blue-light-activated histidine kinase with a light, oxygen, or voltage domain) between aerobic and anaerobic conditions in spheroplasts of the aerobic photosynthetic bacterium Erythrobacter litoralis. The spheroplasts conducted photosynthesis under red light but not under blue light. All three blue-light-responsive genes showed higher expression under aerobic conditions than under anaerobic conditions under blue light. In contrast, under red light, although the expression level of appA was higher in the presence of oxygen than in the absence of oxygen, the expression levels of ppsR and EL368 were similar in the presence and absence of oxygen. Our findings demonstrate that the expression of blue-light-responsive genes is strongly affected by oxygen in E. litoralis spheroplasts.

Molecular cloning of a putative gene encoding isopentenyltransferase from pingyitiancha (Malus hupehensis) and characterization of its response to nitrate.

PubMed

Peng, Jing; Peng, Futian; Zhu, Chunfu; Wei, Shaochong

2008-06-01

A putative isopentenyltransferase (IPT) encoding gene was identified from a pingyitiancha (Malus hupehensis Rehd.) expressed sequence tag database, and the full-length gene was cloned by RACE. Based on expression profile and sequence alignment, the nucleotide sequence of the clone, named MhIPT3, was most similar to AtIPT3, an IPT gene in Arabidopsis. The full-length cDNA contained a 963-bp open reading frame encoding a protein of 321 amino acids with a molecular mass of 37.3 kDa. Sequence analysis of genomic DNA revealed the absence of introns in the frame. Quantitative real-time PCR analysis demonstrated that the gene was expressed in roots, stems and leaves. Application of nitrate to roots of nitrogen-deprived seedlings strongly induced expression of MhIPT3 and was accompanied by the accumulation of cytokinins, whereas MhIPT3 expression was little affected by ammonium application to roots of nitrogen-deprived seedlings. Application of nitrate to leaves also up-regulated the expression of MhIPT3 and corresponded closely with the accumulation of isopentyladenine and isopentyladenosine in leaves.

Differential Expression of Three α-Galactosidase Genes and a Single β-Galactosidase Gene from Aspergillus niger

PubMed Central

de Vries, Ronald P.; van den Broeck, Hetty C.; Dekkers, Ester; Manzanares, Paloma; de Graaff, Leo H.; Visser, Jaap

1999-01-01

A gene encoding a third α-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other α-galactosidases revealed that it belongs to a subfamily of α-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic α-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger β-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins. PMID:10347026

Regulation of Bacteriocin Production in Streptococcus mutans by the Quorum-Sensing System Required for Development of Genetic Competence

PubMed Central

van der Ploeg, Jan R.

2005-01-01

In Streptococcus mutans, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by comC). Here, it is shown that the same system is also required for expression of the nlmAB genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional nlmAB′-lacZ fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the comDE genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the nlmAB promoter region led to a decrease in CSP-dependent expression of nlmAB′-lacZ. In agreement with these results, a comDE mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci. PMID:15937160

Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

DTIC Science & Technology

1988-10-31

00 0 Cloning and Expression of Genes for Dengue Virus (Type-2 Encoded-Antigens for Rapid ODiagnosis and Vaccine DevelopmentN| ANNUAL PROGRESS REPORT...11. TITLE (include Security Classification) Cloning and Expression of Genes f or Dengue Virus Type 2 Fncoded Antigens for Rapid Diagnosis and Vaccine ...epidemics in Central and South Americas and the Caribbean is a cause of major concern. An effective vaccine is not available to protect individuals

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

ERIC Educational Resources Information Center

Mokin, Maxim; Keifer, Joyce

2005-01-01

Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

Effect of Roux-en-Y gastric bypass on the distribution and hormone expression of small-intestinal enteroendocrine cells in obese patients with type 2 diabetes.

PubMed

Rhee, Nicolai A; Wahlgren, Camilla D; Pedersen, Jens; Mortensen, Brynjulf; Langholz, Ebbe; Wandall, Erik P; Friis, Steffen U; Vilmann, Peter; Paulsen, Sarah J; Kristiansen, Viggo B; Jelsing, Jacob; Dalbøge, Louise S; Poulsen, Steen S; Holst, Jens J; Vilsbøll, Tina; Knop, Filip K

2015-10-01

We studied the impact of Roux-en-Y gastric bypass (RYGB) on the density and hormonal gene expression of small-intestinal enteroendocrine cells in obese patients with type 2 diabetes. Twelve patients with diabetes and 11 age- and BMI-matched controls underwent RYGB followed by enteroscopy ~10 months later. Mucosal biopsies taken during surgery and enteroscopy were immunohistochemically stained for glucagon-like peptide-1 (GLP-1), peptide YY (PYY), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP) and prohormone convertase 2 (PC2) and the expression of GCG (encoding preproglucagon), PYY, CCK, GIP, GHRL (encoding ghrelin), SCT (encoding secretin), NTS (encoding neurotensin) and NR1H4 (encoding farnesoid X receptor) was evaluated. The density of cells immunoreactive for GLP-1, CCK and GIP increased in patients after RYGB and the density of those immunoreactive for GLP-1, PYY, CCK and PC2 increased in controls. In both groups, GHRL, SCT and GIP mRNA was reduced after RYGB while PYY, CCK, NTS and NR1H4 gene expression was unaltered. GCG mRNA was upregulated in both groups. Numerous alterations in the distribution of enteroendocrine cells and their expression of hormonal genes are seen after RYGB and include increased density of GLP-1-, PYY-, CCK-, GIP- and PC2-positive cells, reduced gene expression of GHRL, SCT and GIP and increased expression of GCG.

Mobile genetic element-encoded cytolysin connects virulence to methicillin resistance in MRSA.

PubMed

Queck, Shu Y; Khan, Burhan A; Wang, Rong; Bach, Thanh-Huy L; Kretschmer, Dorothee; Chen, Liang; Kreiswirth, Barry N; Peschel, Andreas; Deleo, Frank R; Otto, Michael

2009-07-01

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.

Similarity and functional analyses of expressed parasitism genes in Heterodera schachtii and Heterodera glycines

USDA-ARS?s Scientific Manuscript database

The secreted proteins encoded by “parasitism genes” expressed within the esophageal glands cells of cyst nematodes play important roles in plant parasitism. Homologous transcripts and encoded proteins of the Heterodera glycines pioneer parasitism genes Hgsyv46, Hg4e02 and Hg5d08 were identified and ...

Influence of energy supply on expression of genes encoding for lipogenic enzymes and regulatory proteins in growing beef steers

USDA-ARS?s Scientific Manuscript database

Forty crossbred beef steers were used to determine the effects metabolizable energy (ME) intake and of site and complexity of carbohydrate (CHO) infusion on expression of genes encoding lipogenic enzymes and regulatory proteins in subcutaneous (SC), mesenteric (MES) and omental (OM) adipose. Treatm...

Expression profiles of genes for mitochondrial respiratory energy-dissipating systems and antioxidant enzymes in wheat leaves during de-etiolation.

PubMed

Garmash, Elena V; Velegzhaninov, Ilya O; Grabelnych, Olga I; Borovik, Olga A; Silina, Ekaterina V; Voinikov, Victor K; Golovko, Tamara K

2017-08-01

Mitochondrial respiratory components participate in the maintenance of chloroplast functional activity. This study investigates the effects 48h de-etiolation of spring wheat seedlings (Triticum aestivum L., var. Irgina) on the expression of genes that encode energy-dissipating respiratory components and antioxidant enzymes under continuous light conditions. The expression of AOX1a following the prolonged darkness exhibited a pattern indicating a prominent dependence on light. The expression of other respiratory genes, including NDA2, NDB2, and UCP1b, increased during de-etiolation and dark-to-light transition; however, changes in the expression of these genes occurred later than those in AOX1a expression. A high expression of NDA1 was detected after 12h of de-etiolation. The suppression of AOX1a, NDA2, NDB2, and UCP1b was observed 24h after de-etiolation when the photosynthetic apparatus and its defence systems against excess light were completely developed. The expression patterns of the respiratory genes and several genes encoding antioxidant enzymes (MnSOD, Cu-ZnSOD, t-APX, GR, and GRX) were quite similar. Our data indicate that the induction of nuclear genes encoding respiratory and antioxidant enzymes allow the plants to control reactive oxygen species (ROS) production and avoid oxidative stress during de-etiolation. Copyright © 2017 Elsevier GmbH. All rights reserved.

Global differential gene expression in response to growth temperature alteration in group A Streptococcus.

PubMed

Smoot, L M; Smoot, J C; Graham, M R; Somerville, G A; Sturdevant, D E; Migliaccio, C A; Sylva, G L; Musser, J M

2001-08-28

Pathogens are exposed to different temperatures during an infection cycle and must regulate gene expression accordingly. However, the extent to which virulent bacteria alter gene expression in response to temperatures encountered in the host is unknown. Group A Streptococcus (GAS) is a human-specific pathogen that is responsible for illnesses ranging from superficial skin infections and pharyngitis to severe invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS survives and multiplies at different temperatures during human infection. DNA microarray analysis was used to investigate the influence of temperature on global gene expression in a serotype M1 strain grown to exponential phase at 29 degrees C and 37 degrees C. Approximately 9% of genes were differentially expressed by at least 1.5-fold at 29 degrees C relative to 37 degrees C, including genes encoding transporter proteins, proteins involved in iron homeostasis, transcriptional regulators, phage-associated proteins, and proteins with no known homologue. Relatively few known virulence genes were differentially expressed at this threshold. However, transcription of 28 genes encoding proteins with predicted secretion signal sequences was altered, indicating that growth temperature substantially influences the extracellular proteome. TaqMan real-time reverse transcription-PCR assays confirmed the microarray data. We also discovered that transcription of genes encoding hemolysins, and proteins with inferred roles in iron regulation, transport, and homeostasis, was influenced by growth at 40 degrees C. Thus, GAS profoundly alters gene expression in response to temperature. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many unforeseen lines of pathogenesis investigation.

Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE)

PubMed Central

Sharon, Dror; Blackshaw, Seth; Cepko, Constance L.; Dryja, Thaddeus P.

2002-01-01

We used the serial analysis of gene expression (SAGE) technique to catalogue and measure the relative levels of expression of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium (RPE) from one or both of two humans, aged 88 and 44 years. The cone photoreceptor contribution to all transcription in the retina was found to be similar in the macula versus the retinal periphery, whereas the rod contribution was greater in the periphery versus the macula. Genes encoding structural proteins for axons were found to be expressed at higher levels in the macula versus the retinal periphery, probably reflecting the large proportion of ganglion cells in the central retina. In comparison with the younger eye, the peripheral retina of the older eye had a substantially higher proportion of mRNAs from genes encoding proteins involved in iron metabolism or protection against oxidative damage and a substantially lower proportion of mRNAs from genes encoding proteins involved in rod phototransduction. These differences may reflect the difference in age between the two donors or merely interindividual variation. The RPE library had numerous previously unencountered tags, suggesting that this cell type has a large, idiosyncratic repertoire of expressed genes. Comparison of these libraries with 100 reported nonocular SAGE libraries revealed 89 retina-specific or enriched genes expressed at substantial levels, of which 14 are known to cause a retinal disease and 53 are RPE-specific genes. We expect that these libraries will serve as a resource for understanding the relative expression levels of genes in the retina and the RPE and for identifying additional disease genes. PMID:11756676

Metadata Analysis of Phanerochaete chrysosporium Gene Expression Data Identified Common CAZymes Encoding Gene Expression Profiles Involved in Cellulose and Hemicellulose Degradation.

PubMed

Kameshwar, Ayyappa Kumar Sista; Qin, Wensheng

2017-01-01

In literature, extensive studies have been conducted on popular wood degrading white rot fungus, Phanerochaete chrysosporium about its lignin degrading mechanisms compared to the cellulose and hemicellulose degrading abilities. This study delineates cellulose and hemicellulose degrading mechanisms through large scale metadata analysis of P. chrysosporium gene expression data (retrieved from NCBI GEO) to understand the common expression patterns of differentially expressed genes when cultured on different growth substrates. Genes encoding glycoside hydrolase classes commonly expressed during breakdown of cellulose such as GH-5,6,7,9,44,45,48 and hemicellulose are GH-2,8,10,11,26,30,43,47 were found to be highly expressed among varied growth conditions including simple customized and complex natural plant biomass growth mediums. Genes encoding carbohydrate esterase class enzymes CE (1,4,8,9,15,16) polysaccharide lyase class enzymes PL-8 and PL-14, and glycosyl transferases classes GT (1,2,4,8,15,20,35,39,48) were differentially expressed in natural plant biomass growth mediums. Based on these results, P. chrysosporium, on natural plant biomass substrates was found to express lignin and hemicellulose degrading enzymes more than cellulolytic enzymes except GH-61 (LPMO) class enzymes, in early stages. It was observed that the fate of P. chrysosporium transcriptome is significantly affected by the wood substrate provided. We believe, the gene expression findings in this study plays crucial role in developing genetically efficient microbe with effective cellulose and hemicellulose degradation abilities.

Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus

PubMed Central

2013-01-01

Background Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. Results In this study, genes encoding putative enzymes from carbon metabolism were identified and their expression was studied in different growth stages of A. bisporus. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies. Conclusions The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples. PMID:24074284

Plasmid-Encoded Tetracycline Efflux Pump Protein Alters Bacterial Stress Responses and Ecological Fitness of Acinetobacter oleivorans

PubMed Central

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment. PMID:25229538

Plasmid-encoded tetracycline efflux pump protein alters bacterial stress responses and ecological fitness of Acinetobacter oleivorans.

PubMed

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment.

Alternative RNA processing events in human calcitonin/calcitonin gene-related peptide gene expression.

PubMed Central

Jonas, V; Lin, C R; Kawashima, E; Semon, D; Swanson, L W; Mermod, J J; Evans, R M; Rosenfeld, M G

1985-01-01

Two mRNAs generated as a consequence of alternative RNA processing events in expression of the human calcitonin gene encode the protein precursors of either calcitonin or calcitonin gene-related peptide (CGRP). Both calcitonin and CGRP RNAs and their encoded peptide products are expressed in the human pituitary and in medullary thyroid tumors. On the basis of sequence comparison, it is suggested that both the calcitonin and CGRP exons arose from a common primordial sequence, suggesting that duplication and rearrangement events are responsible for the generation of this complex transcription unit. Images PMID:3872459

A second cistron in the CACNA1A gene encodes a transcription factor that mediates cerebellar development and SCA6

PubMed Central

Du, Xiaofei; Wang, Jun; Zhu, Haipeng; Rinaldo, Lorenzo; Lamar, Kay-Marie; Palmenberg, Ann C.; Hansel, Christian; Gomez, Christopher M.

2014-01-01

SUMMARY The CACNA1A gene, encoding the voltage-gated calcium channel subunit α1A, is involved in pre- and postsynaptic Ca2+ signaling, gene expression, and several genetic neurological disorders. We found that CACNA1A employs a novel strategy to directly coordinate a gene expression program, using a bicistronic mRNA bearing a cryptic internal ribosomal entry site (IRES). The first cistron encodes the well-characterized α1A subunit. The second expresses a newly-recognized transcription factor, α1ACT, that coordinates expression of a program of genes involved in neural and Purkinje cell development. α1ACT also contains the polyglutamine (polyQ) tract that, when expanded, causes spinocerebellar ataxia type 6 (SCA6). When expressed as an independent polypeptide, α1ACT, bearing an expanded polyQ tract, lacks transcription factor function and neurite outgrowth properties, causes cell death in culture, and leads to ataxia and cerebellar atrophy in transgenic mice. Suppression of CACNA1A IRES function in SCA6 may be a potential therapeutic strategy. PMID:23827678

Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

PubMed Central

Baumann, Sascha; Sander, Adrianne; Gurnon, James R.; Yanai-Balser, Giane; VanEtten, James L.; Piotrowski, Markus

2007-01-01

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+- binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses. PMID:17101165

Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

USDA-ARS?s Scientific Manuscript database

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells.

PubMed

Minchenko, O H; Riabovol, O O; Tsymbal, D O; Minchenko, D O; Ratushna, O O

2016-01-01

We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2), malic enzyme 2 (ME2), mitochondrial aspartate aminotransferase (GOT2), and subunit B of succinate dehydrogenase (SDHB) in control (transfected by empty vector) glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) and subunit D of succinate dehydrogenase (SDHD) genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

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

The effect of diet on the expression of lipase genes in the midgut of the lightbrown apple moth (Epiphyas postvittana Walker; Tortricidae).

PubMed

Christeller, J T; Poulton, J; Markwick, N M; Simpson, R M

2010-02-01

We have identified lipase-like genes from an Epiphyas postvittana larval midgut EST library. Of the 10 pancreatic lipase family genes, six appear to encode active lipases and four encode inactive lipases, based on the presence/absence of essential catalytic residues. The four gastric lipase family genes appear to encode active proteins. Phylogenetic analysis of 54 lepidopteran pancreatic lipase proteins resolved the clade into five groups of midgut origin and a sixth of non-midgut lipases. The inactive proteins formed two separate groups with highly conserved mutations. The lepidopteran midgut lipases formed a ninth subfamily of pancreatic lipases. Eighteen insect and human gastric lipases were analysed phylogenetically with only very weak support for any groupings. Gene expression was measured in the larval midgut following feeding on five artificial diets and on apple leaves. The artificial diets contained different levels of triacylglycerol, linoleic acid and cholesterol. Significant changes in gene expression (more than 100-fold for active pancreatic lipases) were observed. All the inactive lipases were also highly expressed. The gastric lipase genes were expressed at lower levels and suppressed in larvae feeding on leaves. Together, protein motif analysis and the gene expression data suggest that, in phytophagous lepidopteran larvae, the pancreatic lipases may function in vivo as galactolipases and phospholipases whereas the gastric lipases may function as triacylglycerol hydrolases.

Frequency and expression of mutacin biosynthesis genes in isolates of Streptococcus mutans with different mutacin-producing phenotypes.

PubMed

Kamiya, Regianne Umeko; Höfling, José Francisco; Gonçalves, Reginaldo Bruno

2008-05-01

The aim of this study was to analyse the frequency and expression of biosynthesis genes in 47 Streptococcus mutans isolates with different mutacin-producing phenotypes. Detection of the frequency and expression of genes encoding mutacin types I, II, III and IV were carried out by PCR and semi-quantitative RT-PCR, respectively, using primers specific for each type of biosynthesis gene. In addition, a further eight genes encoding putative bacteriocins, designated bsm 283, bsm 299, bsm 423, bsm 1889c, bsm 1892c, bsm 1896, bsm 1906c and bsm 1914, were also screened. There was a high phenotypic diversity; some Streptococcus mutans isolates presented broad antimicrobial spectra against other Streptococcus mutans clinical isolates, including bacteria resistant to common antibiotics, as well as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Streptococcus pyogenes. The expression frequency of the bsm gene was higher than that of the previously characterized mutacins (I-IV). There was no positive correlation between the number of indicator strains inhibited (antimicrobial spectra) and the number of biosynthesis genes expressed (Spearman correlation test, r=-0.03, P>0.05). In conclusion, the high diversity of mutacin-producing phenotypes, associated with high frequency of expression of the biosynthesis genes screened, reveals a broad repertoire of genetic determinants encoding antimicrobial peptides that can act in different combinations.

High-level expression of a synthetic gene encoding a sweet protein, monellin, in Escherichia coli.

PubMed

Chen, Zhongjun; Cai, Heng; Lu, Fuping; Du, Lianxiang

2005-11-01

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE.

Transcriptional analysis of immune-related gene expression in p53-deficient mice with increased susceptibility to influenza A virus infection.

PubMed

Yan, Wenjun; Wei, Jianchao; Deng, Xufang; Shi, Zixue; Zhu, Zixiang; Shao, Donghua; Li, Beibei; Wang, Shaohui; Tong, Guangzhi; Ma, Zhiyong

2015-08-18

p53 is a tumor suppressor that contributes to the host immune response against viral infections in addition to its well-established protective role against cancer development. In response to influenza A virus (IAV) infection, p53 is activated and plays an essential role in inhibiting IAV replication. As a transcription factor, p53 regulates the expression of a range of downstream responsive genes either directly or indirectly in response to viral infection. We compared the expression profiles of immune-related genes between IAV-infected wild-type p53 (p53WT) and p53-deficient (p53KO) mice to gain an insight into the basis of p53-mediated antiviral response. p53KO and p53WT mice were infected with influenza A/Puerto Rico/8/1934 (PR8) strain. Clinical symptoms and body weight changes were monitored daily. Lung specimens of IAV-infected mice were collected for analysis of virus titers and gene expression profiles. The difference in immune-related gene expression levels between IAV-infected p53KO and p53WT mice was comparatively determined using microarray analysis and confirmed by quantitative real-time reverse transcription polymerase chain reaction. p53KO mice showed an increased susceptibility to IAV infection compared to p53WT mice. Microarray analysis of gene expression profiles in the lungs of IAV-infected mice indicated that the increased susceptibility was associated with significantly changed expression levels in a range of immune-related genes in IAV-infected p53KO mice. A significantly attenuated expression of Ifng (encoding interferon (IFN)-gamma), Irf7 (encoding IFN regulator factor 7), and antiviral genes, such as Mx2 and Eif2ak2 (encoding PKR), were observed in IAV-infected p53KO mice, suggesting an impaired IFN-mediated immune response against IAV infection in the absence of p53. In addition, dysregulated expression levels of proinflammatory cytokines and chemokines, such as Ccl2 (encoding MCP-1), Cxcl9, Cxcl10 (encoding IP-10), and Tnf, were detected in IAV-infected p53KO mice during early IAV infection, reflecting an aberrant inflammatory response. Lack of p53 resulted in the impaired expression of genes involved in IFN signaling and the dysregulated expression of cytokine and chemokine genes in IAV-infected mice, suggesting an essential role of p53 in the regulation of antiviral and inflammatory responses during IAV infection.

The Gene YALI0E20207g from Yarrowia lipolytica Encodes an N-Acetylglucosamine Kinase Implicated in the Regulated Expression of the Genes from the N-Acetylglucosamine Assimilatory Pathway

PubMed Central

Flores, Carmen-Lisset; Gancedo, Carlos

2015-01-01

The non-conventional yeast Yarrowia lipolytica possesses an ORF, YALI0E20207g, which encodes a protein with an amino acid sequence similar to hexokinases from different organisms. We have cloned that gene and determined several enzymatic properties of its encoded protein showing that it is an N-acetylglucosamine (NAGA) kinase. This conclusion was supported by the lack of growth in NAGA of a strain carrying a YALI0E20207g deletion. We named this gene YlNAG5. Expression of YlNAG5 as well as that of the genes encoding the enzymes of the NAGA catabolic pathway—identified by a BLAST search—was induced by this sugar. Deletion of YlNAG5 rendered that expression independent of the presence of NAGA in the medium and reintroduction of the gene restored the inducibility, indicating that YlNag5 participates in the transcriptional regulation of the NAGA assimilatory pathway genes. Expression of YlNAG5 was increased during sporulation and homozygous Ylnag5/Ylnag5 diploid strains sporulated very poorly as compared with a wild type isogenic control strain pointing to a participation of the protein in the process. Overexpression of YlNAG5 allowed growth in glucose of an Ylhxk1glk1 double mutant and produced, in a wild type background, aberrant morphologies in different media. Expression of the gene in a Saccharomyces cerevisiae hxk1 hxk2 glk1 triple mutant restored ability to grow in glucose. PMID:25816199

Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l.

PubMed Central

2013-01-01

Background Hydrophobins are small secreted cysteine-rich proteins that play diverse roles during different phases of fungal life cycle. In basidiomycetes, hydrophobin-encoding genes often form large multigene families with up to 40 members. The evolutionary forces driving hydrophobin gene expansion and diversification in basidiomycetes are poorly understood. The functional roles of individual genes within such gene families also remain unclear. The relationship between the hydrophobin gene number, the genome size and the lifestyle of respective fungal species has not yet been thoroughly investigated. Here, we present results of our survey of hydrophobin gene families in two species of wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l. We have also investigated the regulatory pattern of hydrophobin-encoding genes from H. annosum s.s. during saprotrophic growth on pine wood as well as on culture filtrate from Phlebiopsis gigantea using micro-arrays. These data are supplemented by results of the protein structure modeling for a representative set of hydrophobins. Results We have identified hydrophobin genes from the genomes of two wood-degrading species of basidiomycetes, Heterobasidion irregulare, representing one of the microspecies within the aggregate H. annosum s.l., and Phlebia brevispora. Although a high number of hydrophobin-encoding genes were observed in H. irregulare (16 copies), a remarkable expansion of these genes was recorded in P. brevispora (26 copies). A significant expansion of hydrophobin-encoding genes in other analyzed basidiomycetes was also documented (1–40 copies), whereas contraction through gene loss was observed among the analyzed ascomycetes (1–11 copies). Our phylogenetic analysis confirmed the important role of gene duplication events in the evolution of hydrophobins in basidiomycetes. Increased number of hydrophobin-encoding genes appears to have been linked to the species’ ecological strategy, with the non-pathogenic fungi having increased numbers of hydrophobins compared with their pathogenic counterparts. However, there was no significant relationship between the number of hydrophobin-encoding genes and genome size. Furthermore, our results revealed significant differences in the expression levels of the 16 H. annosum s.s. hydrophobin-encoding genes which suggest possible differences in their regulatory patterns. Conclusions A considerable expansion of the hydrophobin-encoding genes in basidiomycetes has been observed. The distribution and number of hydrophobin-encoding genes in the analyzed species may be connected to their ecological preferences. Results of our analysis also have shown that H. annosum s.l. hydrophobin-encoding genes may be under positive selection. Our gene expression analysis revealed differential expression of H. annosum s.s. hydrophobin genes under different growth conditions, indicating their possible functional diversification. PMID:24188142

Cloning and characterization of the nagA gene that encodes beta-n-acetylglucosaminidase from Aspergillus nidulans and its expression in Aspergillus oryzae.

PubMed

Kim, Sunhwa; Matsuo, Ichiro; Ajisaka, Katsumi; Nakajima, Harushi; Kitamoto, Katsuhiko

2002-10-01

We isolated a beta-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed beta-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of beta-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture.

Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631.

PubMed

Yamada, Osamu; Sakamoto, Kazutoshi; Tominaga, Mihoko; Nakayama, Tasuku; Koseki, Takuya; Fujita, Akiko; Akita, Osamu

2005-03-01

We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.

Sexual selection, genetic conflict, selfish genes, and the atypical patterns of gene expression in spermatogenic cells.

PubMed

Kleene, Kenneth C

2005-01-01

This review proposes that the peculiar patterns of gene expression in spermatogenic cells are the consequence of powerful evolutionary forces known as sexual selection. Sexual selection is generally characterized by intense competition of males for females, an enormous variety of the strategies to maximize male reproductive success, exaggerated male traits at all levels of biological organization, co-evolution of sexual traits in males and females, and conflict between the sexual advantage of the male trait and the reproductive fitness of females and the individual fitness of both sexes. In addition, spermatogenesis is afflicted by selfish genes that promote their transmission to progeny while causing deleterious effects. Sexual selection, selfish genes, and genetic conflict provide compelling explanations for many atypical features of gene expression in spermatogenic cells including the gross overexpression of certain mRNAs, transcripts encoding truncated proteins that cannot carry out basic functions of the proteins encoded by the same genes in somatic cells, the large number of gene families containing paralogous genes encoding spermatogenic cell-specific isoforms, the large number of testis-cancer-associated genes that are expressed only in spermatogenic cells and malignant cells, and the overbearing role of Sertoli cells in regulating the number and quality of spermatozoa.

Field Evaluation of a Fluorogenic Probe-Based PCR Assay for Identification of a Visceral Leishmaniasis Gene Target

DTIC Science & Technology

2004-06-01

encodes protein required for amastigote development, which can ultimately be expressed in humans as VL (3, 4, 5). The leishmaniasises are also expressed ...Leishmania surveillance at Tallil Air Base, south central Iraq, expressed concern of a potential leishmaniasis outbreak situation. In response, we...site. That L. donovani promastigote-to-amastigote development, and VL pathogenesis, requires an A2 gene family encoded factor defines this protein

Expression profiles of key phenylpropanoid genes during Vanilla planifolia pod development reveal a positive correlation between PAL gene expression and vanillin biosynthesis.

PubMed

Fock-Bastide, Isabelle; Palama, Tony Lionel; Bory, Séverine; Lécolier, Aurélie; Noirot, Michel; Joët, Thierry

2014-01-01

In Vanilla planifolia pods, development of flavor precursors is dependent on the phenylpropanoid pathway. The distinctive vanilla aroma is produced by numerous phenolic compounds of which vanillin is the most important. Because of the economic importance of vanilla, vanillin biosynthetic pathways have been extensively studied but agreement has not yet been reached on the processes leading to its accumulation. In order to explore the transcriptional control exerted on these pathways, five key phenylpropanoid genes expressed during pod development were identified and their mRNA accumulation profiles were evaluated during pod development and maturation using quantitative real-time PCR. As a prerequisite for expression analysis using qRT-PCR, five potential reference genes were tested, and two genes encoding Actin and EF1 were shown to be the most stable reference genes for accurate normalization during pod development. For the first time, genes encoding a phenylalanine ammonia-lyase (VpPAL1) and a cinnamate 4-hydroxylase (VpC4H1) were identified in vanilla pods and studied during maturation. Among phenylpropanoid genes, differential regulation was observed from 3 to 8 months after pollination. VpPAL1 was gradually up-regulated, reaching the maximum expression level at maturity. In contrast, genes encoding 4HBS, C4H, OMT2 and OMT3 did not show significant increase in expression levels after the fourth month post-pollination. Expression profiling of these key phenylpropanoid genes is also discussed in light of accumulation patterns for key phenolic compounds. Interestingly, VpPAL1 gene expression was shown to be positively correlated to maturation and vanillin accumulation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Two pheromone precursor genes are transcriptionally expressed in the homothallic ascomycete Sordaria macrospora.

PubMed

Pöggeler, S

2000-06-01

In order to analyze the involvement of pheromones in cell recognition and mating in a homothallic fungus, two putative pheromone precursor genes, named ppg1 and ppg2, were isolated from a genomic library of Sordaria macrospora. The ppg1 gene is predicted to encode a precursor pheromone that is processed by a Kex2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. The ppg2 gene encodes a 24-amino-acid polypeptide that contains a putative farnesylated and carboxy methylated C-terminal cysteine residue. The sequences of the predicted pheromones display strong structural similarity to those encoded by putative pheromones of heterothallic filamentous ascomycetes. Both genes are expressed during the life cycle of S. macrospora. This is the first description of pheromone precursor genes encoded by a homothallic fungus. Southern-hybridization experiments indicated that ppg1 and ppg2 homologues are also present in other homothallic ascomycetes.

Innate responses to gene knockouts impact overlapping gene networks and vary with respect to resistance to viral infection.

PubMed

Liu, Yonghong; Liu, Yuanyuan; Wu, Jiaming; Roizman, Bernard; Zhou, Grace Guoying

2018-04-03

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: ( i ) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. ( ii ) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica

PubMed Central

Blackman, Leila M.; Cullerne, Darren P.; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R.

2015-01-01

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response. PMID:26332397

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica.

PubMed

Blackman, Leila M; Cullerne, Darren P; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R

2015-01-01

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response.

Environmental conditions affect transcription of the pectinase genes of Erwinia chrysanthemi 3937.

PubMed Central

Hugouvieux-Cotte-Pattat, N; Dominguez, H; Robert-Baudouy, J

1992-01-01

To depolymerize plant pectin, the phytopathogenic enterobacterium Erwinia chrysanthemi produces a series of enzymes which include a pectin-methyl-esterase encoded by the pem gene and five isoenzymes of pectate lyases encoded by the five genes pelA, pelB, pelC, pelD, and pelE. We have constructed transcriptional fusions between the pectinase gene promoters and the uidA gene, encoding beta-glucuronidase, to study the regulation of these E. chrysanthemi pectinase genes individually. The transcription of the pectinase genes is dependent on many environmental conditions. All the fusions were induced by pectic catabolic products and responded, to different degrees, to growth phase, catabolite repression, temperature, and nitrogen starvation. Transcription of pelA, pelD, and pelE was also increased in anaerobic growth conditions. High osmolarity of the culture medium increased expression of pelE but decreased that of pelD; the other pectinase genes were not affected. The level of expression of each gene was different. Transcription of pelA was very low under all growth conditions. The expression of the pelB, pelC, and pem genes was intermediate. The pelE gene had a high basal level of expression. Expression of pelD was generally the most affected by changes in culture conditions and showed a low basal level but very high induced levels. These differences in the expression of the pectinase genes of E. chrysanthemi 3937 presumably reflect their role during infection of plants, because the degradation of pectic polymers of the plant cell walls is the main determinant of tissue maceration caused by soft rot erwiniae. PMID:1447147

Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells.

PubMed

Grove, J R; Deutsch, P J; Price, D J; Habener, J F; Avruch, J

1989-11-25

Plasmids that encode a bioactive amino-terminal fragment of the heat-stable inhibitor of the cAMP-dependent protein kinase, PKI(1-31), were employed to characterize the role of this protein kinase in the control of transcriptional activity mediated by three DNA regulatory elements in the JEG-3 human placental cell line. The 5'-flanking sequence of the human collagenase gene contains the heptameric sequence, 5'-TGAGTCA-3', previously identified as a "phorbol ester" response element. Reporter genes containing either the intact 1.2-kilobase 5'-flanking sequence from the human collagenase gene or just the 7-base pair (bp) response element, when coupled to an enhancerless promoter, each exhibit both cAMP and phorbol ester-stimulated expression in JEG-3 cells. Cotransfection of either construct with plasmids encoding PKI(1-31) inhibits cAMP-stimulated but not basal- or phorbol ester-stimulated expression. Pretreatment of cells with phorbol ester for 1 or 2 days abrogates completely the response to rechallenge with phorbol ester but does not alter the basal expression of either construct; cAMP-stimulated expression, while modestly inhibited, remains vigorous. The 5'-flanking sequence of the human chorionic gonadotropin-alpha subunit (HCG alpha) gene has two copies of the sequence, 5'-TGACGTCA-3', contained in directly adjacent identical 18-bp segments, previously identified as a cAMP-response element. Reporter genes containing either the intact 1.5 kilobase of 5'-flanking sequence from the HCG alpha gene, or just the 36-bp tandem repeat cAMP response element, when coupled to an enhancerless promoter, both exhibit a vigorous cAMP stimulation of expression but no response to phorbol ester in JEG-3 cells. Cotransfection with plasmids encoding PKI(1-31) inhibits both basal and cAMP-stimulated expression in a parallel fashion. The 5'-flanking sequence of the human enkephalin gene mediates cAMP-stimulated expression of reporter genes in both JEG-3 and CV-1 cells. Plasmids encoding PKI(1-31) inhibit the expression that is stimulated by the addition of cAMP analogs in both cell lines; basal expression, however, is inhibited by PKI(1-31) only in the JEG-3 cell line and not in the CV-1 cells. These observations indicate that, in JEG-3 cells, PKI(1-31) is a specific inhibitor of kinase A-mediated gene transcription, but it does not modify kinase C-directed transcription.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of the nuclear gene TaF(A)d is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm.

PubMed

Xu, Pei; Yang, Yuwen; Zhang, Zhengzhi; Chen, Weihua; Zhang, Caiqin; Zhang, Lixia; Zou, Sixiang; Ma, Zhengqiang

2008-01-01

Alterations of mitochondrial-encoded subunits of the F(o)F(1)-ATP synthase are frequently associated with cytoplasmic male sterility (CMS) in plants; however, little is known about the relationship of the nuclear encoded subunits of this enzyme with CMS. In the present study, the full cDNA of the gene TaF(A)d that encodes the putative F(A)d subunit of the F(o)F(1)-ATP synthase was isolated from the wheat (Triticum aestivum) fertility restorer '2114' for timopheevii cytoplasm-based CMS. The deduced 238 amino acid polypeptide is highly similar to its counterparts in dicots and other monocots but has low homology to its mammalian equivalents. TaF(A)d is a single copy gene in wheat and maps to the short arm of the group 6 chromosomes. Transient expression of the TaF(A)d-GFP fusion in onion epidermal cells demonstrated TaF(A)d's mitochondrial location. TaF(A)d was expressed abundantly in stem, leaf, anther, and ovary tissues of 2114. Nevertheless, its expression was repressed in anthers of CMS plants with timopheevii cytoplasm. Genic male sterility did not affect its expression in anthers. The expression of the nuclear gene encoding the 20 kDa subunit of F(o) was down-regulated in a manner similar to TaF(A)d in the T-CMS anthers while that of genes encoding the 6 kDa subunit of F(o) and the gamma subunit of F(1) was unaffected. These observations implied that TaF(A)d is under mitochondrial retrograde regulation in the anthers of CMS plants with timopheevii cytoplasm.

Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function

USDA-ARS?s Scientific Manuscript database

This study is focused on the characterization and expression of genes in the red flour beetle, Tribolium castaneum, encoding proteins that possess six-cysteine-containing chitin-binding domains (CBDs) related to the peritrophin A domain (ChtBD2). An exhaustive bioinformatics search of the genome of...

Impact assessment of bisphenol A on lignin-modifying enzymes by basidiomycete Trametes versicolor.

PubMed

Takamiya, Minako; Magan, Naresh; Warner, Philip J

2008-06-15

The impact of different concentrations of bisphenol A (BPA) was evaluated on growth of the white-rot basidiomycete, Trametes versicolor, and on the expression of genes encoding lignin-modifying enzyme (LME) activities. Effective doses (EDs) were obtained from fungal growth rate to monitor LME activities and the expression levels of their encoding genes. The fungus showed mycelial growth at concentrations of up to 300 microg ml(-1) of BPA with an ED50 value of 185 microg ml(-1). The LME activities were stimulated by BPA concentrations up to 300 microg ml(-1). The lignin peroxidase (LIP) encoding gene may be sensitive to BPA stress.

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

PubMed

Ruíz, Teresa; De la Rosa, José M; Domínguez, Angel; Rodríguez, Luis

2003-05-01

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.

Massive Collection of Full-Length Complementary DNA Clones and Microarray Analyses:. Keys to Rice Transcriptome Analysis

NASA Astrophysics Data System (ADS)

Kikuchi, Shoshi

2009-02-01

Completion of the high-precision genome sequence analysis of rice led to the collection of about 35,000 full-length cDNA clones and the determination of their complete sequences. Mapping of these full-length cDNA sequences has given us information on (1) the number of genes expressed in the rice genome; (2) the start and end positions and exon-intron structures of rice genes; (3) alternative transcripts; (4) possible encoded proteins; (5) non-protein-coding (np) RNAs; (6) the density of gene localization on the chromosome; (7) setting the parameters of gene prediction programs; and (8) the construction of a microarray system that monitors global gene expression. Manual curation for rice gene annotation by using mapping information on full-length cDNA and EST assemblies has revealed about 32,000 expressed genes in the rice genome. Analysis of major gene families, such as those encoding membrane transport proteins (pumps, ion channels, and secondary transporters), along with the evolution from bacteria to higher animals and plants, reveals how gene numbers have increased through adaptation to circumstances. Family-based gene annotation also gives us a new way of comparing organisms. Massive amounts of data on gene expression under many kinds of physiological conditions are being accumulated in rice oligoarrays (22K and 44K) based on full-length cDNA sequences. Cluster analyses of genes that have the same promoter cis-elements, that have similar expression profiles, or that encode enzymes in the same metabolic pathways or signal transduction cascades give us clues to understanding the networks of gene expression in rice. As a tool for that purpose, we recently developed "RiCES", a tool for searching for cis-elements in the promoter regions of clustered genes.

Diverse expression levels of two codon-optimized genes that encode human papilloma virus type 16 major protein L1 in Hansenula polymorpha.

PubMed

Liu, Cunbao; Yang, Xu; Yao, Yufeng; Huang, Weiwei; Sun, Wenjia; Ma, Yanbing

2014-05-01

Two versions of an optimized gene that encodes human papilloma virus type 16 major protein L1 were designed according to the codon usage frequency of Pichia pastoris. Y16 was highly expressed in both P. pastoris and Hansenula polymorpha. M16 expression was as efficient as that of Y16 in P. pastoris, but merely detectable in H. polymorpha even though transcription levels of M16 and Y16 were similar. H. polymorpha had a unique codon usage frequency that contains many more rare codons than Saccharomyces cerevisiae or P. pastoris. These findings indicate that even codon-optimized genes that are expressed well in S. cerevisiae and P. pastoris may be inefficiently expressed in H. polymorpha; thus rare codons must be avoided when universal optimized gene versions are designed to facilitate expression in a variety of yeast expression systems, especially H. polymorpha is involved.

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex.

PubMed

Łopieńska-Biernat, E; Zaobidna, E A; Dmitryjuk, M

2015-01-01

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen-trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)-in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes.

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex

PubMed Central

Łopieńska-Biernat, E.; Zaobidna, E. A.; Dmitryjuk, M.

2015-01-01

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen—trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)—in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes. PMID:26783451

Differential expression of the nuclear-encoded mitochondrial transcriptome in pediatric septic shock.

PubMed

Weiss, Scott L; Cvijanovich, Natalie Z; Allen, Geoffrey L; Thomas, Neal J; Freishtat, Robert J; Anas, Nick; Meyer, Keith; Checchia, Paul A; Shanley, Thomas P; Bigham, Michael T; Fitzgerald, Julie; Banschbach, Sharon; Beckman, Eileen; Howard, Kelli; Frank, Erin; Harmon, Kelli; Wong, Hector R

2014-11-19

Increasing evidence supports a role for mitochondrial dysfunction in organ injury and immune dysregulation in sepsis. Although differential expression of mitochondrial genes in blood cells has been reported for several diseases in which bioenergetic failure is a postulated mechanism, there are no data about the blood cell mitochondrial transcriptome in pediatric sepsis. We conducted a focused analysis using a multicenter genome-wide expression database of 180 children ≤ 10 years of age with septic shock and 53 healthy controls. Using total RNA isolated from whole blood within 24 hours of PICU admission for septic shock, we evaluated 296 nuclear-encoded mitochondrial genes using a false discovery rate of 1%. A series of bioinformatic approaches were applied to compare differentially expressed genes across previously validated gene expression-based subclasses (groups A, B, and C) of pediatric septic shock. In total, 118 genes were differentially regulated in subjects with septic shock compared to healthy controls, including 48 genes that were upregulated and 70 that were downregulated. The top scoring canonical pathway was oxidative phosphorylation, with general downregulation of the 51 genes corresponding to the electron transport system (ETS). The top two gene networks were composed primarily of mitochondrial ribosomal proteins highly connected to ETS complex I, and genes encoding for ETS complexes I, II, and IV that were highly connected to the peroxisome proliferator activated receptor (PPAR) family. There were 162 mitochondrial genes differentially regulated between groups A, B, and C. Group A, which had the highest maximum number of organ failures and mortality, exhibited a greater downregulation of mitochondrial genes compared to groups B and C. Based on a focused analysis of a pediatric septic shock transcriptomic database, nuclear-encoded mitochondrial genes were differentially regulated early in pediatric septic shock compared to healthy controls, as well as across genotypic and phenotypic distinct pediatric septic shock subclasses. The nuclear genome may be an important mechanism contributing to alterations in mitochondrial bioenergetic function and outcomes in pediatric sepsis.

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.

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

Function of Protein Phosphatase 2A in Control of Proliferation: Isolation and Analysis of Dominant-Defective Mutants

DTIC Science & Technology

1999-06-01

subunits are expressed ubiquitously and appear to be encoded by small and quite homogeneous gene families. In plants , however, A and C subunit gene...1996). In both plants and animals, different B subunit isoforms are encoded by two or more unrelated gene families, some of which are expressed in a...PP2A functions in whole plants and in mammalian tissue culture cells. This genetic system may also prove useful for analyzing interactions between

Cloning and expression of clt genes encoding milk-clotting proteases from Myxococcus xanthus 422.

PubMed

Poza, M; Prieto-Alcedo, M; Sieiro, C; Villa, T G

2004-10-01

The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.

The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds.

PubMed

de Vries, Ronald P; vanKuyk, Patricia A; Kester, Harry C M; Visser, Jaap

2002-04-15

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin.

The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds.

PubMed Central

de Vries, Ronald P; vanKuyk, Patricia A; Kester, Harry C M; Visser, Jaap

2002-01-01

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin. PMID:11931668

Identification of InuR, a new Zn(II)2Cys6 transcriptional activator involved in the regulation of inulinolytic genes in Aspergillus niger.

PubMed

Yuan, Xiao-Lian; Roubos, Johannes A; van den Hondel, Cees A M J J; Ram, Arthur F J

2008-01-01

The expression of inulinolytic genes in Aspergillus niger is co-regulated and induced by inulin and sucrose. We have identified a positive acting transcription factor InuR, which is required for the induced expression of inulinolytic genes. InuR is a member of the fungal specific class of transcription factors of the Zn(II)2Cys6 type. Involvement of InuR in inulin and sucrose metabolism was suspected because of the clustering of inuR gene with sucB, which encodes an intracellular invertase with transfructosylation activity and a putative sugar transporter encoding gene (An15g00310). Deletion of the inuR gene resulted in a strain displaying a severe reduction in growth on inulin and sucrose medium. Northern analysis revealed that expression of inulinolytic and sucrolytic genes, e.g., inuE, inuA, sucA, as well as the putative sugar transporter gene (An15g00310) is dependent on InuR. Genome-wide expression analysis revealed, three additional putative sugar transporters encoding genes (An15g04060, An15g03940 and An17g01710), which were strongly induced by sucrose in an InuR dependent way. In silico analysis of the promoter sequences of strongly InuR regulated genes suggests that InuR might bind as dimer to two CGG triplets, which are separated by eight nucleotides.

Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

PubMed

Cui, Hao-Ran; Zhang, Zheng-Rong; Lv, Wei; Xu, Jia-Ning; Wang, Xiao-Yun

2015-08-01

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

Protein tyrosine phosphatase encoded in Cotesia plutellae bracovirus suppresses a larva-to-pupa metamorphosis of the diamondback moth, Plutella xylostella.

PubMed

Kim, Jiwan; Hepat, Rahul; Lee, Daeweon; Kim, Yonggyun

2013-09-01

Parasitization by an endoparasitoid wasp, Cotesia plutellae, inhibits a larva-to-pupa metamorphosis of the diamondback moth, Plutella xylostella. This study tested an inhibitory effect of C. plutellae bracovirus (CpBV) on the metamorphosis of P. xylostella. Parasitized P. xylostella exhibited significantly reduced prothoracic gland (PTG) development at the last instar compared to nonparasitized larvae. Expression of the ecdysone receptor (EcR) was markedly suppressed during the last instar larvae parasitized by C. plutellae. By contrast, expression of the insulin receptor (InR) significantly increased in the parasitized larvae. Microinjection of CpBV significantly inhibited the larva-to-pupa metamorphosis of nonparasitized larvae in a dose-dependent manner. Injection of CpBV also inhibited the expression of the EcR and increased the expression of the InR. Individual CpBV segments were transiently expressed in its encoded genes in nonparasitized larvae and screened to determine antimetamorphic viral gene(s). Out of 21 CpBV segments, two viral segments (CpBV-S22 and CpBV-S27) were proved to inhibit larva-to-pupa metamorphosis by transient expression assay. RNA interference of each gene encoded in the viral segments was applied to determine antimetamorphic gene(s). Protein tyrosine phosphatase, early expressed gene, and four hypothetical genes were selected to be associated with the antimetamorphic activity of CpBV. These results suggest that antimetamorphosis of P. xylostella parasitized by C. plutellae is induced by inhibiting PTG development and subsequent ecdysteroid signaling with viral factors of CpBV. Copyright © 2013 Elsevier Inc. All rights reserved.

A Brassica oleracea gene expressed in a variety-specific manner may encode a novel plant transmembrane receptor.

PubMed

Palmer, J E; Dikeman, D A; Fujinuma, T; Kim, B; Jones, J I; Denda, M; Martínez-Zapater, J M; Cruz-Alvarez, M

2001-04-01

The species Brassica oleracea includes several agricultural varieties characterized by the proliferation of different types of meristems. Using a combination of subtractive hybridization and PCR (polymerase chain reaction) techniques we have identified several genes which are expressed in the reproductive meristems of the cauliflower curd (B. oleracea var. botrytis) but not in the vegetative meristems of Brussels sprouts (B. oleracea var. gemmifera) axillary buds. One of the cloned genes, termed CCE1 (CAULIFLOWER CURD EXPRESSION 1) shows specific expression in the botrytis variety. Preferential expression takes place in this variety in the meristems of the curd and in the stem throughout the vegetative and reproductive stages of plant growth. CCE1 transcripts are not detected in any of the organs of other B. oleracea varieties analyzed. Based on the nucleotide sequence of a cDNA encompassing the complete coding region, we predict that this gene encodes a transmembrane protein, with three transmembrane domains. The deduced amino acid sequence includes motifs conserved in G-protein-coupled receptors (GPCRs) from yeast and animal species. Our results suggest that the cloned gene encodes a protein belonging to a new, so far unidentified, family of transmembrane receptors in plants. The expression pattern of the gene suggests that the receptor may be involved in the control of meristem development/arrest that takes place in cauliflower.

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.

The significance of alternative transcripts for Caenorhabditis elegans transcription factor genes, based on expression pattern analysis

PubMed Central

2013-01-01

Background Sequence-specific DNA-binding proteins, with their paramount importance in the regulation of expression of the genetic material, are encoded by approximately 5% of the genes in an animal’s genome. But it is unclear to what extent alternative transcripts from these genes may further increase the complexity of the transcription factor complement. Results Of the 938 potential C. elegans transcription factor genes, 197 were annotated in WormBase as encoding at least two distinct isoforms. Evaluation of prior evidence identified, with different levels of confidence, 50 genes with alternative transcript starts, 23 with alternative transcript ends, 35 with alternative splicing and 34 with alternative transcripts generated by a combination of mechanisms, leaving 55 that were discounted. Expression patterns were determined for transcripts for a sample of 29 transcription factor genes, concentrating on those with alternative transcript starts for which the evidence was strongest. Seamless fosmid recombineering was used to generate reporter gene fusions with minimal modification to assay expression of specific transcripts while maintaining the broad genomic DNA context and alternative transcript production. Alternative transcription factor gene transcripts were typically expressed with identical or substantially overlapping distributions rather than in distinct domains. Conclusions Increasingly sensitive sequencing technologies will reveal rare transcripts but many of these are clearly non-productive. The majority of the transcription factor gene alternative transcripts that are productive may represent tolerable noise rather than encoding functionally distinct isoforms. PMID:23586691

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

PubMed

Iuchi, S; Cole, S T; Lin, E C

1990-01-01

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.

Effect of long-term actual spaceflight on the expression of key genes encoding serotonin and dopamine system

NASA Astrophysics Data System (ADS)

Popova, Nina; Shenkman, Boris; Naumenko, Vladimir; Kulikov, Alexander; Kondaurova, Elena; Tsybko, Anton; Kulikova, Elisabeth; Krasnov, I. B.; Bazhenova, Ekaterina; Sinyakova, Nadezhda

The effect of long-term spaceflight on the central nervous system represents important but yet undeveloped problem. The aim of our work was to study the effect of 30-days spaceflight of mice on Russian biosatellite BION-M1 on the expression in the brain regions of key genes of a) serotonin (5-HT) system (main enzymes in 5-HT metabolism - tryptophan hydroxylase-2 (TPH-2), monoamine oxydase A (MAO A), 5-HT1A, 5-HT2A and 5-HT3 receptors); b) pivotal enzymes in DA metabolism (tyrosine hydroxylase, COMT, MAO A, MAO B) and D1, D2 receptors. Decreased expression of genes encoding the 5-HT catabolism (MAO A) and 5-HT2A receptor in some brain regions was shown. There were no differences between “spaceflight” and control mice in the expression of TPH-2 and 5-HT1A, 5-HT3 receptor genes. Significant changes were found in genetic control of DA system. Long-term spaceflight decreased the expression of genes encoding the enzyme in DA synthesis (tyrosine hydroxylase in s.nigra), DA metabolism (MAO B in the midbrain and COMT in the striatum), and D1 receptor in hypothalamus. These data suggested that 1) microgravity affected genetic control of 5-HT and especially the nigrostriatal DA system implicated in the central regulation of muscular tonus and movement, 2) the decrease in the expression of genes encoding key enzyme in DA synthesis, DA degradation and D1 receptor contributes to the movement impairment and dyskinesia produced by the spaceflight. The study was supported by Russian Foundation for Basic Research grant No. 14-04-00173.

AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000

USDA-ARS?s Scientific Manuscript database

Plant pathogenic bacteria are able to integrate information about their environment and adjust gene expression to provide adaptive functions. AlgU, an ECF sigma factor encoded by Pseudomonas syringae, controls expression of genes for alginate biosynthesis and is active while the bacteria are associa...

The Yersinia pestis gcvB gene encodes two small regulatory RNA molecules

PubMed Central

McArthur, Sarah D; Pulvermacher, Sarah C; Stauffer, George V

2006-01-01

Background In recent years it has become clear that small non-coding RNAs function as regulatory elements in bacterial virulence and bacterial stress responses. We tested for the presence of the small non-coding GcvB RNAs in Y. pestis as possible regulators of gene expression in this organism. Results In this study, we report that the Yersinia pestis KIM6 gcvB gene encodes two small RNAs. Transcription of gcvB is activated by the GcvA protein and repressed by the GcvR protein. The gcvB-encoded RNAs are required for repression of the Y. pestis dppA gene, encoding the periplasmic-binding protein component of the dipeptide transport system, showing that the GcvB RNAs have regulatory activity. A deletion of the gcvB gene from the Y. pestis KIM6 chromosome results in a decrease in the generation time of the organism as well as a change in colony morphology. Conclusion The results of this study indicate that the Y. pestis gcvB gene encodes two small non-coding regulatory RNAs that repress dppA expression. A gcvB deletion is pleiotropic, suggesting that the sRNAs are likely involved in controlling genes in addition to dppA. PMID:16768793

Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication.

PubMed

van der Ley, P

1988-11-01

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.

Gene Expression in Class 2 Integrons Is SOS-Independent and Involves Two Pc Promoters.

PubMed

Jové, Thomas; Da Re, Sandra; Tabesse, Aurore; Gassama-Sow, Amy; Ploy, Marie-Cécile

2017-01-01

Integrons are powerful bacterial genetic elements that permit the expression and dissemination of antibiotic-resistance gene cassettes. They contain a promoter Pc that allows the expression of gene cassettes captured through site-specific recombination catalyzed by IntI, the integron-encoded integrase. Class 1 and 2 integrons are found in both clinical and environmental settings. The regulation of intI and of Pc promoters has been extensively studied in class 1 integrons and the regulatory role of the SOS response on intI expression has been shown. Here we investigated class 2 integrons. We characterized the P intI2 promoter and showed that intI2 expression is not regulated via the SOS response. We also showed that, unlike class 1 integrons, class 2 integrons possess not one but two active Pc promoters that are located within the attI2 region that seem to contribute equally to gene cassette expression. Class 2 integrons mostly encode an inactive truncated integrase, but the rare class 2 integrons that encode an active integrase are associated with less efficient Pc2 promoter variants. We propose an evolutionary model for class 2 integrons in which the absence of repression of the integrase gene expression led to mutations resulting in either inactive integrase or Pc variants of weaker activity, thereby reducing the potential fitness cost of these integrons.

Cloning and characterization of a mouse gene with homology to the human von Hippel-Lindau disease tumor suppressor gene: implications for the potential organization of the human von Hippel-Lindau disease gene.

PubMed

Gao, J; Naglich, J G; Laidlaw, J; Whaley, J M; Seizinger, B R; Kley, N

1995-02-15

The human von Hippel-Lindau disease (VHL) gene has recently been identified and, based on the nucleotide sequence of a partial cDNA clone, has been predicted to encode a novel protein with as yet unknown functions [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. The length of the encoded protein and the characteristics of the cellular expressed protein are as yet unclear. Here we report the cloning and characterization of a mouse gene (mVHLh1) that is widely expressed in different mouse tissues and shares high homology with the human VHL gene. It predicts a protein 181 residues long (and/or 162 amino acids, considering a potential alternative start codon), which across a core region of approximately 140 residues displays a high degree of sequence identity (98%) to the predicted human VHL protein. High stringency DNA and RNA hybridization experiments and protein expression analyses indicate that this gene is the most highly VHL-related mouse gene, suggesting that it represents the mouse VHL gene homologue rather than a related gene sharing a conserved functional domain. These findings provide new insights into the potential organization of the VHL gene and nature of its encoded protein.

Regulation of the expression of plant resistance gene SNC1 by a protein with a conserved BAT2 domain.

PubMed

Li, Yingzhong; Tessaro, Mark J; Li, Xin; Zhang, Yuelin

2010-07-01

Plant Resistance (R) genes encode immune receptors that recognize pathogens and activate defense responses. Because of fitness costs associated with maintaining R protein-mediated resistance, expression levels of R genes have to be tightly regulated. However, mechanisms on how R-gene expression is regulated are poorly understood. Here we show that MODIFIER OF snc1, 1 (MOS1) regulates the expression of SUPPRESSOR OF npr1-1, CONSTITUTIVE1 (SNC1), which encodes a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat type of R protein in Arabidopsis (Arabidopsis thaliana). In the mos1 loss-of-function mutant plants, snc1 expression is repressed and constitutive resistance responses mediated by snc1 are lost. The repression of snc1 expression in mos1 is released by knocking out DECREASE IN DNA METHYLATION1. In mos1 mutants, DNA methylation in a region upstream of SNC1 is altered. Furthermore, expression of snc1 transgenes using the native promoter does not require MOS1, indicating that regulation of SNC1 expression by MOS1 is at the chromatin level. Map-based cloning of MOS1 revealed that it encodes a novel protein with a HLA-B ASSOCIATED TRANSCRIPT2 (BAT2) domain that is conserved in plants and animals. Our study on MOS1 suggests that BAT2 domain-containing proteins may function in regulation of gene expression at chromatin level.

Differential gene expression in Staphylococcus aureus exposed to Orange II and Sudan III azo dyes

PubMed Central

Pan, Hongmiao; Xu, Joshua; Kweon, Oh-Gew; Zou, Wen; Feng, Jinhui; He, Gui-Xin; Cerniglia, Carl E.

2018-01-01

We previously demonstrated the effects of azo dyes and their reduction metabolites on bacterial cell growth and cell viability. In this report, the effects of Orange II and Sudan III on gene expression profiling in Staphylococcus aureus ATCC BAA 1556 were analyzed using microarray and quantitative RT-PCR technology. Upon exposure to 6 μg/ml Orange II for 18 h, 21 genes were found to be differently expressed. Among them, 8 and 13 genes were up- and down-regulated, respectively. Most proteins encoded by these differentially expressed genes involve stress response caused by drug metabolism, oxidation, and alkaline shock indicating that S. aureus could adapt to Orange II exposure through a balance between up and down regulated gene expression. Whereas, after exposure to 6 μg/ml Sudan III for 18 h, 57 genes were differentially expressed. In which, 51 genes were up-regulated and 6 were down-regulated. Most proteins encoded by these differentially expressed genes involve in cell wall/membrane biogenesis and biosynthesis, nutrient uptake, transport and metabolite, and stress response, suggesting that Sudan III damages the bacterial cell wall or/and membrane due to binding of the dye. Further analysis indicated that all differentially expressed genes encoded membrane proteins were up-regulated and most of them serve as transporters. The result suggested that these genes might contribute to survival, persistence and growth in the presence of Sudan III. Only one gene msrA, which plays an important role in oxidative stress resistance, was found to be down-regulated after exposure to both Orange II and Sudan III. The present results suggested that both these two azo dyes can cause stress in S. aureus and the response of the bacterium to the stress is mainly related to characteristics of the azo dyes. PMID:25720844

Gene expression profiling of Listeria monocytogenes strain F2365 during growth in ultrahigh-temperature-processed skim milk.

PubMed

Liu, Yanhong; Ream, Amy

2008-11-01

To study how Listeria monocytogenes survives and grows in ultrahigh-temperature-processed (UHT) skim milk, microarray technology was used to monitor the gene expression profiles of strain F2365 in UHT skim milk. Total RNA was isolated from strain F2365 in UHT skim milk after 24 h of growth at 4 degrees C, labeled with fluorescent dyes, and hybridized to "custom-made" commercial oligonucleotide (35-mers) microarray chips containing the whole genome of L. monocytogenes strain F2365. Compared to L. monocytogenes grown in brain heart infusion (BHI) broth for 24 h at 4 degrees C, 26 genes were upregulated (more-than-twofold increase) in UHT skim milk, whereas 14 genes were downregulated (less-than-twofold decrease). The upregulated genes included genes encoding transport and binding proteins, transcriptional regulators, proteins in amino acid biosynthesis and energy metabolism, protein synthesis, cell division, and hypothetical proteins. The downregulated genes included genes that encode transport and binding proteins, protein synthesis, cellular processes, cell envelope, energy metabolism, a transcriptional regulator, and an unknown protein. The gene expression changes determined by microarray assays were confirmed by real-time reverse transcriptase PCR analyses. Furthermore, cells grown in UHT skim milk displayed the same sensitivity to hydrogen peroxide as cells grown in BHI, demonstrating that the elevated levels of expression of genes encoding manganese transporter complexes in UHT skim milk did not result in changes in the oxidative stress sensitivity. To our knowledge, this report represents a novel study of global transcriptional gene expression profiling of L. monocytogenes in a liquid food.

Molecular and functional characterization of novel fructosyltransferases and invertases from Agave tequilana.

PubMed

Cortés-Romero, Celso; Martínez-Hernández, Aída; Mellado-Mojica, Erika; López, Mercedes G; Simpson, June

2012-01-01

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants.

Molecular and Functional Characterization of Novel Fructosyltransferases and Invertases from Agave tequilana

PubMed Central

Cortés-Romero, Celso; Martínez-Hernández, Aída; Mellado-Mojica, Erika; López, Mercedes G.; Simpson, June

2012-01-01

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants. PMID:22558253

Regulation of HSD17B1 and SRD5A1 in lymphocytes.

PubMed

Zhou, Z; Speiser, P W

1999-11-01

We previously reported lymphocyte expression of genes encoding enzymes required for steroid metabolism; however, only 17beta-HSD and 5alpha-reductase showed significant enzyme activity. We now investigate regulation of lymphocyte expression for genes encoding 17beta-HSD and 5alpha-reductase. Cultured human T and B lymphoid cell lines and peripheral blood mononuclear cells were treated with known regulators of steroidogenic gene expression including forskolin, PMA, ionomycin, various steroids, interleukin (IL)-4, and IL-6. Treatment with 10 or 50 microM forskolin resulted in a 20-60% reduction of expression for HSD17B1 (encoding 17beta-HSD I) in T and B lymphoid cell lines and peripheral blood mononuclear cells, although such a change was not observed in the expression of SRD5A1 (encoding 5alpha-reductase I). No significant changes were found when cells were treated for 24 h with various concentrations of PMA or ionomycin. Incubation with 10(-9) to 10(-7) M androstenedione or estradiol increased expression of HSD17B1, while testosterone decreased the expression of this gene. SRD5A1 expression was increased in the presence of 5alpha-DHT although no consistent changes were observed when the cells were treated with testosterone. Other steroids, including dexamethasone, progesterone, and 6-hydroxypregnanolone, produced no effects on expression of either HSD17B1 or SRD5A1. Treatment with 0.1-10 ng/ml of IL-4 or IL-6 also did not effect significant changes in gene expression. These data implicate the involvement of the cAMP-protein kinase signal transduction pathway in regulating lymphocyte expression of HSD17B1. Furthermore, it appears that lymphocyte HSD17B1 and SRD5A1 are regulated to some extent by specific steroids. Copyright 1999 Academic Press.

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus.

PubMed

Qiu, T; Lu, R H; Zhang, J; Zhu, Z Y

2001-07-01

The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mu1 of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mu1.

Paralogous ALT1 and ALT2 Retention and Diversification Have Generated Catalytically Active and Inactive Aminotransferases in Saccharomyces cerevisiae

PubMed Central

Peñalosa-Ruiz, Georgina; Aranda, Cristina; Ongay-Larios, Laura; Colon, Maritrini; Quezada, Hector; Gonzalez, Alicia

2012-01-01

Background Gene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase. Principal Findings Our results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function. Conclusions ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1 repressor. Presented results indicate that alanine could act as ALT2 Nrg1-co-repressor. PMID:23049841

Comparative differential gene expression analysis of nucleus-encoded proteins for Rafflesia cantleyi against Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Ng, Siuk-Mun; Lee, Xin-Wei; Wan, Kiew-Lian; Firdaus-Raih, Mohd

2015-09-01

Regulation of functional nucleus-encoded proteins targeting the plastidial functions was comparatively studied for a plant parasite, Rafflesia cantleyi versus a photosynthetic plant, Arabidopsis thaliana. This study involved two species of different feeding modes and different developmental stages. A total of 30 nucleus-encoded proteins were found to be differentially-regulated during two stages in the parasite; whereas 17 nucleus-encoded proteins were differentially-expressed during two developmental stages in Arabidopsis thaliana. One notable finding observed for the two plants was the identification of genes involved in the regulation of photosynthesis-related processes where these processes, as expected, seem to be present only in the autotroph.

Nuclear-encoded mitochondrial complex I gene expression is restored to normal levels by inhibition of unedited ATP9 transgene expression in Arabidopsis thaliana.

PubMed

Busi, María V; Gómez-Casati, Diego F; Perales, Mariano; Araya, Alejandro; Zabaleta, Eduardo

2006-01-01

Mitochondria play an important role during sporogenesis in plants. The steady state levels of the nuclear-encoded mitochondrial complex I (nCI), PSST, TYKY and NADHBP transcripts increase in flowers of male-sterile plants with impairment of mitochondrial function generated by the expression of the unedited version of ATP9 (u-ATP9). This suggests a nuclear control of nCI genes in response to the mitochondrial flaw. To evaluate this hypothesis, transgenic plants carrying the GUS reporter gene, under the control of the PSST, TYKY and NADHBP promoters, were constructed. We present evidence that suppression by antisense strategy of the expression of u-ATP9 restores the normal levels of three nCI transcripts, indicating that the increase in PSST, TYKY and NADHBP in plants with a mitochondrial flaw occurs at the transcriptional level. The data presented here support the hypothesis that a mitochondrial dysfunction triggers a retrograde signaling which induce some nuclear-encoded mitochondrial genes. Moreover, these results demonstrate that this is a valuable experimental model for studying nucleus-mitochondria cross-talk events.

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

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F. William; Dubendorff, John W.

1998-01-01

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages

DOEpatents

Studier, F. William; Dubendorff, John W.

1998-01-01

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

PubMed

Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

2015-09-30

Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

Identification and Analysis of the Biosynthetic Gene Cluster Encoding the Thiopeptide Antibiotic Cyclothiazomycin in Streptomyces hygroscopicus 10-22▿ †

PubMed Central

Wang, Jiang; Yu, Yi; Tang, Kexuan; Liu, Wen; He, Xinyi; Huang, Xi; Deng, Zixin

2010-01-01

Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from Streptomyces hygroscopicus 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in Streptomyces lividans 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, cltBCDEFG flanking the structural gene cltA, were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, cltMN, were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides. PMID:20154110

Identification and Characterization of Genes Required for Early Myxococcus xanthus Developmental Gene Expression

PubMed Central

Guo, Dongchuan; Wu, Yun; Kaplan, Heidi B.

2000-01-01

Starvation and cell density regulate the developmental expression of Myxococcus xanthus gene 4521. Three classes of mutants allow expression of this developmental gene during growth on nutrient agar, such that colonies of strains containing a Tn5 lac Ω4521 fusion are Lac+. One class of these mutants inactivates SasN, a negative regulator of 4521 expression; another class activates SasS, a sensor kinase-positive regulator of 4521 expression; and a third class blocks lipopolysaccharide (LPS) O-antigen biosynthesis. To identify additional positive regulators of 4521 expression, 11 Lac− TnV.AS transposon insertion mutants were isolated from a screen of 18,000 Lac+ LPS O-antigen mutants containing Tn5 lac Ω4521 (Tcr). Ten mutations identified genes that could encode positive regulators of 4521 developmental expression based on their ability to abolish 4521 expression during development in the absence of LPS O antigen and in an otherwise wild-type background. Eight of these mutations mapped to the sasB locus, which encodes the known 4521 regulators SasS and SasN. One mapped to sasS, whereas seven identified new genes. Three mutations mapped to a gene encoding an NtrC-like response regulator homologue, designated sasR, and four others mapped to a gene designated sasP. One mutation, designated ssp10, specifically suppressed the LPS O-antigen defect; the ssp10 mutation had no effect on 4521 expression in an otherwise wild-type background but reduced 4521 developmental expression in the absence of LPS O antigen to a level close to that of the parent strain. All of the mutations except those in sasP conferred defects during growth and development. These data indicate that a number of elements are required for 4521 developmental expression and that most of these are necessary for normal growth and fruiting body development. PMID:10913090

Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.

PubMed

Fairley, D J; Wang, G; Rensing, C; Pepper, I L; Larkin, M J

2006-12-01

Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.

The candidate histocompatibility locus of a Basal chordate encodes two highly polymorphic proteins.

PubMed

Nydam, Marie L; Netuschil, Nikolai; Sanders, Erin; Langenbacher, Adam; Lewis, Daniel D; Taketa, Daryl A; Marimuthu, Arumugapradeep; Gracey, Andrew Y; De Tomaso, Anthony W

2013-01-01

The basal chordate Botryllus schlosseri undergoes a natural transplantation reaction governed by a single, highly polymorphic locus called the fuhc. Our initial characterization of this locus suggested it encoded a single gene alternatively spliced into two transcripts: a 555 amino acid-secreted form containing the first half of the gene, and a full-length, 1008 amino acid transmembrane form, with polymorphisms throughout the ectodomain determining outcome. We have now found that the locus encodes two highly polymorphic genes which are separated by a 227 bp intergenic region: first, the secreted form as previously described, and a second gene encoding a 531 amino acid membrane-bound gene containing three extracellular immunoglobulin domains. While northern blotting revealed only these two mRNAs, both PCR and mRNA-seq detect a single capped and polyadenylated transcript that encodes processed forms of both genes linked by the intergenic region, as well as other transcripts in which exons of the two genes are spliced together. These results might suggest that the two genes are expressed as an operon, during which both genes are co-transcribed and then trans-spliced into two separate messages. This type of transcriptional regulation has been described in tunicates previously; however, the membrane-bound gene does not encode a typical Splice Leader (SL) sequence at the 5' terminus that usually accompanies trans-splicing. Thus, the presence of stable transcripts encoding both genes may suggest a novel mechanism of regulation, or conversely may be rare but stable transcripts in which the two mRNAs are linked due to a small amount of read-through by RNA polymerase. Both genes are highly polymorphic and co-expressed on tissues involved in histocompatibility. In addition, polymorphisms on both genes correlate with outcome, although we have found a case in which it appears that the secreted form may be major allorecognition determinant.

Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

PubMed

Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

2015-11-01

Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Topographical cone photopigment gene expression in deutan-type red-green color vision defects.

PubMed

Bollinger, Kathryn; Sjoberg, Stacy A; Neitz, Maureen; Neitz, Jay

2004-01-01

Eye donors were identified who had X-chromosome photopigment gene arrays like those of living deuteranomalous men; the arrays contained two genes encoding long-wavelength sensitive (L) pigments as well as genes to encode middle-wavelength sensitive (M) photopigment. Ultrasensitive methods failed to detect the presence of M photopigment mRNA in the retinas of these deutan donors. This provides direct evidence that deuteranomaly is caused by the complete absence of M pigment mRNA. Additionally, for those eyes with mRNA corresponding to two different L-type photopigments, the ratio of mRNA from the first vs. downstream L genes was analyzed across the retinal topography. Results show that the pattern of first relative to downstream L gene expression in the deuteranomalous retina is similar to the pattern of L vs. M gene expression found in normal retinas.

Chlorophyll metabolism in pollinated vs. parthenocarpic fig fruits throughout development and ripening.

PubMed

Rosianskey, Yogev; Dahan, Yardena; Yadav, Sharawan; Freiman, Zohar E; Milo-Cochavi, Shira; Kerem, Zohar; Eyal, Yoram; Flaishman, Moshe A

2016-08-01

Expression of 13 genes encoding chlorophyll biosynthesis and degradation was evaluated. Chlorophyll degradation was differentially regulated in pollinated and parthenocarpic fig fruits, leading to earlier chlorophyll degradation in parthenocarpic fruits. Varieties of the common fig typically yield a commercial summer crop that requires no pollination, although it can be pollinated. Fig fruit pollination results in larger fruit size, greener skin and darker interior inflorescence color, and slows the ripening process compared to non-pollinated fruits. We evaluated the effect of pollination on chlorophyll content and levels of transcripts encoding enzymes of the chlorophyll metabolism in fruits of the common fig 'Brown Turkey'. We cloned and evaluated the expression of 13 different genes. All 13 genes showed high expression in the fruit skin, inflorescences and leaves, but extremely low expression in roots. Pollination delayed chlorophyll breakdown in the ripening fruit skin and inflorescences. This was correlated with the expression of genes encoding enzymes in the chlorophyll biosynthesis and degradation pathways. Expression of pheophorbide a oxygenase (PAO) was strongly negatively correlated with chlorophyll levels during ripening in pollinated fruits; along with its high expression levels in yellow leaves, this supports a pivotal role for PAO in chlorophyll degradation in figs. Normalizing expression levels of all chlorophyll metabolism genes in the pollinated and parthenocarpic fruit skin and inflorescences showed three synthesis (FcGluTR1, FcGluTR2 and FcCLS1) and three degradation (FcCLH1, FcCLH2 and FcRCCR1) genes with different temporal expression in the pollinated vs. parthenocarpic fruit skin and inflorescences. FcCAO also showed different expressions in the parthenocarpic fruit skin. Thus, chlorophyll degradation is differentially regulated in the pollinated and parthenocarpic fruit skin and inflorescences, leading to earlier and more sustained chlorophyll degradation in the parthenocarpic fruit.

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

PubMed

de Souza, Alessandra A; Takita, Marco A; Coletta-Filho, Helvécio D; Caldana, Camila; Yanai, Giane M; Muto, Nair H; de Oliveira, Regina C; Nunes, Luiz R; Machado, Marcos A

2004-08-15

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family.

PubMed

Pearce, Stephen; Huttly, Alison K; Prosser, Ian M; Li, Yi-dan; Vaughan, Simon P; Gallova, Barbora; Patil, Archana; Coghill, Jane A; Dubcovsky, Jorge; Hedden, Peter; Phillips, Andrew L

2015-06-05

The gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis. The wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1β-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp. The comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD enzymes of the GA pathway in wheat and barley will provide the basis for a better understanding of GA-regulated development in these species. This analysis revealed the existence of a novel, endosperm-specific GA 1-oxidase in wheat and a related GA 3,18-dihydroxylase enzyme in barley that may play important roles during grain expansion and development.

Transcriptome Analysis Revealed Highly Expressed Genes Encoding Secondary Metabolite Pathways and Small Cysteine-Rich Proteins in the Sclerotium of Lignosus rhinocerotis

PubMed Central

Yap, Hui-Yeng Y.; Chooi, Yit-Heng; Fung, Shin-Yee; Ng, Szu-Ting; Tan, Chon-Seng; Tan, Nget-Hong

2015-01-01

Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications. PMID:26606395

Using RNA-seq data to select reference genes for normalizing gene expression in apple roots.

PubMed

Zhou, Zhe; Cong, Peihua; Tian, Yi; Zhu, Yanmin

2017-01-01

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.

Using RNA-seq data to select reference genes for normalizing gene expression in apple roots

PubMed Central

Zhou, Zhe; Cong, Peihua; Tian, Yi

2017-01-01

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization. PMID:28934340

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.

Development of B cells expressing surface immunoglobulin molecules that lack V(D)J-encoded determinants in the avian embryo bursa of Fabricius

PubMed Central

Sayegh, Camil E.; Demaries, Sandra L.; Iacampo, Sandra; Ratcliffe, Michael J. H.

1999-01-01

Immunoglobulin gene rearrangement in avian B cell precursors generates surface Ig receptors of limited diversity. It has been proposed that specificities encoded by these receptors play a critical role in B lineage development by recognizing endogenous ligands within the bursa of Fabricius. To address this issue directly we have introduced a truncated surface IgM, lacking variable region domains, into developing B precursors by retroviral gene transfer in vivo. Cells expressing this truncated receptor lack endogenous surface IgM, and the low level of endogenous Ig rearrangements that have occurred within this population of cells has not been selected for having a productive reading frame. Such cells proliferate rapidly within bursal epithelial buds of normal morphology. In addition, despite reduced levels of endogenous light chain rearrangement, those light chain rearrangements that have occurred have undergone variable region diversification by gene conversion. Therefore, although surface expression of an Ig receptor is required for bursal colonization and the induction of gene conversion, the specificity encoded by the prediversified receptor is irrelevant and, consequently, there is no obligate ligand for V(D)J-encoded determinants of prediversified avian cell surface IgM receptor. PMID:10485907

The ANGULATA7 gene encodes a DnaJ-like zinc finger-domain protein involved in chloroplast function and leaf development in Arabidopsis.

PubMed

Muñoz-Nortes, Tamara; Pérez-Pérez, José Manuel; Ponce, María Rosa; Candela, Héctor; Micol, José Luis

2017-03-01

The characterization of mutants with altered leaf shape and pigmentation has previously allowed the identification of nuclear genes that encode plastid-localized proteins that perform essential functions in leaf growth and development. A large-scale screen previously allowed us to isolate ethyl methanesulfonate-induced mutants with small rosettes and pale green leaves with prominent marginal teeth, which were assigned to a phenotypic class that we dubbed Angulata. The molecular characterization of the 12 genes assigned to this phenotypic class should help us to advance our understanding of the still poorly understood relationship between chloroplast biogenesis and leaf morphogenesis. In this article, we report the phenotypic and molecular characterization of the angulata7-1 (anu7-1) mutant of Arabidopsis thaliana, which we found to be a hypomorphic allele of the EMB2737 gene, which was previously known only for its embryonic-lethal mutations. ANU7 encodes a plant-specific protein that contains a domain similar to the central cysteine-rich domain of DnaJ proteins. The observed genetic interaction of anu7-1 with a loss-of-function allele of GENOMES UNCOUPLED1 suggests that the anu7-1 mutation triggers a retrograde signal that leads to changes in the expression of many genes that normally function in the chloroplasts. Many such genes are expressed at higher levels in anu7-1 rosettes, with a significant overrepresentation of those required for the expression of plastid genome genes. Like in other mutants with altered expression of plastid-encoded genes, we found that anu7-1 exhibits defects in the arrangement of thylakoidal membranes, which appear locally unappressed. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Gene Expression by the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough Grown on an Iron Electrode under Cathodic Protection Conditions▿ †

PubMed Central

Caffrey, Sean M.; Park, Hyung Soo; Been, Jenny; Gordon, Paul; Sensen, Christoph W.; Voordouw, Gerrit

2008-01-01

The genome sequence of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was reanalyzed to design unique 70-mer oligonucleotide probes against 2,824 probable protein-coding regions. These included three genes not previously annotated, including one that encodes a c-type cytochrome. Using microarrays printed with these 70-mer probes, we analyzed the gene expression profile of wild-type D. vulgaris grown on cathodic hydrogen, generated at an iron electrode surface with an imposed negative potential of −1.1 V (cathodic protection conditions). The gene expression profile of cells grown on cathodic hydrogen was compared to that of cells grown with gaseous hydrogen bubbling through the culture. Relative to the latter, the electrode-grown cells overexpressed two hydrogenases, the hyn-1 genes for [NiFe] hydrogenase 1 and the hyd genes, encoding [Fe] hydrogenase. The hmc genes for the high-molecular-weight cytochrome complex, which allows electron flow from the hydrogenases across the cytoplasmic membrane, were also overexpressed. In contrast, cells grown on gaseous hydrogen overexpressed the hys genes for [NiFeSe] hydrogenase. Cells growing on the electrode also overexpressed genes encoding proteins which promote biofilm formation. Although the gene expression profiles for these two modes of growth were distinct, they were more closely related to each other than to that for cells grown in a lactate- and sulfate-containing medium. Electrochemically measured corrosion rates were lower for iron electrodes covered with hyn-1, hyd, and hmc mutant biofilms than for wild-type biofilms. This confirms the importance, suggested by the gene expression studies, of the corresponding gene products in D. vulgaris-mediated iron corrosion. PMID:18310429

Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme.

PubMed

Moriau, L; Michelet, B; Bogaerts, P; Lambert, L; Michel, A; Oufattole, M; Boutry, M

1999-07-01

The plasma membrane H+-ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+-ATPase) genes. Four of these are classified into two gene subfamilies, pma1-2-3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5' end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma-gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell-type specific manner. Expression in these organs was confirmed at the protein level, using subfamily-specific antibodies. (ii) pma4-gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+-ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+-ATPases with different enzymatic properties.

Expression of AmGR10 of the Gustatory Receptor Family in Honey Bee Is Correlated with Nursing Behavior.

PubMed

Paerhati, Yisilahaiti; Ishiguro, Shinichi; Ueda-Matsuo, Risa; Yang, Ping; Yamashita, Tetsuro; Ito, Kikukatsu; Maekawa, Hideaki; Tani, Hiroko; Suzuki, Koichi

2015-01-01

We investigated the association between the expression of a gene encoding gustatory receptor (G10) and division of labor in the honey bee, Apis mellifera. Among 10 GR genes encoding proteins 15% ~ 99% amino acid identity in the honey bee, we found that AmGR10 with 99% identity is involved in nursing or brood care. Expression of AmGR10 was restricted to organs of the hypopharyngeal gland, brain, and ovary in the nurse bee phase. Members of an extended nursing caste under natural conditions continued to express this gene. RNAi knockdown of AmGR10 accelerated the transition to foraging. Our findings demonstrate that this one gene has profound effects on the division of labor associated with the development and physiology of honeybee society.

Regulation of sugar transport and metabolism by the Candida albicans Rgt1 transcriptional repressor.

PubMed

Sexton, Jessica A; Brown, Victoria; Johnston, Mark

2007-10-01

The ability of the fungal pathogen Candida albicans to cause systemic infections depends in part on the function of Hgt4, a cell surface sugar sensor. The orthologues of Hgt4 in Saccharomyces cerevisiae, Snf3 and Rgt2, initiate a signalling cascade that inactivates Rgt1, a transcriptional repressor of genes encoding hexose transporters. To determine whether Hgt4 functions similarly through the C. albicans orthologue of Rgt1, we analysed Cargt1 deletion mutants. We found that Cargt1 mutants are sensitive to the glucose analogue 2-deoxyglucose, a phenotype probably due to uncontrolled expression of genes encoding glucose transporters. Indeed, transcriptional profiling revealed that expression of about two dozen genes, including multiple HGT genes encoding hexose transporters, is increased in the Cargt1 mutant in the absence of sugars, suggesting that CaRgt1 represses expression of several HGT genes under this condition. Some of the HGT genes (probably encoding high-affinity transporters) are also repressed by high levels of glucose, and we show that this repression is mediated by CaMig1, the orthologue of the major glucose-activated repressor in S. cerevisiae, but not by its paralogue CaMig2. Therefore, CaRgt1 and CaMig1 collaborate to control expression of C. albicans hexose transporters in response to different levels of sugars. We were surprised to find that CaRgt1 also regulates expression of GAL1, suggesting that regulation of galactose metabolism in C. albicans is unconventional. Finally, Cargt1 mutations cause cells to hyperfilament, and suppress the hypofilamented phenotype of an hgt4 mutant, indicating that the Hgt4 glucose sensor may affect filamentation by modulating sugar import and metabolism via CaRgt1. Copyright 2007 John Wiley & Sons, Ltd.

Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia through Control of Genes by the SsuR Transcription Factor▿

PubMed Central

Łochowska, Anna; Iwanicka-Nowicka, Roksana; Zielak, Agata; Modelewska, Anna; Thomas, Mark S.; Hryniewicz, Monika M.

2011-01-01

The genome of Burkholderia cenocepacia contains two genes encoding closely related LysR-type transcriptional regulators, CysB and SsuR, involved in control of sulfur assimilation processes. In this study we show that the function of SsuR is essential for the utilization of a number of organic sulfur sources of either environmental or human origin. Among the genes upregulated by SsuR identified here are the tauABC operon encoding a predicted taurine transporter, three tauD-type genes encoding putative taurine dioxygenases, and atsA encoding a putative arylsulfatase. The role of SsuR in expression of these genes/operons was characterized through (i) construction of transcriptional reporter fusions to candidate promoter regions and analysis of their expression in the presence/absence of SsuR and (ii) testing the ability of SsuR to bind SsuR-responsive promoter regions. We also demonstrate that expression of SsuR-activated genes is not repressed in the presence of inorganic sulfate. A more detailed analysis of four SsuR-responsive promoter regions indicated that ∼44 bp of the DNA sequence preceding and/or overlapping the predicted −35 element of such promoters is sufficient for SsuR binding. The DNA sequence homology among SsuR “recognition motifs” at different responsive promoters appears to be limited. PMID:21317335

Involvement of the ornithine decarboxylase gene in acid stress response in probiotic Lactobacillus delbrueckii UFV H2b20.

PubMed

Ferreira, A B; Oliveira, M N V de; Freitas, F S; Paiva, A D; Alfenas-Zerbini, P; Silva, D F da; Queiroz, M V de; Borges, A C; Moraes, C A de

2015-01-01

Amino acid decarboxylation is important for the maintenance of intracellular pH under acid stress. This study aims to carry out phylogenetic and expression analysis by real-time PCR of two genes that encode proteins involved in ornithine decarboxylation in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress. Sequencing and phylogeny analysis of genes encoding ornithine decarboxylase and amino acid permease in L. delbrueckii UFV H2b20 showed their high sequence identity (99%) and grouping with those of L. delbrueckii subsp. bulgaricus ATCC 11842. Exposure of L. delbrueckii UFV H2b20 cells in MRS pH 3.5 for 30 and 60 min caused a significant increase in expression of the gene encoding ornithine decarboxylase (up to 8.1 times higher when compared to the control treatment). Increased expression of the ornithine decarboxylase gene demonstrates its involvement in acid stress response in L. delbrueckii UFV H2b20, evidencing that the protein encoded by that gene could be involved in intracellular pH regulation. The results obtained show ornithine decarboxylation as a possible mechanism of adaptation to an acidic environmental condition, a desirable and necessary characteristic for probiotic cultures and certainly important to the survival and persistence of the L. delbrueckii UFV H2b20 in the human gastrointestinal tract.

Molecular Cloning and Tissue-Specific Expression of an Anionic Peroxidase in Zucchini1

PubMed Central

Carpin, Sabine; Crèvecoeur, Michèle; Greppin, Hubert; Penel, Claude

1999-01-01

A calcium-pectate-binding anionic isoperoxidase (APRX) from zucchini (Cucurbita pepo) was purified and subjected to N-terminal amino acid microsequencing. The cDNA encoding this enzyme was obtained by reverse transcriptase polymerase chain reaction from a cDNA library. It encoded a mature protein of 309 amino acids exhibiting all of the sequence characteristics of a plant peroxidase. Despite the presence of a C-terminal propeptide, APRX was found in the apoplast. APRX protein and mRNA were found in the root, hypocotyls, and cotyledons. In situ hybridization showed that the APRX-encoding gene was expressed in many different tissues. The strongest expression was observed in root epidermis and in some cells of the stele, in differentiating tracheary elements of hypocotyl, in the lower and upper epidermis, in the palisade parenchyma of cotyledons, and in lateral and adventitious root primordia. In the hypocotyl hook there was an asymmetric expression, with the inner part containing more transcripts than the outer part. Treatment with 2,3,5-triiodobenzoic acid reduced the expression of the APRX-encoding gene in the lower part of the hypocotyl. Our observations suggest that APRX could be involved in lignin formation and that the transcription of its gene was related to auxin level. PMID:10398715

Alternative intronic promoters in development and disease.

PubMed

Vacik, Tomas; Raska, Ivan

2017-05-01

Approximately 20,000 mammalian genes are estimated to encode between 250 thousand and 1 million different proteins. This enormous diversity of the mammalian proteome is caused by the ability of a single-gene locus to encode multiple protein isoforms. Protein isoforms encoded by one gene locus can be functionally distinct, and they can even have antagonistic functions. One of the mechanisms involved in creating this proteome complexity is alternative promoter usage. Alternative intronic promoters are located downstream from their canonical counterparts and drive the expression of alternative RNA isoforms that lack upstream exons. These upstream exons can encode some important functional domains, and proteins encoded by alternative mRNA isoforms can be thus functionally distinct from the full-length protein encoded by canonical mRNA isoforms. Since any misbalance of functionally distinct protein isoforms is likely to have detrimental consequences for the cell and the whole organism, their expression must be precisely regulated. Misregulation of alternative intronic promoters is frequently associated with various developmental defects and diseases including cancer, and it is becoming increasingly clear that this phenomenon deserves more attention.

L-rhamnose induction of Aspergillus nidulans α-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake.

PubMed

Tamayo-Ramos, Juan A; Flipphi, Michel; Pardo, Ester; Manzanares, Paloma; Orejas, Margarita

2012-02-21

Little is known about the structure and regulation of fungal α-L-rhamnosidase genes despite increasing interest in the biotechnological potential of the enzymes that they encode. Whilst the paradigmatic filamentous fungus Aspergillus nidulans growing on L-rhamnose produces an α-L-rhamnosidase suitable for oenological applications, at least eight genes encoding putative α-L-rhamnosidases have been found in its genome. In the current work we have identified the gene (rhaE) encoding the former activity, and characterization of its expression has revealed a novel regulatory mechanism. A shared pattern of expression has also been observed for a second α-L-rhamnosidase gene, (AN10277/rhaA). Amino acid sequence data for the oenological α-L-rhamnosidase were determined using MALDI-TOF mass spectrometry and correspond to the amino acid sequence deduced from AN7151 (rhaE). The cDNA of rhaE was expressed in Saccharomyces cerevisiae and yielded pNP-rhamnohydrolase activity. Phylogenetic analysis has revealed this eukaryotic α-L-rhamnosidase to be the first such enzyme found to be more closely related to bacterial rhamnosidases than other α-L-rhamnosidases of fungal origin. Northern analyses of diverse A. nidulans strains cultivated under different growth conditions indicate that rhaA and rhaE are induced by L-rhamnose and repressed by D-glucose as well as other carbon sources, some of which are considered to be non-repressive growth substrates. Interestingly, the transcriptional repression is independent of the wide domain carbon catabolite repressor CreA. Gene induction and glucose repression of these rha genes correlate with the uptake, or lack of it, of the inducing carbon source L-rhamnose, suggesting a prominent role for inducer exclusion in repression. The A. nidulans rhaE gene encodes an α-L-rhamnosidase phylogenetically distant to those described in filamentous fungi, and its expression is regulated by a novel CreA-independent mechanism. The identification of rhaE and the characterization of its regulation will facilitate the design of strategies to overproduce the encoded enzyme - or homologs from other fungi - for industrial applications. Moreover, A. nidulans α-L-rhamnosidase encoding genes could serve as prototypes for fungal genes coding for plant cell wall degrading enzymes regulated by a novel mechanism of CCR.

L-Rhamnose induction of Aspergillus nidulans α-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake

PubMed Central

2012-01-01

Background Little is known about the structure and regulation of fungal α-L-rhamnosidase genes despite increasing interest in the biotechnological potential of the enzymes that they encode. Whilst the paradigmatic filamentous fungus Aspergillus nidulans growing on L-rhamnose produces an α-L-rhamnosidase suitable for oenological applications, at least eight genes encoding putative α-L-rhamnosidases have been found in its genome. In the current work we have identified the gene (rhaE) encoding the former activity, and characterization of its expression has revealed a novel regulatory mechanism. A shared pattern of expression has also been observed for a second α-L-rhamnosidase gene, (AN10277/rhaA). Results Amino acid sequence data for the oenological α-L-rhamnosidase were determined using MALDI-TOF mass spectrometry and correspond to the amino acid sequence deduced from AN7151 (rhaE). The cDNA of rhaE was expressed in Saccharomyces cerevisiae and yielded pNP-rhamnohydrolase activity. Phylogenetic analysis has revealed this eukaryotic α-L-rhamnosidase to be the first such enzyme found to be more closely related to bacterial rhamnosidases than other α-L-rhamnosidases of fungal origin. Northern analyses of diverse A. nidulans strains cultivated under different growth conditions indicate that rhaA and rhaE are induced by L-rhamnose and repressed by D-glucose as well as other carbon sources, some of which are considered to be non-repressive growth substrates. Interestingly, the transcriptional repression is independent of the wide domain carbon catabolite repressor CreA. Gene induction and glucose repression of these rha genes correlate with the uptake, or lack of it, of the inducing carbon source L-rhamnose, suggesting a prominent role for inducer exclusion in repression. Conclusions The A. nidulans rhaE gene encodes an α-L-rhamnosidase phylogenetically distant to those described in filamentous fungi, and its expression is regulated by a novel CreA-independent mechanism. The identification of rhaE and the characterization of its regulation will facilitate the design of strategies to overproduce the encoded enzyme - or homologs from other fungi - for industrial applications. Moreover, A. nidulans α-L-rhamnosidase encoding genes could serve as prototypes for fungal genes coding for plant cell wall degrading enzymes regulated by a novel mechanism of CCR. PMID:22353731

Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes.

PubMed

Youssef, Reham M; MacDonald, Margaret H; Brewer, Eric P; Bauchan, Gary R; Kim, Kyung-Hwan; Matthews, Benjamin F

2013-04-25

The gene encoding PAD4 (PHYTOALEXIN-DEFICIENT4) is required in Arabidopsis for expression of several genes involved in the defense response to Pseudomonas syringae pv. maculicola. AtPAD4 (Arabidopsis thaliana PAD4) encodes a lipase-like protein that plays a regulatory role mediating salicylic acid signaling. We expressed the gene encoding AtPAD4 in soybean roots of composite plants to test the ability of AtPAD4 to deter plant parasitic nematode development. The transformed roots were challenged with two different plant parasitic nematode genera represented by soybean cyst nematode (SCN; Heterodera glycines) and root-knot nematode (RKN; Meloidogyne incognita). Expression of AtPAD4 in soybean roots decreased the number of mature SCN females 35 days after inoculation by 68 percent. Similarly, soybean roots expressing AtPAD4 exhibited 77 percent fewer galls when challenged with RKN. Our experiments show that AtPAD4 can be used in an economically important crop, soybean, to provide a measure of resistance to two different genera of nematodes.

Gene Expression Profiling of the Cephalothorax and Eyestalk in Penaeus Monodon during Ovarian Maturation

PubMed Central

Brady, Philip; Elizur, Abigail; Williams, Richard; Cummins, Scott F.; Knibb, Wayne

2012-01-01

In crustaceans, a range of physiological processes involved in ovarian maturation occurs in organs of the cephalothorax including the hepatopancrease, mandibular and Y-organ. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites within the eyestalk. Reproductive dysfunction in captive-reared prawns, Penaeus monodon, is believed to be due to deficiencies in these factors. In this study, we investigated the expression of gene transcripts in the cephalothorax and eyestalk from wild-caught and captive-reared animals throughout ovarian maturation using custom oligonucleotide microarray screening. We have isolated numerous transcripts that appear to be differentially expressed throughout ovarian maturation and between wild-caught and captive-reared animals. In the cephalothorax, differentially expressed genes included the 1,3-β-D-glucan-binding high-density lipoprotein, 2/3-oxoacyl-CoA thiolase and vitellogenin. In the eyestalk, these include gene transcripts that encode a protein that modulates G-protein coupled receptor activity and another that encodes an architectural transcription factor. Each may regulate the expression of reproductive neuropeptides, such as the crustacean hyperglycaemic hormone and molt-inhibiting hormone. We could not identify differentially expressed transcripts encoding known reproductive neuropeptides in the eyestalk of either wild-caught or captive-reared prawns at any ovarian maturation stage, however, this result may be attributed to low relative expression levels of these transcripts. In summary, this study provides a foundation for the study of target genes involved in regulating penaeid reproduction. PMID:22355268

Cooperative activity of GABP with PU.1 or C/EBPε regulates lamin B receptor gene expression, implicating their roles in granulocyte nuclear maturation1

PubMed Central

Malu, Krishnakumar; Garhwal, Rahul; Pelletier, Margery G. H.; Gotur, Deepali; Halene, Stephanie; Zwerger, Monika; Yang, Zhong-Fa; Rosmarin, Alan G.; Gaines, Peter

2016-01-01

Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted ETS factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils. PMID:27342846

Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect L-Lysine Production in Corynebacterium glutamicum.

PubMed

Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

2016-03-09

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in L-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport--NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885--were also expressed at significantly higher levels in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, L-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

Application of myostatin in sheep breeding programs: A review

PubMed Central

Miar, Younes; Salehi, Abdolreza; Kolbehdari, Davood; Aleyasin, Seyed Ahmad

2014-01-01

Plasma membrane H+-ATPase is a major integral membrane protein with a role in various physiological processes including abiotic stress response. To study the effect of NaCl on the expression pattern of a gene encoding the plasma membrane H+-ATPase, an experiment was carried out in a completely random design with three replications. A pair of specific primers was designed based on the sequence of the gene encoding plasma membrane H+-ATPase in Aeluropus littoralis to amplify a 259 bp fragment from the target gene by PCR. A gene encoding actin was used as reference gene to normalize the expression level of the target gene. A pair of specific primers was designed to amplify a 157 bp fragment from the actin gene by PCR. Plants were treated with different concentrations of NaCl, 0, 50, 100, 150, 200, 250, 500 and 1000 mM, for two days. Our results showed that the expression level of the plasma membrane H+-ATPase gene increased dramatically at 500 mM and then decreased with increasing concentrations of NaCl. The results also indicated that the leaves of plants, were treated with high concentrations of NaCl changed morphologically, but those grown under low concentrations of NaCl as well as the control plants did not show morphological changes in their leaves. Our results suggest a relation between morphological changes of treated plants and the expression level of the plasma membrane H+-ATPase gene in Aeluropus littoralis. PMID:27843975

Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli.

PubMed

Mercer, Ryan; Nguyen, Oanh; Ou, Qixing; McMullen, Lynn; Gänzle, Michael G

2017-10-15

The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae , including pathogenic strains of Salmonella enterica and Escherichia coli The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1 GI , yfdX2 , hdeD GI , orf11 , trx GI , kefB , and psiE GI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript "GI" [genomic island] if an ortholog of the same gene is present in genomes of E. coli ) LHR-encoded heat shock proteins sHSP20, ClpK GI , and sHSP GI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trx GI , kefB , and psiE GI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food. Copyright © 2017 American Society for Microbiology.

Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli

PubMed Central

Mercer, Ryan; Nguyen, Oanh; Ou, Qixing; McMullen, Lynn

2017-01-01

ABSTRACT The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae, including pathogenic strains of Salmonella enterica and Escherichia coli. The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1GI, yfdX2, hdeDGI, orf11, trxGI, kefB, and psiEGI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript “GI” [genomic island] if an ortholog of the same gene is present in genomes of E. coli.) LHR-encoded heat shock proteins sHSP20, ClpKGI, and sHSPGI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trxGI, kefB, and psiEGI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA. In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food. PMID:28802266

Truncation of a mannanase from Trichoderma harzianum improves its enzymatic properties and expression efficiency in Trichoderma reesei.

PubMed

Wang, Juan; Zeng, Desheng; Liu, Gang; Wang, Shaowen; Yu, Shaowen

2014-01-01

To obtain high expression efficiency of a mannanase gene, ThMan5A, cloned from Trichoderma harzianum MGQ2, both the full-length gene and a truncated gene (ThMan5AΔCBM) that contains only the catalytic domain, were expressed in Trichoderma reesei QM9414 using the strong constitutive promoter of the gene encoding pyruvate decarboxylase (pdc), and purified to homogeneity, respectively. We found that truncation of the gene improved its expression efficiency as well as the enzymatic properties of the encoded protein. The recombinant strain expressing ThMan5AΔCBM produced 2,460 ± 45.1 U/ml of mannanase activity in the culture supernatant; 2.3-fold higher than when expressing the full-length ThMan5A gene. In addition, the truncated mannanase had superior thermostability compared with the full-length enzyme and retained 100 % of its activity after incubation at 60 °C for 48 h. Our results clearly show that the truncated ThMan5A enzyme exhibited improved characteristics both in expression efficiency and in its thermal stability. These characteristics suggest that ThMan5AΔCBM has potential applications in the food, feed, paper, and pulp industries.

Characterization of a Gene Encoding Clathrin Heavy Chain in Maize Up-Regulated by Salicylic Acid, Abscisic Acid and High Boron Supply

PubMed Central

Zeng, Mu-Heng; Liu, Sheng-Hong; Yang, Miao-Xian; Zhang, Ya-Jun; Liang, Jia-Yong; Wan, Xiao-Rong; Liang, Hong

2013-01-01

Clathrin, a three-legged triskelion composed of three clathrin heavy chains (CHCs) and three light chains (CLCs), plays a critical role in clathrin-mediated endocytosis (CME) in eukaryotic cells. In this study, the genes ZmCHC1 and ZmCHC2 encoding clathrin heavy chain in maize were cloned and characterized for the first time in monocots. ZmCHC1 encodes a 1693-amino acid-protein including 29 exons and 28 introns, and ZmCHC2 encodes a 1746-amino acid-protein including 28 exons and 27 introns. The high similarities of gene structure, protein sequences and 3D models among ZmCHC1, and Arabidopsis AtCHC1 and AtCHC2 suggest their similar functions in CME. ZmCHC1 gene is predominantly expressed in maize roots instead of ubiquitous expression of ZmCHC2. Consistent with a typical predicted salicylic acid (SA)-responsive element and four predicted ABA-responsive elements (ABREs) in the promoter sequence of ZmCHC1, the expression of ZmCHC1 instead of ZmCHC2 in maize roots is significantly up-regulated by SA or ABA, suggesting that ZmCHC1 gene may be involved in the SA signaling pathway in maize defense responses. The expressions of ZmCHC1 and ZmCHC2 genes in maize are down-regulated by azide or cold treatment, further revealing the energy requirement of CME and suggesting that CME in plants is sensitive to low temperatures. PMID:23880865

Oxalate-Metabolising Genes of the White-Rot Fungus Dichomitus squalens Are Differentially Induced on Wood and at High Proton Concentration

PubMed Central

de Vries, Ronald P.; Timonen, Sari; Hildén, Kristiina

2014-01-01

Oxalic acid is a prevalent fungal metabolite with versatile roles in growth and nutrition, including degradation of plant biomass. However, the toxicity of oxalic acid makes regulation of its intra- and extracellular concentration crucial. To increase the knowledge of fungal oxalate metabolism, a transcriptional level study on oxalate-catabolising genes was performed with an effective lignin-degrading white-rot fungus Dichomitus squalens, which has demonstrated particular abilities in production and degradation of oxalic acid. The expression of oxalic-acid decomposing oxalate decarboxylase (ODC) and formic-acid decomposing formate dehydrogenase (FDH) encoding genes was followed during the growth of D. squalens on its natural spruce wood substrate. The effect of high proton concentration on the regulation of the oxalate-catabolising genes was determined after addition of organic acid (oxalic acid) and inorganic acid (hydrochloric acid) to the liquid cultures of D. squalens. In order to evaluate the co-expression of oxalate-catabolising and manganese peroxidase (MnP) encoding genes, the expression of one MnP encoding gene, mnp1, of D. squalens was also surveyed in the solid state and liquid cultures. Sequential action of ODC and FDH encoding genes was detected in the studied cultivations. The odc1, fdh2 and fdh3 genes of D. squalens showed constitutive expression, whereas ODC2 and FHD1 most likely are the main responsible enzymes for detoxification of high concentrations of oxalic and formic acids. The results also confirmed the central role of ODC1 when D. squalens grows on coniferous wood. Phylogenetic analysis revealed that fungal ODCs have evolved from at least two gene copies whereas FDHs have a single ancestral gene. As a conclusion, the multiplicity of oxalate-catabolising genes and their differential regulation on wood and in acid-amended cultures of D. squalens point to divergent physiological roles for the corresponding enzymes. PMID:24505339

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F.W.; Dubendorff, J.W.

1998-10-20

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

DOEpatents

Studier, F.W.; Dubendorff, J.W.

1998-11-03

This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

Digital Gene Expression Analysis Provides Insight into the Transcript Profile of the Genes Involved in Aporphine Alkaloid Biosynthesis in Lotus (Nelumbo nucifera)

PubMed Central

Yang, Mei; Zhu, Lingping; Li, Ling; Li, Juanjuan; Xu, Liming; Feng, Ji; Liu, Yanling

2017-01-01

The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (NMCH), and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This transcriptomic database provides new directions for future studies on clarifying the aporphine alkaloid pathway. PMID:28197160

DNA Gyrase Is the Target for the Quinolone Drug Ciprofloxacin in Arabidopsis thaliana*

PubMed Central

Evans-Roberts, Katherine M.; Mitchenall, Lesley A.; Wall, Melisa K.; Leroux, Julie; Mylne, Joshua S.; Maxwell, Anthony

2016-01-01

The Arabidopsis thaliana genome contains four genes that were originally annotated as potentially encoding DNA gyrase: ATGYRA, ATGYRB1, ATGYRB2, and ATGYRB3. Although we subsequently showed that ATGYRB3 does not encode a gyrase subunit, the other three genes potentially encode subunits of a plant gyrase. We also showed evidence for the existence of supercoiling activity in A. thaliana and that the plant is sensitive to quinolone and aminocoumarin antibiotics, compounds that target DNA gyrase in bacteria. However, it was not possible at that time to show whether the A. thaliana genes encoded an active gyrase enzyme, nor whether that enzyme is indeed the target for the quinolone and aminocoumarin antibiotics. Here we show that an A. thaliana mutant resistant to the quinolone drug ciprofloxacin has a point mutation in ATGYRA. Moreover we show that, as in bacteria, the quinolone-sensitive (wild-type) allele is dominant to the resistant gene. Further we have heterologously expressed ATGYRA and ATGYRB2 in a baculovirus expression system and shown supercoiling activity of the partially purified enzyme. Expression/purification of the quinolone-resistant A. thaliana gyrase yields active enzyme that is resistant to ciprofloxacin. Taken together these experiments now show unequivocally that A. thaliana encodes an organelle-targeted DNA gyrase that is the target of the quinolone drug ciprofloxacin; this has important consequences for plant physiology and the development of herbicides. PMID:26663076

Developmental and Spatial Expression of sir2 Genes in the Cellular Slime Mold Dictyostelium discoideum.

PubMed

Katayama, Takahiro; Yasukawa, Hiro

2008-01-01

The cellular slime mold Dictyostelium discoideum grows as unicellular free-living amoebae in the presence of nutrients. Upon starvation, the amoebae aggregate and form multicellular structures that each consist of a stalk and spores. D. discoideum encodes at least four proteins (Sir2A, Sir2B, Sir2C, and Sir2D) homologous to human SIRT. RT-PCR and WISH analyses showed that the genes for Sir2A, Sir2C, and Sir2D were expressed at high levels in growing cells but at decreased levels in developing cells, whereas the gene encoding Sir2B was expressed in the prestalk-cell region in the developmental phase.

A transcriptional approach to unravel the connection between phospholipases A₂ and D and ABA signal in citrus under water stress.

PubMed

Romero, Paco; Lafuente, M Teresa; Alférez, Fernando

2014-07-01

The effect of water stress on the interplay between phospholipases (PL) A2 and D and ABA signalling was investigated in fruit and leaves from the sweet orange Navelate and its fruit-specific ABA-deficient mutant Pinalate by studying simultaneously expression of 5 PLD and 3 PLA2-encoding genes. In general, expression levels of PLD-encoding genes were higher at harvest in the flavedo (coloured outer part of the peel) from Pinalate. Moreover, a higher and transient increase in expression of CsPLDα, CsPLDβ, CsPLDδ and CsPLDζ was observed in the mutant as compared to Navelate fruit under water stress, which may reflect a mechanism of acclimation to water stress influenced by ABA deficiency. An early induction in CsPLDγ gene expression, when increase in peel damage during fruit storage was most evident, suggested a role for this gene in membrane degradation processes during water stress. Exogenous ABA on mutant fruit modified the expression of all PLD genes and reduced the expression of CsPLDα and CsPLDβ by 1 week to levels similar to those of Navelate, suggesting a repressor role of ABA on these genes. In general, CssPLA2α and β transcript levels were lower in flavedo from Pinalate than from Navelate fruit during the first 3 weeks of storage, suggesting that expression of these genes also depends at least partially on ABA levels. Patterns of expression of PLD and PLA2-encoding genes were very similar in Navelate and Pinalate leaves, which have similar ABA levels, when comparing both RH conditions. Results comparison with other from previous works in the same experimental systems helped to decipher the effect of the stress severity on the differential response of some of these genes under dehydration conditions and pointed out the interplay between PLA2 and PLD families and their connection with ABA signalling in citrus. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Evolution and Variation of Renin Genes in Mice

PubMed Central

Dickinson, Douglas P.; Gross, Kenneth W.; Piccini, Nina; Wilson, Carol M.

1984-01-01

Inbred strains of mice carry Ren-1, a gene encoding the thermostable Renin-1 isozyme. Ren-1 is expressed at relatively low levels in mouse submandibular gland and kidney. Some strains also carry Ren-2, a gene encoding the thermolabile Renin-2 isozyme. Ren-2 is expressed at high levels in the mouse submandibular gland and at very low levels, if at all, in the kidney. Ren-1 and Ren-2 are closely linked on mouse chromosome 1, show extensive homology in coding and noncoding regions and provide a model for studying the regulation of gene expression. An investigation of renin genes and enzymatic activity in wild-derived mice identified several restriction site polymorphisms as well as putative variants in renin gene expression and protein structure. The number of renin genes carried by different subpopulations of wild-derived mice is consistent with the occurrence of a gene duplication event prior to the divergence of M. spretus (2.75–5.5 million yr ago). This conclusion is in agreement with a prior estimate based upon comparative sequence analysis of Ren-1 and Ren-2 from inbred laboratory mice. PMID:6389258

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.

Involvement of Retinoblastoma Protein and HBP1 in Histone H10 Gene Expression

PubMed Central

Lemercier, Claudie; Duncliffe, Kym; Boibessot, Isabelle; Zhang, Hui; Verdel, André; Angelov, Dimitar; Khochbin, Saadi

2000-01-01

The histone H10-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H10 promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H10 gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H10 H4 box were therefore expected to link differentiation-dependent expression of H10 to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H10 H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H10 gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H10, HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells. PMID:10958660

[Construction and expression of recombinant human serum albumin-EPO fusion protein].

PubMed

Huang, Ying-Chun; Gou, Xing-Hua; Han, Lei; Li, De-Hua; Zhao, Lan-Ying; Wu, Qia-Qing

2011-05-01

OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

Gene expression patterns during somatic embryo development and germination in maize Hi II callus cultures.

PubMed

Che, Ping; Love, Tanzy M; Frame, Bronwyn R; Wang, Kan; Carriquiry, Alicia L; Howell, Stephen H

2006-09-01

Gene expression patterns were profiled during somatic embryogenesis in a regeneration-proficient maize hybrid line, Hi II, in an effort to identify genes that might be used as developmental markers or targets to optimize regeneration steps for recovering maize plants from tissue culture. Gene expression profiles were generated from embryogenic calli induced to undergo embryo maturation and germination. Over 1,000 genes in the 12,060 element arrays showed significant time variation during somatic embryo development. A substantial number of genes were downregulated during embryo maturation, largely histone and ribosomal protein genes, which may result from a slowdown in cell proliferation and growth during embryo maturation. The expression of these genes dramatically recovered at germination. Other genes up-regulated during embryo maturation included genes encoding hydrolytic enzymes (nucleases, glucosidases and proteases) and a few storage genes (an alpha-zein and caleosin), which are good candidates for developmental marker genes. Germination is accompanied by the up-regulation of a number of stress response and membrane transporter genes, and, as expected, greening is associated with the up-regulation of many genes encoding photosynthetic and chloroplast components. Thus, some, but not all genes typically associated with zygotic embryogenesis are significantly up or down-regulated during somatic embryogenesis in Hi II maize line regeneration. Although many genes varied in expression throughout somatic embryo development in this study, no statistically significant gene expression changes were detected between total embryogenic callus and callus enriched for transition stage somatic embryos.

Evolution and Distribution of Teleost myomiRNAs: Functionally Diversified myomiRs in Teleosts.

PubMed

Siddique, Bhuiyan Sharmin; Kinoshita, Shigeharu; Wongkarangkana, Chaninya; Asakawa, Shuichi; Watabe, Shugo

2016-06-01

Myosin heavy chain (MYH) genes belong to a multigene family, and the regulated expression of each member determines the physiological and contractile muscle properties. Among these, MYH6, MYH7, and MYH14 occupy unique positions in the mammalian MYH gene family because of their specific expression in slow/cardiac muscles and the existence of intronic micro(mi) RNAs. MYH6, MYH7, and MYH14 encode miR-208a, miR-208b, and miR-499, respectively. These MYH encoded miRNAs are designated as myomiRs because of their muscle-specific expression and functions. In mammals, myomiRs and host MYHs form a transcription network involved in muscle fiber-type specification; thus, genomic positions and expression patterns of them are well conserved. However, our previous studies revealed divergent distribution and expression of MYH14/miR-499 among teleosts, suggesting the unique evolution of myomiRs and host MYHs in teleosts. Here, we examined distribution and expression of myomiRs and host MYHs in various teleost species. The major cardiac MYH isoforms in teleosts are an intronless gene, atrial myosin heavy chain (amhc), and ventricular myosin heavy chain (vmhc) gene that encodes an intronic miRNA, miR-736. Phylogenetic analysis revealed that vmhc/miR-736 is a teleost-specific myomiR that differed from tetrapoda MYH6/MYH7/miR-208s. Teleost genomes also contain species-specific orthologs in addition to vmhc and amhc, indicating complex gene duplication and gene loss events during teleost evolution. In medaka and torafugu, miR-499 was highly expressed in slow/cardiac muscles whereas the expression of miR-736 was quite low and not muscle specific. These results suggest functional diversification of myomiRs in teleost with the diversification of host MYHs.

Improved Innate and Adaptive Immunostimulation by Genetically Modified HIV-1 Protein Expressing NYVAC Vectors

PubMed Central

Quakkelaar, Esther D.; Redeker, Anke; Haddad, Elias K.; Harari, Alexandre; McCaughey, Stella Mayo; Duhen, Thomas; Filali-Mouhim, Abdelali; Goulet, Jean-Philippe; Loof, Nikki M.; Ossendorp, Ferry; Perdiguero, Beatriz; Heinen, Paul; Gomez, Carmen E.; Kibler, Karen V.; Koelle, David M.; Sékaly, Rafick P.; Sallusto, Federica; Lanzavecchia, Antonio; Pantaleo, Giuseppe; Esteban, Mariano; Tartaglia, Jim; Jacobs, Bertram L.; Melief, Cornelis J. M.

2011-01-01

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines. PMID:21347234

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta).

PubMed

Zhang, YiChen; Jiang, Peng; Gao, JiangTao; Liao, JianMin; Sun, ShiJing; Shen, ZiLong; Qin, Song

2008-12-01

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

PubMed

Kast, Alene; Voges, Raphael; Schroth, Michael; Schaffrath, Raffael; Klassen, Roland; Meinhardt, Friedhelm

2015-05-01

Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

Comparative and Evolutionary Analysis of Grass Pollen Allergens Using Brachypodium distachyon as a Model System.

PubMed

Sharma, Akanksha; Sharma, Niharika; Bhalla, Prem; Singh, Mohan

2017-01-01

Comparative genomics have facilitated the mining of biological information from a genome sequence, through the detection of similarities and differences with genomes of closely or more distantly related species. By using such comparative approaches, knowledge can be transferred from the model to non-model organisms and insights can be gained in the structural and evolutionary patterns of specific genes. In the absence of sequenced genomes for allergenic grasses, this study was aimed at understanding the structure, organisation and expression profiles of grass pollen allergens using the genomic data from Brachypodium distachyon as it is phylogenetically related to the allergenic grasses. Combining genomic data with the anther RNA-Seq dataset revealed 24 pollen allergen genes belonging to eight allergen groups mapping on the five chromosomes in B. distachyon. High levels of anther-specific expression profiles were observed for the 24 identified putative allergen-encoding genes in Brachypodium. The genomic evidence suggests that gene encoding the group 5 allergen, the most potent trigger of hay fever and allergic asthma originated as a pollen specific orphan gene in a common grass ancestor of Brachypodium and Triticiae clades. Gene structure analysis showed that the putative allergen-encoding genes in Brachypodium either lack or contain reduced number of introns. Promoter analysis of the identified Brachypodium genes revealed the presence of specific cis-regulatory sequences likely responsible for high anther/pollen-specific expression. With the identification of putative allergen-encoding genes in Brachypodium, this study has also described some important plant gene families (e.g. expansin superfamily, EF-Hand family, profilins etc) for the first time in the model plant Brachypodium. Altogether, the present study provides new insights into structural characterization and evolution of pollen allergens and will further serve as a base for their functional characterization in related grass species.

Molecular cloning, sequence identification and tissue expression profile of three novel sheep (Ovis aries) genes - BCKDHA, NAGA and HEXA.

PubMed

Liu, G Y; Gao, S Z

2009-01-01

The complete coding sequences of three sheep genes- BCKDHA, NAGA and HEXA were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR), based on the conserved sequence information of the mouse or other mammals. The nucleotide sequences of these three genes revealed that the sheep BCKDHA gene encodes a protein of 313 amino acids which has high homology with the BCKDHA gene that encodes a protein of 447 amino acids that has high homology with the Branched chain keto acid dehydrogenase El, alpha polypeptide (BCKDHA) of five species chimpanzee (93%), human (96%), crab-eating macaque (93%), bovine (98%) and mouse (91%). The sheep NAGA gene encodes a protein of 411 amino acids that has high homology with the alpha-N-acetylgalactosaminidase (NAGA) of five species human (85%), bovine (94%), mouse (91%), rat (83%) and chicken (74%). The sheep HEXA gene encodes a protein of 529 amino acids that has high homology with the hexosaminidase A(HEXA) of five species bovine (98%), human (84%), Bornean orangután (84%), rat (80%) and mouse (81%). Finally these three novel sheep genes were assigned to GenelDs: 100145857, 100145858 and 100145856. The phylogenetic tree analysis revealed that the sheep BCKDHA, NAGA, and HEXA all have closer genetic relationships to the BCKDHA, NAGA, and HEXA of bovine. Tissue expression profile analysis was also carried out and results revealed that sheep BCKDHA, NAGA and HEXA genes were differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, small and large intestine. Our experiment is the first to establish the primary foundation for further research on these three sheep genes.

Heterogenic expression of genes encoding secreted proteins at the periphery of Aspergillus niger colonies.

PubMed

Vinck, Arman; de Bekker, Charissa; Ossin, Adam; Ohm, Robin A; de Vries, Ronald P; Wösten, Han A B

2011-01-01

Colonization of a substrate by fungi starts with the invasion of exploring hyphae. These hyphae secrete enzymes that degrade the organic material into small molecules that can be taken up by the fungus to serve as nutrients. We previously showed that only part of the exploring hyphae of Aspergillus niger highly express the glucoamylase gene glaA. This was an unexpected finding since all exploring hyphae are exposed to the same environmental conditions. Using GFP as a reporter, we here demonstrate that the acid amylase gene aamA, the α-glucuronidase gene aguA, and the feruloyl esterase gene faeA of A. niger are also subject to heterogenic expression within the exploring mycelium. Coexpression studies using GFP and dTomato as reporters showed that hyphae that highly express one of these genes also highly express the other genes encoding secreted proteins. Moreover, these hyphae also highly express the amylolytic regulatory gene amyR, and the glyceraldehyde-3-phosphate dehydrogenase gene gpdA. In situ hybridization demonstrated that the high expressers are characterized by a high 18S rRNA content. Taken together, it is concluded that two subpopulations of hyphae can be distinguished within the exploring mycelium of A. niger. The experimental data indicate that these subpopulations differ in their transcriptional and translational activity. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

cDNA microarray analyses reveal candidate marker genes for the detection of ascidian disease in Korea.

PubMed

Azumi, Kaoru; Usami, Takeshi; Kamimura, Akiko; Sabau, Sorin V; Miki, Yasufumi; Fujie, Manabu; Jung, Sung-Ju; Kitamura, Shin-Ichi; Suzuki, Satoru; Yokosawa, Hideyoshi

2007-12-01

A serious disease of the ascidian Halocynthia roretzi has been spread extensively among Korean aquaculture sites. To reveal the cause of the disease and establish a monitoring system for it, we constructed a cDNA microarray spotted with 2,688 cDNAs derived from H. roretzi hemocyte cDNA libraries to detect genes differentially expressed in hemocytes between diseased and non-diseased ascidians. We detected 21 genes showing increased expression and 16 genes showing decreased expression in hemocytes from diseased ascidians compared with those from non-diseased ascidians. RT-PCR analyses confirmed that the expression levels of genes encoding astacin, lysozyme, ribosomal protein PO, and ubiquitin-ribosomal protein L40e fusion protein were increased in hemocytes from diseased ascidians, while those of genes encoding HSP40, HSP70, fibronectin, carboxypeptidase and lactate dehydrogenase were decreased. These genes were expressed not only in hemocytes but also in various other tissues in ascidians. Furthermore, the expression of glutathione-S transferase omega, which is known to be up-regulated in H. roretzi hemocytes during inflammatory responses, was strongly increased in hemocytes from diseased ascidians. These gene expression profiles suggest that immune and inflammatory reactions occur in the hemocytes of diseased ascidians. These genes will be good markers for detecting and monitoring this disease of ascidians in Korean aquaculture sites.

Characterization of Chicken Spleen Transcriptome after Infection with Salmonella enterica Serovar Enteritidis

PubMed Central

Matulova, Marta; Rajova, Jana; Vlasatikova, Lenka; Volf, Jiri; Stepanova, Hana; Havlickova, Hana; Sisak, Frantisek; Rychlik, Ivan

2012-01-01

In this study we were interested in identification of new markers of chicken response to Salmonella Enteritidis infection. To reach this aim, gene expression in the spleens of naive chickens and those intravenously infected with S. Enteritidis with or without previous oral vaccination was determined by 454 pyrosequencing of splenic mRNA/cDNA. Forty genes with increased expression at the level of transcription were identified. The most inducible genes encoded avidin (AVD), extracellular fatty acid binding protein (EXFABP), immune responsive gene 1 (IRG1), chemokine ah221 (AH221), trappin-6-like protein (TRAP6) and serum amyloid A (SAA). Using cDNA from sorted splenic B-lymphocytes, macrophages, CD4, CD8 and γδ T-lymphocytes, we found that the above mentioned genes were preferentially expressed in macrophages. AVD, EXFABP, IRG1, AH221, TRAP6 and SAA were induced also in the cecum of chickens orally infected with S. Enteritidis on day 1 of life or day 42 of life. Unusual results were obtained for the immunoglobulin encoding transcripts. Prior to the infection, transcripts coding for the constant parts of IgM, IgY, IgA and Ig light chain were detected in B-lymphocytes. However, after the infection, immunoglobulin encoding transcripts were expressed also by T-lymphocytes and macrophages. Expression of AVD, EXFABP, IRG1, AH221, TRAP6, SAA and all immunoglobulin genes can be therefore used for the characterization of the course of S. Enteritidis infection in chickens. PMID:23094107

[Construction of plant expression plasmid of chimera SBR-CT delta A1].

PubMed

Mai, Sui; Ling, Junqi

2003-08-01

The purpose of this study is to construct plant expression plasmid containing the gene encoding chimera SBR-CT delta A1. The target gene fragment P2, including the gene-encoded chimera SBR-CT delta A1 (3,498-5,378 bp), was obtained by standard PCR amplification. The PCR products were ligated with pGEM-easy vector through TA clone to form plasmid pTSC. The plasmid pTSC and plasmid pPOKII were digested by restricted endonuclease BamHI and KpnI, and the digested products were extracted and purified for recombination. Then the purified P2 and plasmid pPOKII were recombined by T4 DNA ligase to form recombinant plasmid pROSC; inserting bar gene into the plasmid and form pROSB plasmid. The recombined plasmids were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing. P2 gene was linked to pPOKII plasmid and formed recombinant plasmid pROSC. The DNA sequence and orientation were corrected. And bar gene was inserted into pPOSC and form recombinant plasmid pROSB. Plant expression vector pROSC and pROSB containing the gene encoding chimera SBR-CT delta A1, which may provide useful experiment foundation for further study on edible vaccine against caries have been successfully constructed.

Comparative analysis of human protein-coding and noncoding RNAs between brain and 10 mixed cell lines by RNA-Seq.

PubMed

Chen, Geng; Yin, Kangping; Shi, Leming; Fang, Yuanzhang; Qi, Ya; Li, Peng; Luo, Jian; He, Bing; Liu, Mingyao; Shi, Tieliu

2011-01-01

In their expression process, different genes can generate diverse functional products, including various protein-coding or noncoding RNAs. Here, we investigated the protein-coding capacities and the expression levels of their isoforms for human known genes, the conservation and disease association of long noncoding RNAs (ncRNAs) with two transcriptome sequencing datasets from human brain tissues and 10 mixed cell lines. Comparative analysis revealed that about two-thirds of the genes expressed between brain and cell lines are the same, but less than one-third of their isoforms are identical. Besides those genes specially expressed in brain and cell lines, about 66% of genes expressed in common encoded different isoforms. Moreover, most genes dominantly expressed one isoform and some genes only generated protein-coding (or noncoding) RNAs in one sample but not in another. We found 282 human genes could encode both protein-coding and noncoding RNAs through alternative splicing in the two samples. We also identified more than 1,000 long ncRNAs, and most of those long ncRNAs contain conserved elements across either 46 vertebrates or 33 placental mammals or 10 primates. Further analysis showed that some long ncRNAs differentially expressed in human breast cancer or lung cancer, several of those differentially expressed long ncRNAs were validated by RT-PCR. In addition, those validated differentially expressed long ncRNAs were found significantly correlated with certain breast cancer or lung cancer related genes, indicating the important biological relevance between long ncRNAs and human cancers. Our findings reveal that the differences of gene expression profile between samples mainly result from the expressed gene isoforms, and highlight the importance of studying genes at the isoform level for completely illustrating the intricate transcriptome.

The human enamel protein gene amelogenin is expressed from both the X and the Y chromosomes

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

Salido, E.C.; Yen, P.H.; Koprivnikar, K.

1992-02-01

Amelogenins, a family of extracellular matrix proteins of the dental enamel, are transiently but abundantly expressed by ameloblasts during tooth development. In this paper the authors report the characterization of the AMGX and AMGY genes on the short arms of the human X and Y chromosomes which encode the amelogenins. Their studies on the expression of the amelogenin genes in male developing tooth buds showed that both the AMGX and AMGY genes are transcriptionally active and encode potentially functional proteins. They have isolated genomic and cDNA clones form both the AMGX and AMGY loci and have studied the sequence organizationmore » of these two genes. Reverse transcriptase (RT)PCR amplification of the 5[prime] portion of the amelogenin transcripts revealed several alternatively spliced products. This information will be useful for studying the molecular basis of X-linked amelogenesis imperfecta, for understanding the evolution and regulation of gene expression on the mammalian sex chromosomes, and for investigating the role of amelogenin genes during tooth development.« less

The 2p21 deletion syndrome: characterization of the transcription content.

PubMed

Parvari, Ruti; Gonen, Yael; Alshafee, Ismael; Buriakovsky, Sophia; Regev, Kfir; Hershkovitz, Eli

2005-08-01

The vast majority of small-deletion syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We have previously identified a homozygous deletion of 179,311 bp on chromosome 2p21 as the cause of a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria. We now present the transcription content of this region: Multiple splicing variants of the genes protein phosphatase 1B (formerly 2C) magnesium-dependent, beta isoform (PPM1B), SLC3A1, and KIAA0436 (approved gene symbol PREPL) were identified and their patterns of expression analyzed. The spliced variants are predicted to have additional functions compared to the known variants and their patterns of expression fit the tissues affected by the syndrome. The first exon of an additional gene (C2orf34) is encoded in the deleted region and the gene is not expressed in the patients. In addition several transcripts with very short open reading frames are also encoded in the deletion. The identification of all transcripts encoded in the region deleted in the patients is the first step in the study of the genotype-phenotype correlation of the 2p21 patients.

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

Method for increasing thermostability in cellulase ennzymes

DOEpatents

Adney, William S.; Thomas, Steven R.; Baker, John O.; Himmel, Michael E.; Chou, Yat-Chen

1998-01-01

The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in Pichia pastoris. A new modified E1 endoglucanase enzyme comprising the catalytic domain of the full size E1 enzyme demonstrates enhanced thermostability and is produced by two methods. The first method of producing the new modified E1 is proteolytic cleavage to remove the cellulose binding domain and linker peptide of the full size E1. The second method of producing the new modified E1 is genetic truncation of the gene encoding the full size E1 so that the catalytic domain is expressed in the expression product.

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

DOE PAGES

Wilson, Charlotte M.; Klingeman, Dawn M.; Schlachter, Caleb; ...

2016-12-21

Organisms regulate gene expression in response to the environment to coordinate metabolic reactions.Clostridium thermocellumexpresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. In one LacI regulator termed GlyR3 inC. thermocellumATCC 27405 we identified a repressor of neighboring genes with repression relieved by laminaribiose (a β-1,3 disaccharide). To better understand the threeC. thermocellumLacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313lacIgenes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Furthermore, growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pHmore » fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2strain, and α-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3strain, upregulation of the two genes adjacent toglyR3in thecelC-glyR3-licAoperon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters. IMPORTANCEUnderstandingC. thermocellumgene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specificC. thermocellumLacI regulons. Finally, the identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation inC. thermocellum.« less

The effect of antimicrobial photodynamic therapy on the expression of novel methicillin resistance markers determined using cDNA-AFLP approach in Staphylococcus aureus.

PubMed

Hoorijani, Mohammad Neshvan; Rostami, Hosein; Pourhajibagher, Maryam; Chiniforush, Nasim; Heidari, Mansour; Pourakbari, Babak; Kazemian, Hossein; Davari, Kambiz; Amini, Vahid; Raoofian, Reza; Bahador, Abbas

2017-09-01

Widespread methicillin resistant Staphylococcus aureus (MRSA) and absence of effective antimicrobial agents has led to limited therapeutic options for treating MRSA infection. We aimed to evaluate the effect of antimicrobial photodynamic therapy (aPDT) on the expression of novel identified methicillin resistance markers (NIMRMs) in S. aureus using complementary DNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) approaches to address the therapeutic alternatives for MRSA infections. We used cDNA-AFLP to compare MRSA and methicillin susceptible S. aureus (MSSA) for identification of target genes implicated in methicillin resistance. To determine the sub-lethal aPDT (sPDT), MRSA and MSSA clinical isolates photosensitized with toluidine blue O (TBO), and then were irradiated with diode laser. After sPDT, the colony forming units/mL was quantified. Antimicrobial susceptibility against methicillin was assessed for cell-surviving aPDT. Effects of sPDT on the expression of NIMRMs were evaluated by real-time quantitative reverse transcription PCR. According to our results, serine hydrolase family protein (Shfp) encoding gene and a gene encoding a conserved hypothetical protein (Chp) were implicated in methicillin resistance in MRSA. sPDT reduced the minimum inhibitory concentrations of methicillin by 3-fold in MRSA. sPDT could lead to about 10- and 6.2- fold suppression of expression of the Chp and Shfp encoding genes, respectively. sPDT would lead to reduction in resistance to methicillin of MRSA in surviving cells by suppressing the expression of the Shfp and Chp encoding genes associated with methicillin resistance. This may have potential implications of aPDT for the treatment of MRSA infections. Copyright © 2017 Elsevier B.V. All rights reserved.

Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties, activity, and expression during tuber dormancy progression

USDA-ARS?s Scientific Manuscript database

The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in meristems isolated from field-g...

Rudimentary expression of RYamide in Drosophila melanogaster relative to other Drosophila species points to a functional decline of this neuropeptide gene.

PubMed

Veenstra, Jan A; Khammassi, Hela

2017-04-01

RYamides are arthropod neuropeptides with unknown function. In 2011 two RYamides were isolated from D. melanogaster as the ligands for the G-protein coupled receptor CG5811. The D. melanogaster gene encoding these neuropeptides is highly unusual, as there are four RYamide encoding exons in the current genome assembly, but an exon encoding a signal peptide is absent. Comparing the D. melanogaster gene structure with those from other species, including D. virilis, suggests that the gene is degenerating. RNAseq data from 1634 short sequence read archives at NCBI containing more than 34 billion spots yielded numerous individual spots that correspond to the RYamide encoding exons, of which a large number include the intron-exon boundary at the start of this exon. Although 72 different sequences have been spliced onto this RYamide encoding exon, none codes for the signal peptide of this gene. Thus, the RNAseq data for this gene reveal only noise and no signal. The very small quantities of peptide recovered during isolation and the absence of credible RNAseq data, indicates that the gene is very little expressed, while the RYamide gene structure in D. melanogaster suggests that it might be evolving into a pseudogene. Yet, the identification of the peptides it encodes clearly shows it is still functional. Using region specific antisera, we could localize numerous neurons and enteroendocrine cells in D. willistoni, D. virilis and D. pseudoobscura, but only two adult abdominal neurons in D. melanogaster. Those two neurons project to and innervate the rectal papillae, suggesting that RYamides may be involved in the regulation of water homeostasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of two distinct dual specificity phosphatases encoded in alternative open reading frames of a single gene located on human chromosome 10q22.2.

PubMed

Chen, Hsu-Hsin; Luche, Ralf; Wei, Bo; Tonks, Nicholas K

2004-10-01

Dual specificity phosphatases (DSPs) are members of the protein-tyrosine phosphatase superfamily that dephosphorylate both phosphotyrosine and phosphoserine/threonine residues in vitro. Many DSPs have been found to play important roles in various aspects of cellular function and to be involved in human disease. We have identified a gene located on human chromosome 10q22.2, which utilizes alternative open reading frames (ORFs) to encode the following two distinct DSPs: the previously described testis and skeletal muscle-specific dual specificity phosphatase (TMDP) and a novel DSP, muscle-restricted dual specificity phosphatase (MDSP). Use of alternative ORFs encoding distinct proteins from a single gene is extremely rare in eukaryotes, and in all previously reported cases the two proteins produced from one gene are unrelated. To our knowledge this is the first example of a gene from which two distinct proteins of the same family are expressed using alternative ORFs. Here we provide evidence that both MDSP and TMDP proteins are expressed in vivo and are restricted to specific tissues, skeletal muscle and testis, respectively. Most interestingly, the protein expression profiles of both MDSP and TMDP during mouse postnatal development are strikingly similar. MDSP is expressed at very low levels in myotubes and early postnatal muscle. TMDP is not detectable in testis lysate in the first 3 weeks of life. The expression of both MDSP and TMDP proteins was markedly increased at approximately the 3rd week after birth and continued to increase gradually into adulthood, implying that the physiological functions of both DSPs are specific to the mature/late-developing organs. The conserved gene structure and the similarity in postnatal expression profile of these two proteins suggest biological significance of the unusual gene arrangement.

The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

PubMed

Streatfield, S J; Weber, A; Kinsman, E A; Häusler, R E; Li, J; Post-Beittenmiller, D; Kaiser, W M; Pyke, K A; Flügge, U I; Chory, J

1999-09-01

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae.

PubMed

Blázquez, M A; Gamo, F J; Gancedo, C

1995-12-18

Yeasts with disruptions in the genes PYC1 and PYC2 encoding the isoenzymes of pyruvate carboxylase cannot grow in a glucose-ammonium medium (Stucka et al. (1991) Mol. Gen. Genet. 229, 307-315). We have isolated a dominant mutation, BPC1-1, that allows growth in this medium of yeasts with interrupted PYC1 and PYC2 genes. The BPC1-1 mutation abolishes catabolite repression of a series of genes and allows expression of the enzymes of the glyoxylate cycle during growth in glucose. A functional glyoxylate cycle is necessary for suppression as a disruption of gene ICL1 encoding isocitrate lyase abolished the phenotypic effect of BPC1-1 on growth in glucose-ammonium. Concurrent expression from constitutive promoters of genes ICL1 and MLS1 (encoding malate synthase) also suppressed the growth phenotype of pyc1 pyc2 mutants. The mutation BPC1-1 is either allelic or closely linked to the mutation DGT1-1.

Regulation of gene expression in the mammalian eye and its relevance to eye disease.

PubMed

Scheetz, Todd E; Kim, Kwang-Youn A; Swiderski, Ruth E; Philp, Alisdair R; Braun, Terry A; Knudtson, Kevin L; Dorrance, Anne M; DiBona, Gerald F; Huang, Jian; Casavant, Thomas L; Sheffield, Val C; Stone, Edwin M

2006-09-26

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F(2) rats generated from an SR/JrHsd x SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (alpha = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5' flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor beta2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.

PUM1 is a biphasic negative regulator of innate immunity genes by suppressing LGP2.

PubMed

Liu, Yonghong; Qu, Linlin; Liu, Yuanyuan; Roizman, Bernard; Zhou, Grace Guoying

2017-08-15

PUM1 is an RNA binding protein shown to regulate the stability and function of mRNAs bearing a specific sequence. We report the following: ( i ) A key function of PUM1 is that of a repressor of key innate immunity genes by repressing the expression of LGP2. Thus, between 12 and 48 hours after transfection of human cells with siPUM1 RNA there was an initial (phase 1) upsurge of transcripts encoding LGP2, CXCL10, IL6, and PKR. This was followed 24 hours later (phase 2) by a significant accumulation of mRNAs encoding RIG-I, SP100, MDA5, IFIT1, PML, STING, and IFNβ. The genes that were not activated encoded HDAC4 and NF-κB1. ( ii ) Simultaneous depletion of PUM1 and LGP2, CXCL10, or IL6 revealed that up-regulation of phase 1 and phase 2 genes was the consequence of up-regulation of LGP2. ( iii ) IFNβ produced 48-72 hours after transfection of siPUM1 was effective in up-regulating LGP2 and phase 2 genes and reducing the replication of HSV-1 in untreated cells. ( iv ) Because only half of genes up-regulated in phase 1 and 2 encode mRNAs containing PUM1 binding sites, the upsurge in gene expression could not be attributed solely to stabilization of mRNAs in the absence of PUM1. ( v ) Lastly, depletion of PUM2 does not result in up-regulation of phase 1 or phase 2 genes. The results of the studies presented here indicate that PUM1 is a negative regulator of LGP2, a master regulator of innate immunity genes expressed in a cascade fashion.

The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

PubMed Central

Poklepovich, Tomas J.; Rinaldi, Mauro A.; Tomazic, Mariela L.; Favale, Nicolas O.; Turkewitz, Aaron P.; Nudel, Clara B.; Nusblat, Alejandro D.

2012-01-01

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. PMID:22982564

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....

Maternal Betaine Supplementation during Gestation Enhances Expression of mtDNA-Encoded Genes through D-Loop DNA Hypomethylation in the Skeletal Muscle of Newborn Piglets.

PubMed

Jia, Yimin; Song, Haogang; Gao, Guichao; Cai, Demin; Yang, Xiaojing; Zhao, Ruqian

2015-11-25

Betaine has been widely used in animal and human nutrition to promote muscle growth and performance, yet it remains unknown whether maternal betaine supplementation during gestation affects the metabolic characteristics of neonatal skeletal muscles. In the present study, feeding sows with betaine-supplemented diets throughout gestation significantly upregulated the expression of mtDNA-encoded OXPHOS genes (p < 0.05), including COX1, COX2, and ND5, in the muscle of newborn piglets, which was associated with enhanced mitochondrial COX enzyme activity (p < 0.05). Concurrently, maternal betaine supplementation increased the plasma betaine concentration and muscle expression of methyl transfer enzymes (p < 0.05), BHMT and GNMT, in offspring piglets. Nevertheless, Dnmt3a was downregulated at the level of both mRNA and protein, which was associated with a hypomethylated mtDNA D-loop region (p < 0.05). These results suggest that maternal betaine supplementation during gestation enhances expression of mtDNA-encoded genes through D-loop DNA hypomethylation in the skeletal muscle of newborn piglets.

Inhibition of Virulence Gene Expression in Staphylococcus aureus by Novel Depsipeptides from a Marine Photobacterium

PubMed Central

Mansson, Maria; Nielsen, Anita; Kjærulff, Louise; Gotfredsen, Charlotte H.; Wietz, Matthias; Ingmer, Hanne; Gram, Lone; Larsen, Thomas O.

2011-01-01

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium. PMID:22363239

Screening and analysis of genes expressed upon infection of broad bean with Clover yellow vein virus causing lethal necrosis.

PubMed

Nakahara, Kenji S; Kitazawa, Hiroaki; Atsumi, Go; Choi, Sun Hee; Suzuki, Yuji; Uyeda, Ichiro

2011-07-18

Clover yellow vein virus (ClYVV) causes lethal systemic necrosis in legumes, including broad bean (Vicia faba) and pea (Pisum sativum). To identify host genes involved in necrotic symptom expression after ClYVV infection, we screened cDNA fragments in which expression was changed in advance of necrotic symptom expression in broad bean (V. faba cv. Wase) using the differential display technique and secondarily with Northern blot analysis. Expression changes were confirmed in 20 genes, and the six that exhibited the most change were analyzed further. These six genes included a gene that encodes a putative nitrate-induced NOI protein (VfNOI), and another was homologous to an Arabidopsis gene that encodes a glycine- and proline-rich protein GPRP (VfGPRP). We recently reported that necrotic symptom development in ClYVV-infected pea is associated with expression of salicylic acid (SA)-dependent pathogenesis-related (PR) proteins and requires SA-dependent host responses. Interestingly, VfNOI and VfGPRP expression was correlated with that of the putative SA-dependent PR proteins in ClYVV-infected broad bean. However, broad bean infected with a recombinant ClYVV expressing the VfGPRP protein showed weaker symptoms and less viral multiplication than that infected with ClYVV expressing the GFP protein. These results imply that VfGPRP plays a role in defense against ClYVV rather than in necrotic symptom expression.

Characterization of HKE2: an ancient antigen encoded in the major histocompatibility complex.

PubMed

Ostrov, D A; Barnes, C L; Smith, L E; Binns, S; Brusko, T M; Brown, A C; Quint, P S; Litherland, S A; Roopenian, D C; Iczkowski, K A

2007-02-01

Genes at the centromeric end of the human leukocyte antigen region influence adaptive autoimmune diseases and cancer. In this study, we characterized protein expression of HKE2, a gene located in the centromeric portion of the class II region of the major histocompatibility complex encoding subunit 6 of prefoldin. Immunohistochemical analysis using an anti-HKE2 antibody indicated that HKE2 protein expression is dramatically upregulated as a consequence of activation. In a tissue microarray and in several tumors, HKE2 was overexpressed in certain cancers compared with normal counterparts. The localization of the HKE2 gene to the class II region, its cytoplasmic expression and putative protein-binding domain suggest that HKE2 may function in adaptive immunity and cancer.

Regression Analysis of Combined Gene Expression Regulation in Acute Myeloid Leukemia

PubMed Central

Li, Yue; Liang, Minggao; Zhang, Zhaolei

2014-01-01

Gene expression is a combinatorial function of genetic/epigenetic factors such as copy number variation (CNV), DNA methylation (DM), transcription factors (TF) occupancy, and microRNA (miRNA) post-transcriptional regulation. At the maturity of microarray/sequencing technologies, large amounts of data measuring the genome-wide signals of those factors became available from Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA). However, there is a lack of an integrative model to take full advantage of these rich yet heterogeneous data. To this end, we developed RACER (Regression Analysis of Combined Expression Regulation), which fits the mRNA expression as response using as explanatory variables, the TF data from ENCODE, and CNV, DM, miRNA expression signals from TCGA. Briefly, RACER first infers the sample-specific regulatory activities by TFs and miRNAs, which are then used as inputs to infer specific TF/miRNA-gene interactions. Such a two-stage regression framework circumvents a common difficulty in integrating ENCODE data measured in generic cell-line with the sample-specific TCGA measurements. As a case study, we integrated Acute Myeloid Leukemia (AML) data from TCGA and the related TF binding data measured in K562 from ENCODE. As a proof-of-concept, we first verified our model formalism by 10-fold cross-validation on predicting gene expression. We next evaluated RACER on recovering known regulatory interactions, and demonstrated its superior statistical power over existing methods in detecting known miRNA/TF targets. Additionally, we developed a feature selection procedure, which identified 18 regulators, whose activities clustered consistently with cytogenetic risk groups. One of the selected regulators is miR-548p, whose inferred targets were significantly enriched for leukemia-related pathway, implicating its novel role in AML pathogenesis. Moreover, survival analysis using the inferred activities identified C-Fos as a potential AML prognostic marker. Together, we provided a novel framework that successfully integrated the TCGA and ENCODE data in revealing AML-specific regulatory program at global level. PMID:25340776

Cloning and Expression of the Benzoate Dioxygenase Genes from Rhodococcus sp. Strain 19070

PubMed Central

Haddad, Sandra; Eby, D. Matthew; Neidle, Ellen L.

2001-01-01

The bopXYZ genes from the gram-positive bacterium Rhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromatic cis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid in Rhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses both meta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl and ben genes, respectively. Open reading frames downstream of bopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences. PMID:11375157

Characteristics of the Lotus japonicus gene repertoire deduced from large-scale expressed sequence tag (EST) analysis.

PubMed

Asamizu, Erika; Nakamura, Yasukazu; Sato, Shusei; Tabata, Satoshi

2004-02-01

To perform a comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 74472 3'-end expressed sequence tags (EST) were generated from cDNA libraries produced from six different organs. Clustering of sequences was performed with an identity criterion of 95% for 50 bases, and a total of 20457 non-redundant sequences, 8503 contigs and 11954 singletons were generated. EST sequence coverage was analyzed by using the annotated L. japonicus genomic sequence and 1093 of the 1889 predicted protein-encoding genes (57.9%) were hit by the EST sequence(s). Gene content was compared to several plant species. Among the 8503 contigs, 471 were identified as sequences conserved only in leguminous species and these included several disease resistance-related genes. This suggested that in legumes, these genes may have evolved specifically to resist pathogen attack. The rate of gene sequence divergence was assessed by comparing similarity level and functional category based on the Gene Ontology (GO) annotation of Arabidopsis genes. This revealed that genes encoding ribosomal proteins, as well as those related to translation, photosynthesis, and cellular structure were more abundantly represented in the highly conserved class, and that genes encoding transcription factors and receptor protein kinases were abundantly represented in the less conserved class. To make the sequence information and the cDNA clones available to the research community, a Web database with useful services was created at http://www.kazusa.or.jp/en/plant/lotus/EST/.

New TFII-I family target genes involved in embryonic development.

PubMed

Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

2009-09-04

Two members of the TFII-I family transcription factor genes, GTF2I and GTF2IRD1, are the prime candidates responsible for the craniofacial and cognitive abnormalities of Williams syndrome patients. We have previously generated mouse lines with targeted disruption of Gtf2i and Gtf2ird1. Microarray analysis revealed significant changes in the expression profile of mutant embryos. Here we described three unknown genes that were dramatically down-regulated in mutants. The 2410018M08Rik/Scand3 gene encodes a protein of unknown function with CHCH and hATC domains. Scand3 is down-regulated during mouse embryonic stem cell (ES) differentiation. 4933436H12Rik is a testis-specific gene, which encodes a protein with no known domains. It is expressed in mouse ES cells. 1110008P08Rik/Kbtbd7 encodes an adapter protein with BTB/POZ, BACK, and Kelch motifs, previously shown to recruit substrates to the enzymatic complexes of the histone modifying or E3 ubiquitin ligase activities. Based on its expression pattern Kbtbd7 may have a specific role in brain development and function. All three genes possess well-conserved TFII-I-binding consensus sites within proximal promoters. Therefore our analysis suggests that these genes can be direct targets of TFII-I proteins and their impaired expression, as a result of the GTF2I and GTF2IRD1 haploinsufficiency, could contribute to the etiology of Williams syndrome.

Mapping gene expression patterns during myeloid differentiation using the EML hematopoietic progenitor cell line.

PubMed

Du, Yang; Campbell, Janee L; Nalbant, Demet; Youn, Hyewon; Bass, Ann C Hughes; Cobos, Everardo; Tsai, Schickwann; Keller, Jonathan R; Williams, Simon C

2002-07-01

The detailed examination of the molecular events that control the early stages of myeloid differentiation has been hampered by the relative scarcity of hematopoietic stem cells and the lack of suitable cell line models. In this study, we examined the expression of several myeloid and nonmyeloid genes in the murine EML hematopoietic stem cell line. Expression patterns for 19 different genes were examined by Northern blotting and RT-PCR in RNA samples from EML, a variety of other immortalized cell lines, and purified murine hematopoietic stem cells. Representational difference analysis (RDA) was performed to identify differentially expressed genes in EML. Expression patterns of genes encoding transcription factors (four members of the C/EBP family, GATA-1, GATA-2, PU.1, CBFbeta, SCL, and c-myb) in EML were examined and were consistent with the proposed functions of these proteins in hematopoietic differentiation. Expression levels of three markers of terminal myeloid differentiation (neutrophil elastase, proteinase 3, and Mac-1) were highest in EML cells at the later stages of differentiation. In a search for genes that were differentially expressed in EML cells during myeloid differentiation, six cDNAs were isolated. These included three known genes (lysozyme, histidine decarboxylase, and tryptophan hydroxylase) and three novel genes. Expression patterns of known genes in differentiating EML cells accurately reflected their expected expression patterns based on previous studies. The identification of three novel genes, two of which encode proteins that may act as regulators of hematopoietic differentiation, suggests that EML is a useful model system for the molecular analysis of hematopoietic differentiation.

Molecular definition of the identity and activation of natural killer cells.

PubMed

Bezman, Natalie A; Kim, Charles C; Sun, Joseph C; Min-Oo, Gundula; Hendricks, Deborah W; Kamimura, Yosuke; Best, J Adam; Goldrath, Ananda W; Lanier, Lewis L

2012-10-01

Using whole-genome microarray data sets of the Immunological Genome Project, we demonstrate a closer transcriptional relationship between NK cells and T cells than between any other leukocytes, distinguished by their shared expression of genes encoding molecules with similar signaling functions. Whereas resting NK cells are known to share expression of a few genes with cytotoxic CD8(+) T cells, our transcriptome-wide analysis demonstrates that the commonalities extend to hundreds of genes, many encoding molecules with unknown functions. Resting NK cells demonstrate a 'preprimed' state compared with naive T cells, which allows NK cells to respond more rapidly to viral infection. Collectively, our data provide a global context for known and previously unknown molecular aspects of NK cell identity and function by delineating the genome-wide repertoire of gene expression of NK cells in various states.

Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida

PubMed Central

2012-01-01

Background Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42. Results We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose. Conclusions We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin. PMID:22272603

Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing.

PubMed

Vannette, Rachel L; Mohamed, Abbas; Johnson, Brian R

2015-11-09

Pollinators, including honey bees, routinely encounter potentially harmful microorganisms and phytochemicals during foraging. However, the mechanisms by which honey bees manage these potential threats are poorly understood. In this study, we examine the expression of antimicrobial, immune and detoxification genes in Apis mellifera and compare between forager and nurse bees using tissue-specific RNA-seq and qPCR. Our analysis revealed extensive tissue-specific expression of antimicrobial, immune signaling, and detoxification genes. Variation in gene expression between worker stages was pronounced in the mandibular and hypopharyngeal gland (HPG), where foragers were enriched in transcripts that encode antimicrobial peptides (AMPs) and immune response. Additionally, forager HPGs and mandibular glands were enriched in transcripts encoding detoxification enzymes, including some associated with xenobiotic metabolism. Using qPCR on an independent dataset, we verified differential expression of three AMP and three P450 genes between foragers and nurses. High expression of AMP genes in nectar-processing tissues suggests that these peptides may contribute to antimicrobial properties of honey or to honey bee defense against environmentally-acquired microorganisms. Together, these results suggest that worker role and tissue-specific expression of AMPs, and immune and detoxification enzymes may contribute to defense against microorganisms and xenobiotic compounds acquired while foraging.

Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing

PubMed Central

Vannette, Rachel L.; Mohamed, Abbas; Johnson, Brian R.

2015-01-01

Pollinators, including honey bees, routinely encounter potentially harmful microorganisms and phytochemicals during foraging. However, the mechanisms by which honey bees manage these potential threats are poorly understood. In this study, we examine the expression of antimicrobial, immune and detoxification genes in Apis mellifera and compare between forager and nurse bees using tissue-specific RNA-seq and qPCR. Our analysis revealed extensive tissue-specific expression of antimicrobial, immune signaling, and detoxification genes. Variation in gene expression between worker stages was pronounced in the mandibular and hypopharyngeal gland (HPG), where foragers were enriched in transcripts that encode antimicrobial peptides (AMPs) and immune response. Additionally, forager HPGs and mandibular glands were enriched in transcripts encoding detoxification enzymes, including some associated with xenobiotic metabolism. Using qPCR on an independent dataset, we verified differential expression of three AMP and three P450 genes between foragers and nurses. High expression of AMP genes in nectar-processing tissues suggests that these peptides may contribute to antimicrobial properties of honey or to honey bee defense against environmentally-acquired microorganisms. Together, these results suggest that worker role and tissue-specific expression of AMPs, and immune and detoxification enzymes may contribute to defense against microorganisms and xenobiotic compounds acquired while foraging. PMID:26549293

Bordetella pertussis risA, but Not risS, Is Required for Maximal Expression of Bvg-Repressed Genes

PubMed Central

Stenson, Trevor H.; Allen, Andrew G.; al-Meer, Jehan A.; Maskell, Duncan; Peppler, Mark S.

2005-01-01

Expression of virulence determinants by Bordetella pertussis, the primary etiological agent of whooping cough, is regulated by the BvgAS two-component regulatory system. The role of a second two-component regulatory system, encoded by risAS, in this process is not defined. Here, we show that mutation of B. pertussis risA does not affect Bvg-activated genes or proteins. However, mutation of risA resulted in greatly diminished expression of Bvg-repressed antigens and decreased transcription of Bvg-repressed genes. In contrast, mutation of risS had no effect on the expression of Bvg-regulated molecules. Mutation of risA also resulted in decreased bacterial invasion in a HeLa cell model. However, decreased invasion could not be attributed to the decreased expression of Bvg-repressed products, suggesting that mutation of risA may affect the expression of a variety of genes. Unlike the risAS operons in B. parapertussis and B. bronchiseptica, B. pertussis risS is a pseudogene that encodes a truncated RisS sensor. Deletion of the intact part of the B. pertussis risS gene does not affect the expression of risA-dependent, Bvg-repressed genes. These observations suggest that RisA activation occurs through cross-regulation by a heterologous system. PMID:16113320

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.

DNA Gyrase Is the Target for the Quinolone Drug Ciprofloxacin in Arabidopsis thaliana.

PubMed

Evans-Roberts, Katherine M; Mitchenall, Lesley A; Wall, Melisa K; Leroux, Julie; Mylne, Joshua S; Maxwell, Anthony

2016-02-12

The Arabidopsis thaliana genome contains four genes that were originally annotated as potentially encoding DNA gyrase: ATGYRA, ATGYRB1, ATGYRB2, and ATGYRB3. Although we subsequently showed that ATGYRB3 does not encode a gyrase subunit, the other three genes potentially encode subunits of a plant gyrase. We also showed evidence for the existence of supercoiling activity in A. thaliana and that the plant is sensitive to quinolone and aminocoumarin antibiotics, compounds that target DNA gyrase in bacteria. However, it was not possible at that time to show whether the A. thaliana genes encoded an active gyrase enzyme, nor whether that enzyme is indeed the target for the quinolone and aminocoumarin antibiotics. Here we show that an A. thaliana mutant resistant to the quinolone drug ciprofloxacin has a point mutation in ATGYRA. Moreover we show that, as in bacteria, the quinolone-sensitive (wild-type) allele is dominant to the resistant gene. Further we have heterologously expressed ATGYRA and ATGYRB2 in a baculovirus expression system and shown supercoiling activity of the partially purified enzyme. Expression/purification of the quinolone-resistant A. thaliana gyrase yields active enzyme that is resistant to ciprofloxacin. Taken together these experiments now show unequivocally that A. thaliana encodes an organelle-targeted DNA gyrase that is the target of the quinolone drug ciprofloxacin; this has important consequences for plant physiology and the development of herbicides. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice.

PubMed

Kwon, Jun Tae; Ham, Sera; Jeon, Suyeon; Kim, Youil; Oh, Seungmin; Cho, Chunghee

2017-01-01

The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea.

PubMed Central

Reith, M; Munholland, J

1993-01-01

Extensive DNA sequencing of the chloroplast genome of the red alga Porphyra purpurea has resulted in the detection of more than 125 genes. Fifty-eight (approximately 46%) of these genes are not found on the chloroplast genomes of land plants. These include genes encoding 17 photosynthetic proteins, three tRNAs, and nine ribosomal proteins. In addition, nine genes encoding proteins related to biosynthetic functions, six genes encoding proteins involved in gene expression, and at least five genes encoding miscellaneous proteins are among those not known to be located on land plant chloroplast genomes. The increased coding capacity of the P. purpurea chloroplast genome, along with other characteristics such as the absence of introns and the conservation of ancestral operons, demonstrate the primitive nature of the P. purpurea chloroplast genome. In addition, evidence for a monophyletic origin of chloroplasts is suggested by the identification of two groups of genes that are clustered in chloroplast genomes but not in cyanobacteria. PMID:12271072

Unraveling flp-11/flp-32 dichotomy in nematodes.

PubMed

Atkinson, Louise E; Miskelly, Iain R; Moffett, Christy L; McCoy, Ciaran J; Maule, Aaron G; Marks, Nikki J; Mousley, Angela

2016-10-01

FMRFamide-like peptide (FLP) signalling systems are core to nematode neuromuscular function. Novel drug discovery efforts associated with nematode FLP/FLP receptor biology are advanced through the accumulation of basic biological data that can reveal subtle complexities within the neuropeptidergic system. This study reports the characterisation of FMRFamide-like peptide encoding gene-11 (flp-11) and FMRFamide-like peptide encoding gene-32 (flp-32), two distinct flp genes which encode the analogous peptide, AMRN(A/S)LVRFamide, in multiple nematode species - the only known example of this phenomenon within the FLPergic system of nematodes. Using bioinformatics, in situ hybridisation, immunocytochemistry and behavioural assays we show that: (i) flp-11 and -32 are distinct flp genes expressed individually or in tandem across multiple nematode species, where they encode a highly similar peptide; (ii) flp-11 does not appear to be the most widely expressed flp in Caenorhabditis elegans; (iii) in species expressing both flp-11 and flp-32, flp-11 displays a conserved, restricted expression pattern across nematode clades and lifestyles; (iv) in species expressing both flp-11 and flp-32, flp-32 expression is more widespread and less conserved than flp-11; (v) in species expressing only flp-11, the flp-11 expression profile is more similar to the flp-32 profile observed in species expressing both; and (vi) FLP-11 peptides inhibit motor function in multiple nematode species. The biological significance and evolutionary origin of flp-11 and -32 peptide duplication remains unclear despite attempts to identify a common ancestor; this may become clearer as the availability of genomic data improves. This work provides insight into the complexity of the neuropeptidergic system in nematodes, and begins to examine how nematodes may compensate for structural neuronal simplicity. From a parasite control standpoint, this work underscores the importance of basic biological data, and has wider implications for the utility of C. elegans as a model for parasite neurobiology. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas.

PubMed

Gu, Keyu; Chiam, Huihui; Tian, Dongsheng; Yin, Zhongchao

2011-04-01

Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a β-subunit of carboxyltransferase (β-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha (Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5' RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa β-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and β-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The C. elegans che-1 gene encodes a zinc finger transcription factor required for specification of the ASE chemosensory neurons.

PubMed

Uchida, Okiko; Nakano, Hiroyuki; Koga, Makoto; Ohshima, Yasumi

2003-04-01

Chemotaxis to water-soluble chemicals such as NaCl is an important behavior of C. elegans when seeking food. ASE chemosensory neurons have a major role in this behavior. We show that che-1, defined by chemotaxis defects, encodes a zinc-finger protein similar to the GLASS transcription factor required for photoreceptor cell differentiation in Drosophila, and that che-1 is essential for specification and function of ASE neurons. Expression of a che-1::gfp fusion construct was predominant in ASE. In che-1 mutants, expression of genes characterizing ASE such as seven-transmembrane receptors, guanylate cyclases and a cyclic-nucleotide gated channel is lost. Ectopic expression of che-1 cDNA induced expression of ASE-specific marker genes, a dye-filling defect in neurons other than ASE and dauer formation.

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

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

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of themore » TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.« less

Characterization and expression of the calpastatin gene in Cyprinus carpio.

PubMed

Chen, W X; Ma, Y

2015-07-03

Calpastatin, an important protein used to regulate meat quality traits in animals, is encoded by the CAST gene. The aim of the present study was to clone the cDNA sequence of the CAST gene and detect the expression of CAST in the tissues of Cyprinus carpio. The cDNA of the C. carpio CAST gene, amplified using rapid amplification of cDNA ends PCR, is 2834 bp in length (accession No. JX275386), contains a 2634-bp open reading frame, and encodes a protein with 877 amino acid residues. The amino acid sequence of the C. carpio CAST gene was 88, 80, and 59% identical to the sequences observed in grass carp, zebrafish, and other fish, respectively. The C. carpio CAST was observed to contain four conserved domains with 54 serine phosphorylation loci, 28 threonine phosphorylation loci, 1 tyrosine phosphorylation loci, and 6 specific protein kinase C phosphorylation loci. The CAST gene showed widespread expression in different tissues of C. carpio. Surprisingly, the relative expression of the CAST transcript in the muscle and heart tissues of C. carpio was significantly higher than in other tissues (P < 0.01).

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

PubMed

Pasion, S G; Brown, G W; Brown, L M; Ray, D S

1994-12-01

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

Effect of hypoxia on the expression of genes encoding insulin-like growth factors and some related proteins in U87 glioma cells without IRE1 function.

PubMed

Minchenko, Dmytro O; Kharkova, A P; Halkin, O V; Karbovskyi, L L; Minchenko, O H

2016-04-01

The aim of the present study was to investigate the effect of hypoxia on the expression of genes encoding insulin-like growth factors (IGF1 and IGF2), their receptor (IGF1R), binding protein-4 (IGFBP4), and stanniocalcin 2 (STC2) in U87 glioma cells in relation to inhibition of endoplasmic reticulum stress signaling mediated by IRE1 (inositol requiring enzyme 1) for evaluation of their possible significance in the control of tumor growth. The expression of IGF1, IGF2, IGF1R, IGFBP4, and STC2 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia was studied by qPCR. The expression of IGF1 and IGF2 genes is down-regulated in glioma cells without IRE1 signaling enzyme function in comparison with the control cells. At the same time, the expression of IGF1R, IGFBP4, and STC2 genes was up-regulated in glioma cells upon inhibition of IRE1, with more significant changes for IGFBP4 and STC2 genes. We also showed that hypoxia does not change significantly the expression of IGF1, IGF2, and IGF1R genes but up-regulated IGFBP4 and STC2 genes expression in control glioma cells. Moreover, the inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells does not change significantly the effect of hypoxia on the expression of IGF1, IGF1R, and IGFBP4 genes but introduces sensitivity of IGF2 gene to hypoxic condition. Thus, the expression of IGF2 gene is resistant to hypoxia only in control glioma cells and significantly down-regulated in cells without functional activity of IRE1 signaling enzyme, which is central mediator of the unfolded protein response and an important component of the tumor growth as well as metabolic diseases. Results of this study demonstrate that the expression of IGF1 and IGF1R genes is resistant to hypoxic condition both in control U87 glioma cells and cells without IRE1 signaling enzyme function. However, hypoxia significantly up-regulates the expression of IGFBP4 gene independently on the inhibition of IRE1 enzyme. These data show that proteins encoded by these genes are resistant to hypoxia except IGFBP4 and participate in the regulation of metabolic and proliferative processes through IRE1 signaling.

Hierarchical regulation of photosynthesis gene expression by the oxygen-responsive PrrBA and AppA-PpsR systems of Rhodobacter sphaeroides.

PubMed

Gomelsky, Larissa; Moskvin, Oleg V; Stenzel, Rachel A; Jones, Denise F; Donohue, Timothy J; Gomelsky, Mark

2008-12-01

In the facultatively phototrophic proteobacterium Rhodobacter sphaeroides, formation of the photosynthetic apparatus is oxygen dependent. When oxygen tension decreases, the response regulator PrrA of the global two-component PrrBA system is believed to directly activate transcription of the puf, puh, and puc operons, encoding structural proteins of the photosynthetic complexes, and to indirectly upregulate the photopigment biosynthesis genes bch and crt. Decreased oxygen also results in inactivation of the photosynthesis-specific repressor PpsR, bringing about derepression of the puc, bch, and crt operons. We uncovered a hierarchical relationship between these two regulatory systems, earlier thought to function independently. We also more accurately assessed the spectrum of gene targets of the PrrBA system. First, expression of the appA gene, encoding the PpsR antirepressor, is PrrA dependent, which establishes one level of hierarchical dominance of the PrrBA system over AppA-PpsR. Second, restoration of the appA transcript to the wild-type level is insufficient for rescuing phototrophic growth impairment of the prrA mutant, whereas inactivation of ppsR is sufficient. This suggests that in addition to controlling appA transcription, PrrA affects the activity of the AppA-PpsR system via an as yet unidentified mechanism(s). Third, PrrA directly activates several bch and crt genes, traditionally considered to be the PpsR targets. Therefore, in R. sphaeroides, the global PrrBA system regulates photosynthesis gene expression (i) by rigorous control over the photosynthesis-specific AppA-PpsR regulatory system and (ii) by extensive direct transcription activation of genes encoding structural proteins of photosynthetic complexes as well as genes encoding photopigment biosynthesis enzymes.

Comparative antennal transcriptome of Apis cerana cerana from four developmental stages.

PubMed

Zhao, Huiting; Peng, Zhu; Du, Yali; Xu, Kai; Guo, Lina; Yang, Shuang; Ma, Weihua; Jiang, Yusuo

2018-06-20

Apis cerana cerana, an important endemic honey bee species in China, possesses valuable characteristics such as a sensitive olfactory system, good foraging ability, and strong resistance to parasitic mites. Here, we performed transcriptome sequencing of the antenna, the major chemosensory organ of the bee, using an Illumina sequencer, to identify typical differentially expressed genes (DEGs) in adult worker bees of different ages, namely, T1 (1 day); T2 (10 days); T3 (15 days); and T4 (25 days). Surprisingly, the expression levels of DEGs changed significantly between the T1 period and the other three periods. All the DEGs were classified into 26 expression profiles by trend analysis. Selected trend clusters were analyzed, and valuable information on gene expression patterns was obtained. We found that the expression levels of genes encoding cuticle proteins declined after eclosion, while those of immunity-related genes increased. In addition, genes encoding venom proteins and major royal jelly proteins were enriched at the T2 stage; small heat shock proteins showed significantly higher expression at the T3 stage; and some metabolism-related genes were more highly expressed at the T4 stage. The DEGs identified in this study may serve as a valuable resource for the characterization of expression patterns of antennal genes in A. cerana cerana. Furthermore, this study provides insights into the relationship between labor division in social bees and gene function. Copyright © 2018. Published by Elsevier B.V.

Adaptive Mutations in RNA Polymerase and the Transcriptional Terminator Rho Have Similar Effects on Escherichia coli Gene Expression.

PubMed

González-González, Andrea; Hug, Shaun M; Rodríguez-Verdugo, Alejandra; Patel, Jagdish Suresh; Gaut, Brandon S

2017-11-01

Modifications to transcriptional regulators play a major role in adaptation. Here, we compared the effects of multiple beneficial mutations within and between Escherichia coli rpoB, the gene encoding the RNA polymerase β subunit, and rho, which encodes a transcriptional terminator. These two genes have harbored adaptive mutations in numerous E. coli evolution experiments but particularly in our previous large-scale thermal stress experiment, where the two genes characterized alternative adaptive pathways. To compare the effects of beneficial mutations, we engineered four advantageous mutations into each of the two genes and measured their effects on fitness, growth, gene expression and transcriptional termination at 42.2 °C. Among the eight mutations, two rho mutations had no detectable effect on relative fitness, suggesting they were beneficial only in the context of epistatic interactions. The remaining six mutations had an average relative fitness benefit of ∼20%. The rpoB mutations affected the expression of ∼1,700 genes; rho mutations affected the expression of fewer genes but most (83%) were a subset of those altered by rpoB mutants. Across the eight mutants, relative fitness correlated with the degree to which a mutation restored gene expression back to the unstressed, 37.0 °C state. The beneficial mutations in the two genes did not have identical effects on fitness, growth or gene expression, but they caused parallel phenotypic effects on gene expression and genome-wide transcriptional termination. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Transcriptional response of mysid crustacean, Americamysis bahia, is affected by subchronic exposure to nonylphenol.

PubMed

Uchida, Masaya; Hirano, Masashi; Ishibashi, Hiroshi; Kobayashi, Jun; Kagami, Yoshihiro; Koyanagi, Akiko; Kusano, Teruhiko; Koga, Minoru; Arizono, Koji

2016-11-01

Nonylphenol (NP) has been classified as an endocrine-disrupting chemical. In this study, we conducted mysid DNA microarray analysis with which has 2240 oligo DNA probes to observe differential gene expressions in mysid crustacean (Americamysis bahia) exposed to 1, 3, 10 and 30 μg/l of NP for 14 days. As a result, we found 31, 27, 39 and 68 genes were differentially expressed in the respective concentrations. Among these genes, the expressions of five particular genes were regulated in a similar manner at all concentrations of the NP exposure. So, we focused on one gene encoding cuticle protein, and another encoding cuticular protein analogous to peritrophins 1-H precursor. These genes were down-regulated by NP exposure in a dose-dependent manner, and it suggested that they were related in a reduction of the number of molting in mysids. Thus, they might become useful molecular biomarker candidates to evaluate molting inhibition in mysids. Copyright © 2016 Elsevier Inc. All rights reserved.

BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS.

PubMed

Creelman, Robert A.; Mullet, John E.

1997-06-01

Jasmonic acid and its derivatives can modulate aspects of fruit ripening, production of viable pollen, root growth, tendril coiling, and plant resistance to insects and pathogens. Jasmonate activates genes involved in pathogen and insect resistance, and genes encoding vegetative storage proteins, but represses genes encoding proteins involved in photosynthesis. Jasmonic acid is derived from linolenic acid, and most of the enzymes in the biosynthetic pathway have been extensively characterized. Modulation of lipoxygenase and allene oxide synthase gene expression in transgenic plants raises new questions about the compartmentation of the biosynthetic pathway and its regulation. The activation of jasmonic acid biosynthesis by cell wall elicitors, the peptide systemin, and other compounds will be related to the function of jasmonates in plants. Jasmonate modulates gene expression at the level of translation, RNA processing, and transcription. Promoter elements that mediate responses to jasmonate have been isolated. This review covers recent advances in our understanding of how jasmonate biosynthesis is regulated and relates this information to knowledge of jasmonate modulated gene expression.

Identification and characterization of Rhox13, a novel X-linked mouse homeobox gene

PubMed Central

Geyer, Christopher B.; Eddy, Edward M.

2008-01-01

Homeobox genes encode transcription factors whose expression organizes programs of development. A number of homeobox genes expressed in reproductive tissues have been identified recently, including a colinear cluster on the X chromosome in mice. This has led to an increased interest in understanding the role(s) of homeobox genes in regulating development of reproductive tissues including the testis, ovary, and placenta. Here we report the identification and characterization of a novel homeobox gene of the paired-like class on the X chromosome distal to the reproductive homeobox (Rhox) cluster in mice. Transcripts are found in the testis and ovary as early as 13.5 days post-coitum (dpc). Transcription ceases in the ovary by 3 days post-partum (dpp), but continues in the testis through adulthood. The Rhox13 gene encodes a 25.3 kDa protein expressed in the adult testis in germ cells at the basal aspect of the seminiferous epithelium. PMID:18675325

Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes

PubMed Central

van der Does, H. Charlotte; Schmidt, Sarah M.; Langereis, Léon; Hughes, Timothy R.

2016-01-01

Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called ‘effectors’. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the ‘pathogenicity’ chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol pathogenicity chromosome may be partially transcriptionally autonomous, but there are also extensive transcriptional connections between core and accessory chromosomes. PMID:27855160

Concurrent host-pathogen gene expression in the lungs of pigs challenged with Actinobacillus pleuropneumoniae.

PubMed

Brogaard, Louise; Klitgaard, Kirstine; Heegaard, Peter M H; Hansen, Mette Sif; Jensen, Tim Kåre; Skovgaard, Kerstin

2015-05-28

Actinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. To obtain in-depth understanding of this infection, the interplay between virulence factors of the pathogen and defense mechanisms of the porcine host needs to be elucidated. However, research has traditionally focused on either bacteriology or immunology; an unbiased picture of the transcriptional responses can be obtained by investigating both organisms in the same biological sample. Host and pathogen responses in pigs experimentally infected with A. pleuropneumoniae were analyzed by high-throughput RT-qPCR. This approach allowed concurrent analysis of selected genes encoding proteins known or hypothesized to be important in the acute phase of this infection. The expression of 17 bacterial and 31 porcine genes was quantified in lung samples obtained within the first 48 hours of infection. This provided novel insight into the early time course of bacterial genes involved in synthesis of pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, lipoprotein) and genes involved in pattern recognition (TLR4, CD14, MD2, LBP, MYD88) in response to A. pleuropneumoniae. Significant up-regulation of proinflammatory cytokines such as IL1B, IL6, and IL8 was observed, correlating with protein levels, infection status and histopathological findings. Host genes encoding proteins involved in iron metabolism, as well as bacterial genes encoding exotoxins, proteins involved in adhesion, and iron acquisition were found to be differentially expressed according to disease progression. By applying laser capture microdissection, porcine expression of selected genes could be confirmed in the immediate surroundings of the invading pathogen. Microbial pathogenesis is the product of interactions between host and pathogen. Our results demonstrate the applicability of high-throughput RT-qPCR for the elucidation of dual-organism gene expression analysis during infection. We showed differential expression of 12 bacterial and 24 porcine genes during infection and significant correlation of porcine and bacterial gene expression. This is the first study investigating the concurrent transcriptional response of both bacteria and host at the site of infection during porcine respiratory infection.

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.

Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog.

PubMed

Lo, Miranda; Murray, Gerald L; Khoo, Chen Ai; Haake, David A; Zuerner, Richard L; Adler, Ben

2010-11-01

Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

Characterization of LeMir, a Root-Knot Nematode-Induced Gene in Tomato with an Encoded Product Secreted from the Root1

PubMed Central

Brenner, Eric D.; Lambert, Kris N.; Kaloshian, Isgouhi; Williamson, Valerie M.

1998-01-01

A tomato gene that is induced early after infection of tomato (Lycopersicon esculentum Mill.) with root-knot nematodes (Meloidogyne javanica) encodes a protein with 54% amino acid identity to miraculin, a flavorless protein that causes sour substances to be perceived as sweet. This gene was therefore named LeMir (L. esculentum miraculin). Sequence similarity places the encoded protein in the soybean trypsin-inhibitor family (Kunitz). LeMir mRNA is found in root, hypocotyl, and flower tissues, with the highest expression in the root. Rapid induction of expression upon nematode infection is localized to root tips. In situ hybridization shows that LeMir is expressed constitutively in the root-cap and root-tip epidermis. The LeMir protein product (LeMir) was produced in the yeast Pichia pastoris for generation of antibodies. Western-blot analysis showed that LeMir expression is up-regulated by nematode infection and by wounding. LeMir is also expressed in tomato callus tissue. Immunoprint analysis revealed that LeMir is expressed throughout the seedling root, but that levels are highest at the root/shoot junction. Analysis of seedling root exudates revealed that LeMir is secreted from the root into the surrounding environment, suggesting that it may interact with soil-borne microorganisms. PMID:9733543

Genetic and Functional Investigation of Zn2Cys6 Transcription Factors RSE2 and RSE3 in Podospora anserina

PubMed Central

Bovier, Elodie; Sellem, Carole H.; Humbert, Adeline

2014-01-01

In Podospora anserina, the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase (aox) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase (pck) and fructose-1,6-biphosphatase (fbp). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3. Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3. It showed that in addition to aox, fbp, and pck, RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn2Cys6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools. PMID:24186951

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

PubMed Central

Schmied, Stéfanie; Affentranger, Sarah; Parvanova, Iana; Kang'a, Simon; Nene, Vishvanath; Katzer, Frank; McKeever, Declan; Müller, Joachim; Bishop, Richard; Pain, Arnab; Dobbelaere, Dirk A. E.

2009-01-01

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins. PMID:19325907

Method for increasing thermostability in cellulase ennzymes

DOEpatents

Adney, W.S.; Thomas, S.R.; Baker, J.O.; Himmel, M.E.; Chou, Y.C.

1998-01-27

The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in Pichia pastoris. A new modified E1 endoglucanase enzyme comprising the catalytic domain of the full size E1 enzyme demonstrates enhanced thermostability and is produced by two methods. The first method of producing the new modified E1 is proteolytic cleavage to remove the cellulose binding domain and linker peptide of the full size E1. The second method of producing the new modified E1 is genetic truncation of the gene encoding the full size E1 so that the catalytic domain is expressed in the expression product. 8 figs.

[Construction and expression of the targeting super-antigen EGF-SEA fusion gene].

PubMed

Xie, Yang; Peng, Shaoping; Liao, Zhiying; Liu, Jiafeng; Liu, Xuemei; Chen, Weifeng

2014-05-01

To construct expression vector for the SEA-EGF fusion gene. Clone the SEA gene and the EGF gene segment with PCR and RT-PCR independently, and connect this two genes by the bridge PCR. Insert the fusion gene EGF-SEA into the expression vector PET-44. Induced the secretion of the fusion protein SEA-EGF by the antileptic. The gene fragment encoding EGF and SEA mature peptide was successfully cloned. The fusion gene EGF-SEA was successfully constructed and was inserted into expression vector. The new recombinant expression vector for fusion gene EGF-SEA is specific for head and neck cancer, laid the foundation for the further study of fusion protein SEA-EGF targeting immune therapy in head and neck tumors.

Control of the synthesis and subcellular targeting of the two GDH genes products in leaves and stems of Nicotiana plumbaginifolia and Arabidopsis thaliana.

PubMed

Fontaine, Jean-Xavier; Saladino, Francesca; Agrimonti, Caterina; Bedu, Magali; Tercé-Laforgue, Thérèse; Tétu, Thierry; Hirel, Bertrand; Restivo, Francesco M; Dubois, Frédéric

2006-03-01

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

Regulation of gene expression in the mammalian eye and its relevance to eye disease

PubMed Central

Scheetz, Todd E.; Kim, Kwang-Youn A.; Swiderski, Ruth E.; Philp, Alisdair R.; Braun, Terry A.; Knudtson, Kevin L.; Dorrance, Anne M.; DiBona, Gerald F.; Huang, Jian; Casavant, Thomas L.; Sheffield, Val C.; Stone, Edwin M.

2006-01-01

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F2 rats generated from an SR/JrHsd × SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (α = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5′ flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor β2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet–Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease. PMID:16983098

Cloning and characterization of an inulinase gene from the marine yeast Candida membranifaciens subsp. flavinogenie W14-3 and its expression in Saccharomyces sp. W0 for ethanol production.

PubMed

Zhang, Lin-Lin; Tan, Mei-Juan; Liu, Guang-Lei; Chi, Zhe; Wang, Guang-Yuan; Chi, Zhen-Ming

2015-04-01

The INU1 gene encoding an exo-inulinase from the marine-derived yeast Candida membranifaciens subsp. flavinogenie W14-3 was cloned and characterized. It had an open reading frame of 1,536 bp long encoding an inulinase. The coding region of it was not interrupted by any intron. The cloned gene encoded 512 amino acid residues of a protein with a putative signal peptide of 23 amino acids and a calculated molecular mass of 57.8 kDa. The protein sequence deduced from the inulinase gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP FS and Q. The protein also had six conserved putative N-glycosylation sites. The deduced inulinase from the yeast strain W14-3 was found to be closely related to that from Candida kutaonensis sp. nov. KRF1, Kluyveromyces marxianus, and Cryptococcus aureus G7a. The inulinase gene with its signal peptide encoding sequence was subcloned into the pMIRSC11 expression vector and expressed in Saccharomyces sp. W0. The recombinant yeast strain W14-3-INU-112 obtained could produce 16.8 U/ml of inulinase activity and 12.5 % (v/v) ethanol from 250 g/l of inulin within 168 h. The monosaccharides were detected after the hydrolysis of inulin with the crude inulinase (the yeast culture). All the results indicated that the cloned gene and the recombinant yeast strain W14-3-INU-112 had potential applications in biotechnology.

Transcription Factors of Lotus: Regulation of Isoflavonoid Biosynthesis Requires Coordinated Changes in Transcription Factor Activity1[W][OA

PubMed Central

Shelton, Dale; Stranne, Maria; Mikkelsen, Lisbeth; Pakseresht, Nima; Welham, Tracey; Hiraka, Hideki; Tabata, Satoshi; Sato, Shusei; Paquette, Suzanne; Wang, Trevor L.; Martin, Cathie; Bailey, Paul

2012-01-01

Isoflavonoids are a class of phenylpropanoids made by legumes, and consumption of dietary isoflavonoids confers benefits to human health. Our aim is to understand the regulation of isoflavonoid biosynthesis. Many studies have shown the importance of transcription factors in regulating the transcription of one or more genes encoding enzymes in phenylpropanoid metabolism. In this study, we coupled bioinformatics and coexpression analysis to identify candidate genes encoding transcription factors involved in regulating isoflavonoid biosynthesis in Lotus (Lotus japonicus). Genes encoding proteins belonging to 39 of the main transcription factor families were examined by microarray analysis of RNA from leaf tissue that had been elicited with glutathione. Phylogenetic analyses of each transcription factor family were used to identify subgroups of proteins that were specific to L. japonicus or closely related to known regulators of the phenylpropanoid pathway in other species. R2R3MYB subgroup 2 genes showed increased expression after treatment with glutathione. One member of this subgroup, LjMYB14, was constitutively overexpressed in L. japonicus and induced the expression of at least 12 genes that encoded enzymes in the general phenylpropanoid and isoflavonoid pathways. A distinct set of six R2R3MYB subgroup 2-like genes was identified. We suggest that these subgroup 2 sister group proteins and those belonging to the main subgroup 2 have roles in inducing isoflavonoid biosynthesis. The induction of isoflavonoid production in L. japonicus also involves the coordinated down-regulation of competing biosynthetic pathways by changing the expression of other transcription factors. PMID:22529285

The prediction of biogenic magnetic nanoparticles biomineralization in human tissues and organs

NASA Astrophysics Data System (ADS)

Medviediev, O.; Gorobets, O. Yu; Gorobets, S. V.; Yadrykhins'ky, V. S.

2017-10-01

In this study, human homologs of magnetosome island proteins basing on pairwise and multiple alignment of amino acid sequences were found. The expression levels of genes, which encode magnetosome island proteins of M. gryphiswaldense MSR-1, that were cultured under oxygen deficiency conditions and also under microaerobic conditions were compared to the expression levels of genes that encode the relevant homologs in human organism. The possibility of BMN biomineralization in human tissues and organs, in which BMN were not experimentally found before, was predicted.

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.

Loss of functional K+ channels encoded by ether-à-go-go-related genes in mouse myometrium prior to labour onset

PubMed Central

Greenwood, I A; Yeung, S Y; Tribe, R M; Ohya, S

2009-01-01

There is a growing appreciation that ion channels encoded by the ether-à-go-go-related gene family have a functional impact in smooth muscle in addition to their accepted role in cardiac myocytes and neurones. This study aimed to assess the expression of ERG1–3 (KCNH1–3) genes in the murine myometrium (smooth muscle layer of the uterus) and determine the functional impact of the ion channels encoded by these genes in pregnant and non-pregnant animals. Quantitative RT-PCR did not detect message for ERG2 and 3 in whole myometrial tissue extracts. In contrast, message for two isoforms of mERG1 were readily detected with mERG1a more abundant than mERG1b. In isometric tension studies of non-pregnant myometrium, the ERG channel blockers dofetilide (1 μm), E4031 (1 μm) and Be-KM1 (100 nm) increased spontaneous contractility and ERG activators (PD118057 and NS1643) inhibited spontaneous contractility. In contrast, neither ERG blockade nor activation had any effect on the inherent contractility in myometrium from late pregnant (19 days gestation) animals. Moreover, dofetilide-sensitive K+ currents with distinctive ‘hooked’ kinetics were considerably smaller in uterine myocytes from late pregnant compared to non-pregnant animals. Expression of mERG1 isoforms did not alter throughout gestation or upon delivery, but the expression of genes encoding auxillary subunits (KCNE) were up-regulated considerably. This study provides the first evidence for a regulation of ERG-encoded K+ channels as a precursor to late pregnancy physiological activity. PMID:19332483

Expression analysis of the N-Myc downstream-regulated gene 1 indicates that myelinating Schwann cells are the primary disease target in hereditary motor and sensory neuropathy-Lom.

PubMed

Berger, Philipp; Sirkowski, Erich E; Scherer, Steven S; Suter, Ueli

2004-11-01

Mutations in the gene encoding N-myc downstream-regulated gene-1 (NDRG1) lead to truncations of the encoded protein and are associated with an autosomal recessive demyelinating neuropathy--hereditary motor and sensory neuropathy-Lom. NDRG1 protein is highly expressed in peripheral nerve and is localized in the cytoplasm of myelinating Schwann cells, including the paranodes and Schmidt-Lanterman incisures. In contrast, sensory and motor neurons as well as their axons lack NDRG1. NDRG1 mRNA levels in developing and injured adult sciatic nerves parallel those of myelin-related genes, indicating that the expression of NDRG1 in myelinating Schwann cells is regulated by axonal interactions. Oligodendrocytes also express NDRG1, and the subtle CNS deficits of affected patients may result from a lack of NDRG1 in these cells. Our data predict that the loss of NDRG1 leads to a Schwann cell autonomous phenotype resulting in demyelination, with secondary axonal loss.

Butyrate modulating effects on pro-inflammatory pathways in human intestinal epithelial cells.

PubMed

Elce, A; Amato, F; Zarrilli, F; Calignano, A; Troncone, R; Castaldo, G; Canani, R B

2017-10-13

Butyrate acts as energy source for intestinal epithelial cells and as key mediator of several immune processes, modulating gene expression mainly through histone deacetylation inhibition. Thanks to these effects, butyrate has been proposed for the treatment of many intestinal diseases. Aim of this study was to investigate the effect of butyrate on the expression of a large series of target genes encoding proteins involved in pro-inflammatory pathways. We performed quantitative real-time-PCR analysis of the expression of 86 genes encoding proteins bearing to pro-inflammatory pathways, before and after butyrate exposure, in primary epithelial cells derived from human small intestine and colon. Butyrate significantly down-regulated the expression of genes involved in inflammatory response, among which nuclear factor kappa beta, interferon-gamma, Toll like 2 receptor and tumour necrosis factor-alpha. Further confirmations of these data, including studies at protein level, would support the use of butyrate as effective therapeutic strategy in intestinal inflammatory disorders.

A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants

PubMed Central

2013-01-01

Background Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. Results RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. Conclusions Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C4 plants. Evolutionary modification of RLSB expression, from a C3 “default” state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C4 plants. PMID:24053212

Characterization and expression analysis of two cDNAs encoding Xa1 and oxysterol binding proteins in sorghum (Sorghum bicolor)

USDA-ARS?s Scientific Manuscript database

Using suppression subtractive hybridization (SSH) and subsequent microarray analysis, expression profiles of sorghum genes responsive to greenbug phloem-feeding were obtained and identified. Among the profiles, two cDNAs designated to MM73 and MM95 were identified to encode Xa1 (Xa1) and oxysterol ...

Two pectin lyase genes, pnl-1 and pnl-2, from Colletotrichum gloeosporioides f. sp. malvae differ in a cellulose-binding domain and in their expression during infection of Malva pusilla.

PubMed

Wei, Yangdou; Shih, Jenny; Li, Jieran; Goodwin, Paul H

2002-07-01

Two pectin lyase genes, designated pnl-1 and pnl-2, were cloned from Colletotrichum gloeosporioides f. sp. malvae, a pathogen of round-leaved mallow (Malva pusilla). pnl-1 was isolated using cDNA from infected plant material; pnl-2 was isolated using cDNA from 3-day-old mycelia grown in mallow-cell-wall extract (MCWE) broth. pnl-1 is the first pectinase gene described thus far to encode a cellulose-binding domain (CBD), which is common in cellulases and xylanases, whereas pnl-2 encodes a pectin lyase that lacks a CBD. In pure culture, pnl-1 expression could be detected when purified pectin or glucose was the sole carbon source, but not when MCWE was the sole carbon source. The lack of pnl-1 expression appeared to be due to gene repression by some unknown factor(s) in the cell-wall extract. In contrast, expression of pnl-2 was detected in cultures when MCWE, but not when purified pectin or glucose, was the sole carbon source. In infected tissue, detection of pnl-1 expression by Northern-blot hybridization and by RT-PCR began with the onset of the necrotrophic phase of infection. Expression ofpnl-2 was not detectable by Northern-blot hybridization, but was observed byRT-PCR in both the biotrophic and necrotrophic phases of infection. The differences between pnl-1 and pnl-2 (i.e. pnl-1 encoding a CBD and differences in the expression patterns of both genes) may be related to the requirements of C. gloeosporioides f. sp. malvae to be able to grow in host tissue under the different conditions present during the biotrophic and necrotrophic phases of infection.

Molecular and biochemical characterisation of two aspartic proteinases TcAP1 and TcAP2 from Theobroma cacao seeds.

PubMed

Laloi, Maryse; McCarthy, James; Morandi, Olivia; Gysler, Christof; Bucheli, Peter

2002-09-01

Aspartic proteinase (EC 3.4.23) activity plays a pivotal role in the degradation of Theobroma cacao L. seed proteins during the fermentation step of cacao bean processing. Therefore, this enzyme is believed to be critical for the formation of the peptide and amino acid cocoa flavor precursors that occurs during fermentation. Using cDNA cloning and northern blot analysis, we show here that there are at least two distinct aspartic proteinase genes ( TcAP1 and TcAP2) expressed during cacao seed development. Both genes are expressed early during seed development and their mRNA levels decrease towards the end of seed maturation. TcAP2 is expressed at a much higher level than TcAP1, although the expression of TcAP1 increases slightly during germination. The proteins encoded by TcAP1 and TcAP2 are relatively different from each other (73% identity). This, and the fact that the two corresponding genes have different expression patterns, suggests that the TcAP1 and TcAP2 proteins may have different functions in the maturing seeds and during germination. Because the TcAP2 gene is expressed at a much higher level during seed development than TcAP1, it is likely that the TcAP2 protein is primarily responsible for the majority of the industrially important protein hydrolysis that occurs during cacao bean fermentation. Finally, TcAP2 has been functionally expressed in the yeast Yarrowia lipolytica. The secreted recombinant protein is able to hydrolyse bovine haemoglobin at acidic pH and is sensitive to pepstatin A, confirming that TcAP2 encodes an aspartic proteinase, and strongly suggests that this gene encodes the well-characterized aspartic proteinase of mature cacao seeds.

Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

DOE PAGES

Zhou, Jilai; Olson, Daniel G.; Lanahan, Anthony A.; ...

2015-09-15

We report that Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. We found that that pyruvate ferredoxin oxidoreductase enzymemore » (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.« less

Identification of a monofunctional aspartate kinase gene of Arabidopsis thaliana with spatially and temporally regulated expression.

PubMed

Yoshioka, Y; Kurei, S; Machida, Y

2001-06-01

We screened a gene trap library of Arabidopsis thaliana and isolated a line in which a gene encoding a homologue of monofunctional aspartate kinase was trapped by the reporter gene. Aspartate kinase (AK) is a key enzyme in the biosynthsis of aspartate family amino acids such as lysine, threonine, isoleucine, and methionine. In plants, two types of AK are known: one is AK which is sensitive to feedback inhibition by threonine and carries both AK and homoserine dehydrogenase (HSD) activities. The other one is monofunctional, sensitive to lysine and synergistically S-adenosylmethionine, and has only AK activity. We concluded that the trapped gene encoded a monofunctional aspartate kinase and designated as AK-lys3, because it lacked the HSD domain and had an amino acid sequence highly similar to those of the monofunctional aspartate kinases ofA. thaliana. AK-lys3 was highly expressed in xylem of leaves and hypocotyls and stele of roots. Significant expression of this gene was also observed in trichomes after bolting. Slight expression of AK-lys3 was detected in vascular bundles and mesophyll cells of cauline leaves, inflorescence stems, sepals, petals, and stigmas. These results indicated that this aspartate kinase gene was not expressed uniformly but in a spatially specific manner.

Cloning and expression profile of ionotropic receptors in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae).

PubMed

Wang, Shan-Ning; Peng, Yong; Lu, Zi-Yun; Dhiloo, Khalid Hussain; Zheng, Yao; Shan, Shuang; Li, Rui-Jun; Zhang, Yong-Jun; Guo, Yu-Yuan

2016-07-01

Ionotropic receptors (IRs) mainly detect the acids and amines having great importance in many insect species, representing an ancient olfactory receptor family in insects. In the present work, we performed RNAseq of Microplitis mediator antennae and identified seventeen IRs. Full-length MmedIRs were cloned and sequenced. Phylogenetic analysis of the Hymenoptera IRs revealed that ten MmedIR genes encoded "antennal IRs" and seven encoded "divergent IRs". Among the IR25a orthologous groups, two genes, MmedIR25a.1 and MmedIR25a.2, were found in M. mediator. Gene structure analysis of MmedIR25a revealed a tandem duplication of IR25a in M. mediator. The tissue distribution and development specific expression of the MmedIR genes suggested that these genes showed a broad expression profile. Quantitative gene expression analysis showed that most of the genes are highly enriched in adult antennae, indicating the candidate chemosensory function of this family in parasitic wasps. Using immunocytochemistry, we confirmed that one co-receptor, MmedIR8a, was expressed in the olfactory sensory neurons. Our data will supply fundamental information for functional analysis of the IRs in parasitoid wasp chemoreception. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nutrient control of gene expression in Drosophila: microarray analysis of starvation and sugar-dependent response

PubMed Central

Zinke, Ingo; Schütz, Christina S.; Katzenberger, Jörg D.; Bauer, Matthias; Pankratz, Michael J.

2002-01-01

We have identified genes regulated by starvation and sugar signals in Drosophila larvae using whole-genome microarrays. Based on expression profiles in the two nutrient conditions, they were organized into different categories that reflect distinct physiological pathways mediating sugar and fat metabolism, and cell growth. In the category of genes regulated in sugar-fed, but not in starved, animals, there is an upregulation of genes encoding key enzymes of the fat biosynthesis pathway and a downregulation of genes encoding lipases. The highest and earliest activated gene upon sugar ingestion is sugarbabe, a zinc finger protein that is induced in the gut and the fat body. Identification of potential targets using microarrays suggests that sugarbabe functions to repress genes involved in dietary fat breakdown and absorption. The current analysis provides a basis for studying the genetic mechanisms underlying nutrient signalling. PMID:12426388

Key gene regulating cell wall biosynthesis and recalcitrance in Populus, gene Y

DOEpatents

Chen, Jay; Engle, Nancy; Gunter, Lee E.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.

2015-12-08

This disclosure provides methods and transgenic plants for improved production of renewable biofuels and other plant-derived biomaterials by altering the expression and/or activity of Gene Y, an O-acetyltransferase. This disclosure also provides expression vectors containing a nucleic acid (Gene Y) which encodes the polypeptide of SEQ ID NO: 1 and is operably linked to a heterologous promoter.

A young root-specific gene (ArMY2) from horseradish encoding a MYR II myrosinase with kinetic preference for the root-specific glucosinolate gluconasturtiin.

PubMed

Loebers, Andreas; Müller-Uri, Frieder; Kreis, Wolfgang

2014-03-01

The pungent taste of horseradish is caused by isothiocyanates which are released from glucosinolates by myrosinases. These enzymes are encoded by genes belonging to one of two subfamilies, termed MYR I and MYR II, respectively. A MYR II-type myrosinase gene was identified for the first time in horseradish. The gene termed ArMY2 was only expressed in young roots. A full-length cDNA encoding a myrosinase termed ArMy2 was isolated and heterologously expressed in Pichia pastoris. The recombinant His-tagged enzyme was characterized biochemically. Substrate affinity was 5 times higher towards gluconasturtiin than towards sinigrin. Gluconasturtiin was found to be the most abundant glucosinolate in young horseradish roots while sinigrin dominated in storage roots and leaves. This indicates that a specialized glucosinolate-myrosinase defense system might be active in young roots. Copyright © 2013 Elsevier Ltd. All rights reserved.

Immune and biochemical responses in skin differ between bovine hosts genetically susceptible and resistant to the cattle tick Rhipicephalus microplus.

PubMed

Franzin, Alessandra Mara; Maruyama, Sandra Regina; Garcia, Gustavo Rocha; Oliveira, Rosane Pereira; Ribeiro, José Marcos Chaves; Bishop, Richard; Maia, Antônio Augusto Mendes; Moré, Daniela Dantas; Ferreira, Beatriz Rossetti; Santos, Isabel Kinney Ferreira de Miranda

2017-01-31

Ticks attach to and penetrate their hosts' skin and inactivate multiple components of host responses in order to acquire a blood meal. Infestation loads with the cattle tick, Rhipicephalus microplus, are heritable: some breeds carry high loads of reproductively successful ticks, whereas in others, few ticks feed and reproduce efficiently. In order to elucidate the mechanisms that result in the different outcomes of infestations with cattle ticks, we examined global gene expression and inflammation induced by tick bites in skins from one resistant and one susceptible breed of cattle that underwent primary infestations with larvae and nymphs of R. microplus. We also examined the expression profiles of genes encoding secreted tick proteins that mediate parasitism in larvae and nymphs feeding on these breeds. Functional analyses of differentially expressed genes in the skin suggest that allergic contact-like dermatitis develops with ensuing production of IL-6, CXCL-8 and CCL-2 and is sustained by HMGB1, ISG15 and PKR, leading to expression of pro-inflammatory chemokines and cytokines that recruit granulocytes and T lymphocytes. Importantly, this response is delayed in susceptible hosts. Histopathological analyses of infested skins showed inflammatory reactions surrounding tick cement cones that enable attachment in both breeds, but in genetically tick-resistant bovines they destabilized the cone. The transcription data provided insights into tick-mediated activation of basophils, which have previously been shown to be a key to host resistance in model systems. Skin from tick-susceptible bovines expressed more transcripts encoding enzymes that detoxify tissues. Interestingly, these enzymes also produce volatile odoriferous compounds and, accordingly, skin rubbings from tick-susceptible bovines attracted significantly more tick larvae than rubbings from resistant hosts. Moreover, transcripts encoding secreted modulatory molecules by the tick were significantly more abundant in larval and in nymphal salivary glands from ticks feeding on susceptible bovines. Compared with tick-susceptible hosts, genes encoding enzymes producing volatile compounds exhibit significantly lower expression in resistant hosts, which may render them less attractive to larvae; resistant hosts expose ticks to an earlier inflammatory response, which in ticks is associated with significantly lower expression of genes encoding salivary proteins that suppress host immunity, inflammation and coagulation.

Protective Vaccination against Blood-Stage Malaria of Plasmodium chabaudi: Differential Gene Expression in the Liver of Balb/c Mice toward the End of Crisis Phase

PubMed Central

Al-Quraishy, Saleh A.; Dkhil, Mohamed A.; Abdel-Baki, Abdel-Azeem A.; Delic, Denis; Wunderlich, Frank

2016-01-01

Protective vaccination induces self-healing of otherwise fatal blood-stage malaria of Plasmodium chabaudi in female Balb/c mice. To trace processes critically involved in self-healing, the liver, an effector against blood-stage malaria, is analyzed for possible changes of its transcriptome in vaccination-protected in comparison to non-protected mice toward the end of the crisis phase. Gene expression microarray analyses reveal that vaccination does not affect constitutive expression of mRNA and lincRNA. However, malaria induces significant (p < 0.01) differences in hepatic gene and lincRNA expression in vaccination-protected vs. non-vaccinated mice toward the end of crisis phase. In vaccination-protected mice, infections induce up-regulations of 276 genes and 40 lincRNAs and down-regulations of 200 genes and 43 lincRNAs, respectively, by >3-fold as compared to the corresponding constitutive expressions. Massive up-regulations, partly by >100-fold, are found for genes as RhD, Add2, Ank1, Ermap, and Slc4a, which encode proteins of erythrocytic surface membranes, and as Gata1 and Gfi1b, which encode transcription factors involved in erythrocytic development. Also, Cldn13 previously predicted to be expressed on erythroblast surfaces is up-regulated by >200-fold, though claudins are known as main constituents of tight junctions acting as paracellular barriers between epithelial cells. Other genes are up-regulated by <100- and >10-fold, which can be subgrouped in genes encoding proteins known to be involved in mitosis, in cell cycle regulation, and in DNA repair. Our data suggest that protective vaccination enables the liver to respond to P. chabaudi infections with accelerated regeneration and extramedullary erythropoiesis during crisis, which contributes to survival of otherwise lethal blood-stage malaria. PMID:27471498

Characterization of EhaJ, a New Autotransporter Protein from Enterohemorrhagic and Enteropathogenic Escherichia coli

PubMed Central

Easton, Donna M.; Totsika, Makrina; Allsopp, Luke P.; Phan, Minh-Duy; Idris, Adi; Wurpel, Daniël J.; Sherlock, Orla; Zhang, Bing; Venturini, Carola; Beatson, Scott A.; Mahony, Timothy J.; Cobbold, Rowland N.; Schembri, Mark A.

2011-01-01

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms have been extensively studied, the factors that mediate host colonization remain to be properly defined. Previously, we identified four genes (ehaA, ehaB, ehaC, and ehaD) from the prototypic EHEC strain EDL933 that encode for proteins that belong to the autotransporter (AT) family. Here we have examined the prevalence of these genes, as well as several other AT-encoding genes, in a collection of EHEC and EPEC strains. We show that the complement of AT-encoding genes in EHEC and EPEC strains is variable, with some AT-encoding genes being highly prevalent. One previously uncharacterized AT-encoding gene, which we have termed ehaJ, was identified in 12/44 (27%) of EHEC and 2/20 (10%) of EPEC strains. The ehaJ gene lies immediately adjacent to a gene encoding a putative glycosyltransferase (referred to as egtA). Western blot analysis using an EhaJ-specific antibody indicated that EhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealed EhaJ is located at the cell surface and in the presence of the egtA glycosyltransferase gene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ also mediated adherence to a range of extracellular matrix proteins, however this occurred independent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-type EPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alter its adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC. Further studies are required to elucidate the function of EhaJ in colonization and virulence. PMID:21687429

Microarray Analyses of Gene Expression during Adventitious Root Development in Pinus contorta1[w

PubMed Central

Brinker, Monika; van Zyl, Leonel; Liu, Wenbin; Craig, Deborah; Sederoff, Ronald R.; Clapham, David H.; von Arnold, Sara

2004-01-01

In order to investigate the gene expression pattern during adventitious root development, RNA of Pinus contorta hypocotyls, pulse-treated with the auxin indole-3-butyric acid and harvested at distinct developmental time points of root development, was hybridized to microarrays containing 2,178 cDNAs from Pinus taeda. Over the period of observation of root development, the transcript levels of 220 genes changed significantly. During the root initiation phase, genes involved in cell replication and cell wall weakening and a transcript encoding a PINHEAD/ZWILLE-like protein were up-regulated, while genes related to auxin transport, photosynthesis, and cell wall synthesis were down-regulated. In addition, there were changes in transcript abundance of genes related to water stress. During the root meristem formation phase the transcript abundances of genes involved in auxin transport, auxin responsive transcription, and cell wall synthesis, and of a gene encoding a B-box zinc finger-like protein, increased, while those encoding proteins involved in cell wall weakening decreased. Changes of transcript abundance of genes related to water stress during the root meristem formation and root formation phase indicate that the plant roots had become functional in water transport. Simultaneously, genes involved in auxin transport were up-regulated, while genes related to cell wall modification were down-regulated. Finally, during the root elongation phase down-regulation of transcripts encoding proteins involved in cell replication and stress occurred. Based on the observed changes in transcript abundances, we suggest hypotheses about the relative importance of various physiological processes during the auxin-induced development of roots in P. contorta. PMID:15247392

BcMF8, a putative arabinogalactan protein-encoding gene, contributes to pollen wall development, aperture formation and pollen tube growth in Brassica campestris

PubMed Central

Lin, Sue; Dong, Heng; Zhang, Fang; Qiu, Lin; Wang, Fangzhan; Cao, Jiashu; Huang, Li

2014-01-01

Background and Aims The arabinogalactan protein (AGP) gene family is involved in plant reproduction. However, little is known about the function of individual AGP genes in pollen development and pollen tube growth. In this study, Brassica campestris male fertility 8 (BcMF8), a putative AGP-encoding gene previously found to be pollen specific in Chinese cabbage (B. campestris ssp. chinensis), was investigated. Methods Real-time reverse transcription–PCR and in situ hybridization were used to analyse the expression pattern of BcMF8 in pistils. Prokaryotic expression and western blots were used to ensure that BcMF8 could encode a protein. Antisense RNA technology was applied to silence gene expression, and morphological and cytological approaches (e.g. scanning electron microscopy and transmission electron microscopy) were used to reveal abnormal phenotypes caused by gene silencing. Key Results The BcMF8 gene encoded a putative AGP protein that was located in the cell wall, and was expressed in pollen grains and pollen tubes. The functional interruption of BcMF8 by antisense RNA technology resulted in slipper-shaped and bilaterally sunken pollen with abnormal intine development and aperture formation. The inhibition of BcMF8 led to a decrease in the percentage of in vitro pollen germination. In pollen that did germinate, the pollen tubes were unstable, abnormally shaped and burst more frequently relative to controls, which corresponded to an in vivo arrest of pollen germination at the stigma surface and retarded pollen tube growth in the stylar transmitting tissues. Conclusions The phenotypic defects of antisense BcMF8 RNA lines (bcmf8) suggest a crucial function of BcMF8 in modulating the physical nature of the pollen wall and in helping in maintaining the integrity of the pollen tube wall matrix. PMID:24489019

CDH4 suppresses the progression of salivary adenoid cystic carcinoma via E-cadherin co-expression.

PubMed

Xie, Jian; Feng, Yan; Lin, Ting; Huang, Xiao-Yu; Gan, Rui-Huan; Zhao, Yong; Su, Bo-Hua; Ding, Lin-Can; She, Lin; Chen, Jiang; Lin, Li-Song; Lin, Xu; Zheng, Da-Li; Lu, You-Guang

2016-12-13

The cadherin-4 gene (CDH4) of the cadherin family encodes non-epithelial R-cadherin (R-cad); however, the function of this gene in different types of cancer remains controversial. In this study, we found higher expression of CDH4 mRNA in a salivary adenoid cystic carcinoma (SACC) cell line with low metastatic potential (SACC-83) than in a cell line with high metastatic potential (SACC-LM). By analyzing 67 samples of SACC tissues and 40 samples of paraneoplastic normal tissues, we found R-cad highly expressed in 100% of normal paraneoplastic tissue but only expressed in 64% of SACC tumor tissues (P<0.001). Knockdown of CDH4 expression in vitro promoted the growth, mobility and invasion of SACC cells, and in vivo experiments showed that decreased CDH4 expression enhanced SACC tumorigenicity. Furthermore, CDH4 suppression resulted in down-regulation of E-cadherin (E-cad), which is encoded by CDH1 gene and is a well-known tumor suppressor gene by inhibition of cell proliferation and migration. These results indicate that CDH4 may play a negative role in the growth and metastasis of SACC via co-expression with E-cadherin.

Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls

NASA Astrophysics Data System (ADS)

Yoshioka, R.; Soga, K.; Wakabayashi, K.; Takeba, G.; Hoson, T.

2003-05-01

Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor ofterpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the α-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.

Transcriptome Profiling of Shewanella oneidensis Gene Expression following Exposure to Acidic and Alkaline pH†

PubMed Central

Leaphart, Adam B.; Thompson, Dorothea K.; Huang, Katherine; Alm, Eric; Wan, Xiu-Feng; Arkin, Adam; Brown, Steven D.; Wu, Liyou; Yan, Tingfen; Liu, Xueduan; Wickham, Gene S.; Zhou, Jizhong

2006-01-01

The molecular response of Shewanella oneidensis MR-1 to variations in extracellular pH was investigated based on genomewide gene expression profiling. Microarray analysis revealed that cells elicited both general and specific transcriptome responses when challenged with environmental acid (pH 4) or base (pH 10) conditions over a 60-min period. Global responses included the differential expression of genes functionally linked to amino acid metabolism, transcriptional regulation and signal transduction, transport, cell membrane structure, and oxidative stress protection. Response to acid stress included the elevated expression of genes encoding glycogen biosynthetic enzymes, phosphate transporters, and the RNA polymerase sigma-38 factor (rpoS), whereas the molecular response to alkaline pH was characterized by upregulation of nhaA and nhaR, which are predicted to encode an Na+/H+ antiporter and transcriptional activator, respectively, as well as sulfate transport and sulfur metabolism genes. Collectively, these results suggest that S. oneidensis modulates multiple transporters, cell envelope components, and pathways of amino acid consumption and central intermediary metabolism as part of its transcriptome response to changing external pH conditions. PMID:16452448

A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana.

PubMed

Zhang, Meixiang; Ahmed Rajput, Nasir; Shen, Danyu; Sun, Peng; Zeng, Wentao; Liu, Tingli; Juma Mafurah, Joseph; Dou, Daolong

2015-06-03

Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses.

Both a PKS and a PPTase are involved in melanin biosynthesis and regulation of Aureobasidium melanogenum XJ5-1 isolated from the Taklimakan desert.

PubMed

Jiang, Hong; Liu, Guang-Lei; Chi, Zhe; Wang, Jian-Ming; Zhang, Ly-Ly; Chi, Zhen-Ming

2017-02-20

A PKS1 gene responsible for the melanin biosynthesis and a NPG1 gene in Aureobasidium melanogenum XJ5-1 were cloned and characterized. An ORF of the PKS1 gene encoding a protein with 2165 amino acids contained 6495bp while an ORF of the NPG1 gene encoding a protein with 340 amino acids had 1076bp. After analysis of their promoters, it was found that expression of both the PKS1 gene and the NPG1 gene was repressed by nitrogen sources and glucose, respectively. The PKS deduced from the cloned gene consisted of one ketosynthase, one acyl transferase, two acyl carrier proteins, one thioesterase and one cyclase while the PPTase belonged to the family Sfp-type. After disruption of the PKS1 gene and the NPG1 gene, expression of the PKS1 gene and the NPG1 gene and the melanin biosynthesis in the disruptants K5 and DP107 disappeared and expression of the PKS1 gene in the disruptant DP107 was also negatively influenced. However, after the NPG1 gene was complemented in the disruptant DP107, the melanin biosynthesis in the complementary strain BP17 was restored and expression of the PKS1 gene and the NPG1 gene was greatly enhanced, suggesting that the PKS was indeed activated and regulated by the PPTase and expression of the PKS1 gene and the NPG1 gene had a coordinate regulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Constitutional downregulation of SEMA5A expression in autism.

PubMed

Melin, M; Carlsson, B; Anckarsater, H; Rastam, M; Betancur, C; Isaksson, A; Gillberg, C; Dahl, N

2006-01-01

There is strong evidence for the importance of genetic factors in idiopathic autism. The results from independent twin and family studies suggest that the disorder is caused by the action of several genes, possibly acting epistatically. We have used cDNA microarray technology for the identification of constitutional changes in the gene expression profile associated with idiopathic autism. Samples were obtained and analyzed from 6 affected subjects belonging to multiplex autism families and from 6 healthy controls. We assessed the expression levels for approximately 7,700 genes by cDNA microarrays using mRNA derived from Epstein-Barr virus-transformed B lymphocytes. The microarray data were analyzed in order to identify up- or downregulation of specific genes. A common pattern with nine downregulated genes was identified among samples derived from individuals with autism when compared to controls. Four of these nine genes encode proteins involved in biological processes associated with brain function or the immune system, and are consequently considered as candidates for genes associated with autism. Quantitative real-time PCR confirms the downregulation of the gene encoding SEMA5A, a protein involved in axonal guidance. Epstein-Barr virus should be considered as a possible source for altered expression, but our consistent results make us suggest SEMA5A as a candidate gene in the etiology of idiopathic autism.

Constitutional downregulation of SEMA5A expression in autism

PubMed Central

Melin, Malin; Carlsson, Birgit; Anckarsäter, Henrik; Rastam, Maria; Betancur, Catalina; Isaksson, Anders; Gillberg, Christopher; Dahl, Niklas

2006-01-01

There is strong evidence for the importance of genetic factors in idiopathic autism. The results from independent twin and family studies suggest that the disorder is caused by the action of several genes, possibly acting epistatically. We have used cDNA microarray technology for the identification of constitutional changes in the gene expression profile associated with idiopathic autism. Samples were obtained and analyzed from six affected subjects belonging to multiplex autism families and from six healthy controls. We assessed the expression levels for approximately 7,700 genes by cDNA microarrays using mRNA derived from Epstein Barr virus (EBV)-transformed B-lymphocytes. The microarray data was analyzed in order to identify up- or down-regulation of specific genes. A common pattern with nine down-regulated genes was identified among samples derived from individuals with autism when compared to controls. Four of these nine genes encode proteins involved in biological processes associated with brain function or the immune system, and are consequently considered as candidates for genes associated with autism. Quantitative realtime PCR confirms the down-regulation of the gene encoding SEMA5A, a protein involved in axonal guidance. EBV should be considered as a possible source for altered expression but our consistent results make us suggest SEMA5A a candidate gene in the etiology of idiopathic autism. PMID:17028446

Discovery of Herpes B Virus-Encoded MicroRNAs▿

PubMed Central

Besecker, Michael I.; Harden, Mallory E.; Li, Guanglin; Wang, Xiu-Jie; Griffiths, Anthony

2009-01-01

Herpes B virus (BV) naturally infects macaque monkeys and is a close relative of herpes simplex virus. BV can zoonotically infect humans to cause a rapidly ascending encephalitis with ∼80% mortality. Therefore, BV is a serious danger to those who come into contact with these monkeys or their tissues and cells. MicroRNAs are regulators of gene expression, and there have been reports of virus-encoded microRNAs. We hypothesize that BV-encoded microRNAs are important for the regulation of viral and cellular genes. Herein, we report the discovery of three herpes B virus-encoded microRNAs. PMID:19144716

Induction of 1-acylglycerophosphocholine acyltransferase genes by fibrates in the liver of rats.

PubMed

Yamazaki, Tohru; Wakabayashi, Michiko; Ikeda, Erika; Tanaka, Shizuyo; Sakamoto, Takeshi; Mitsumoto, Atsushi; Kudo, Naomi; Kawashima, Yoichi

2012-01-01

The effect of fibrates (clofibric acid, bezafibrate and fenofibrate) on the gene expression and activity of 1-acylglycerophosphocholine acyltransferase (LPCAT) was investigated. The administration of 0.1% (w/w) clofibric acid, bezafibrate or fenofibrate in diet for 14 d to rats induced LPCAT activity in hepatic microsomes in the following order: fenofibrate>bezafibrate>clofibric acid. The LPCAT induced by fenofibrate preferred to arachidonoyl-CoA and linoleoyl-CoA to a greater extent than did LPCAT in control microsomes. The treatment with the fibrates resulted in upregulation of the relative expression of mRNAs encoding LPCAT3 and LPCAT4 in the following order: fenofibrate>bezafibrate>clofibric acid. The administration of fibrates did not change the expression of genes encoding either LPCAT1 or LPCAT2. The treatment with fibrates elevated relative levels of both mRNAs encoding Δ6 desaturase (Fads2) and Δ5 desaturase (Fads1) in the order of fenofibrate>bezafibrate>clofibric acid, and the extent of the increase in the level of Δ6 desaturase mRNA was greater than that of Δ5 desaturase. Fatty acid profile in hepatic phosphatidylcholine (PC) was significantly changed by the treatments with fibrates. These results suggest (i) that fibrates induce LPCAT activity in hepatic microsomes by elevating the expression of genes encoding LPCAT3 and LPCAT4, (ii) that the changes in fatty acid profile of hepatic PC are, in part, due to the elevated expression of two isoforms, LPCAT3 and LPCAT4, and (iii) that the ability of fibrates to induce these changes are in the order of fenofibrate>bezafibrate>clofibric acid.

Identification and characterization of a gene encoding for a nucleotidase from Phaseolus vulgaris.

PubMed

Cabello-Díaz, Juan Miguel; Gálvez-Valdivieso, Gregorio; Caballo, Cristina; Lambert, Rocío; Quiles, Francisco Antonio; Pineda, Manuel; Piedras, Pedro

2015-08-01

Nucleotidases are phosphatases that catalyze the removal of phosphate from nucleotides, compounds with an important role in plant metabolism. A phosphatase enzyme, with high affinity for nucleotides monophosphate previously identified and purified in embryonic axes from French bean, has been analyzed by MALDI TOF/TOF and two internal peptides have been obtained. The information of these peptide sequences has been used to search in the genome database and only a candidate gene that encodes for the phosphatase was identified (PvNTD1). The putative protein contains the conserved domains (motif I-IV) for haloacid dehalogenase-like hydrolases superfamily. The residues involved in the catalytic activity are also conserved. A recombinant protein overexpressed in Escherichia coli has shown molybdate resistant phosphatase activity with nucleosides monophosphate as substrate, confirming that the identified gene encodes for the phosphatase with high affinity for nucleotides purified in French bean embryonic axes. The activity of the purified protein was inhibited by adenosine. The expression of PvNTD1 gene was induced at the specific moment of radicle protrusion in embryonic axes. The gene was also highly expressed in young leaves whereas the level of expression in mature tissues was minimal. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

Characterization and expression of genes encoding three small heat shock proteins in Sesamia inferens (Lepidoptera: Noctuidae).

PubMed

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2014-12-12

The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

Characterization and Expression of Genes Encoding Three Small Heat Shock Proteins in Sesamia inferens (Lepidoptera: Noctuidae)

PubMed Central

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2014-01-01

The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens. PMID:25514417

Adaptation of Staphylococcus xylosus to Nutrients and Osmotic Stress in a Salted Meat Model

PubMed Central

Vermassen, Aurore; Dordet-Frisoni, Emilie; de La Foye, Anne; Micheau, Pierre; Laroute, Valérie; Leroy, Sabine; Talon, Régine

2016-01-01

Staphylococcus xylosus is commonly used as starter culture for meat fermentation. Its technological properties are mainly characterized in vitro, but the molecular mechanisms for its adaptation to meat remain unknown. A global transcriptomic approach was used to determine these mechanisms. S. xylosus modulated the expression of about 40–50% of the total genes during its growth and survival in the meat model. The expression of many genes involved in DNA machinery and cell division, but also in cell lysis, was up-regulated. Considering that the S. xylosus population remained almost stable between 24 and 72 h of incubation, our results suggest a balance between cell division and cell lysis in the meat model. The expression of many genes encoding enzymes involved in glucose and lactate catabolism was up-regulated and revealed that glucose and lactate were used simultaneously. S. xylosus seemed to adapt to anaerobic conditions as revealed by the overexpression of two regulatory systems and several genes encoding cofactors required for respiration. In parallel, genes encoding transport of peptides and peptidases that could furnish amino acids were up-regulated and thus concomitantly a lot of genes involved in amino acid synthesis were down-regulated. Several genes involved in glutamate homeostasis were up-regulated. Finally, S. xylosus responded to the osmotic stress generated by salt added to the meat model by overexpressing genes involved in transport and synthesis of osmoprotectants, and Na+ and H+ extrusion. PMID:26903967

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.

Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ.

PubMed

Forsgren, Margareta; Attersand, Anneli; Lake, Staffan; Grünler, Jacob; Swiezewska, Ewa; Dallner, Gustav; Climent, Isabel

2004-09-01

The COQ2 gene in Saccharomyces cerevisiae encodes a Coq2 (p-hydroxybenzoate:polyprenyl transferase), which is required in the biosynthetic pathway of CoQ (ubiquinone). This enzyme catalyses the prenylation of p-hydroxybenzoate with an all-trans polyprenyl group. We have isolated cDNA which we believe encodes the human homologue of COQ2 from a human muscle and liver cDNA library. The clone contained an open reading frame of length 1263 bp, which encodes a polypeptide that has sequence homology with the Coq2 homologues in yeast, bacteria and mammals. The human COQ2 gene, when expressed in yeast Coq2 null mutant cells, rescued the growth of this yeast strain in the absence of a non-fermentable carbon source and restored CoQ biosynthesis. However, the rate of CoQ biosynthesis in the rescued cells was lower when compared with that in cells rescued with the yeast COQ2 gene. CoQ formed when cells were incubated with labelled decaprenyl pyrophosphate and nonaprenyl pyrophosphate, showing that the human enzyme is active and that it participates in the biosynthesis of CoQ.

Role of protein farnesylation events in the ABA-mediated regulation of the Pinoresinol-Lariciresinol Reductase 1 (LuPLR1) gene expression and lignan biosynthesis in flax (Linum usitatissimum L.).

PubMed

Corbin, Cyrielle; Decourtil, Cédric; Marosevic, Djurdjica; Bailly, Marlène; Lopez, Tatiana; Renouard, Sullivan; Doussot, Joël; Dutilleul, Christelle; Auguin, Daniel; Giglioli-Guivarc'h, Nathalie; Lainé, Eric; Lamblin, Frédéric; Hano, Christophe

2013-11-01

A Linum usitatissimum LuERA1 gene encoding a putative ortholog of the ERA1 (Enhanced Response to ABA 1) gene of Arabidopsis thaliana (encoding the beta subunit of a farnesyltransferase) was analyzed in silico and for its expression in flax. The gene and the protein sequences are highly similar to other sequences already characterized in plants and all the features of a farnesyltransferase were detected. Molecular modeling of LuERA1 protein confirmed its farnesyltransferase nature. LuERA1 is expressed in the vegetative organs and also in the outer seedcoat of the flaxseed, where it could modulate the previously observed regulation operated by ABA on lignan synthesis. This effect could be mediated by the regulation of the transcription of a key gene for lignan synthesis in flax, the LuPLR1 gene, encoding a pinoresinol lariciresinol reductase. The positive effect of manumycin A, a specific inhibitor of farnesyltransferase, on lignan biosynthesis in flax cell suspension systems supports the hypothesis of the involvement of such an enzyme in the negative regulation of ABA action. In Arabidopsis, ERA1 is able to negatively regulate the ABA effects and the mutant era1 has an enhanced sensitivity to ABA. When expressed in an Arabidopsis cell suspension (heterologous system) LuERA1 is able to reverse the effect of the era1 mutation. RNAi experiments in flax targeting the farnesyltransferase β-subunit encoded by the LuERA1 gene led to an increase LuPLR1 expression level associated with an increased content of lignan in transgenic calli. Altogether these results strongly suggest a role of the product of this LuERA1 gene in the ABA-mediated upregulation of lignan biosynthesis in flax cells through the activation of LuPLR1 promoter. This ABA signaling pathway involving ERA1 probably acts through the ABRE box found in the promoter sequence of LuPLR1, a key gene for lignan synthesis in flax, as demonstrated by LuPLR1 gene promoter-reporter experiments in flax cells using wild type and mutated promoter sequences. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Whole exome sequencing with genomic triangulation implicates CDH2-encoded N-cadherin as a novel pathogenic substrate for arrhythmogenic cardiomyopathy.

PubMed

Turkowski, Kari L; Tester, David J; Bos, J Martijn; Haugaa, Kristina H; Ackerman, Michael J

2017-03-01

Arrhythmogenic cardiomyopathy (ACM) is a heritable disease characterized by fibrofatty replacement of cardiomyocytes, has a prevalence of approximately 1 in 5000 individuals, and accounts for approximately 20% of sudden cardiac death in the young (≤35 years). ACM is most often inherited as an autosomal dominant trait with incomplete penetrance and variable expression. While mutations in several genes that encode key desmosomal proteins underlie about half of all ACM, the remainder is elusive genetically. Here, whole exome sequencing (WES) was performed with genomic triangulation in an effort to identify a novel explanation for a phenotype-positive, genotype-negative multi-generational pedigree with a presumed autosomal dominant, maternal inheritance of ACM. WES and genomic triangulation was performed on a symptomatic 14-year-old female proband, her affected mother and affected sister, and her unaffected father to elucidate a novel ACM-susceptibility gene for this pedigree. Following variant filtering using Ingenuity® Variant Analysis, gene priority ranking was performed on the candidate genes using ToppGene and Endeavour. The phylogenetic and physiochemical properties of candidate mutations were assessed further by 6 in silico prediction tools. Species alignment and amino acid conservation analysis was performed using the Uniprot Consortium. Tissue expression data was abstracted from Expression Atlas. Following WES and genomic triangulation, CDH2 emerged as a novel, autosomal dominant, ACM-susceptibility gene. The CDH2-encoded N-cadherin is a cell-cell adhesion protein predominately expressed in the heart. Cardiac dysfunction has been demonstrated in prior CDH2 knockout and over-expression animal studies. Further in silico mutation prediction, species conservation, and protein expression analysis supported the ultra-rare (minor allele frequency <0.005%) p.Asp407Asn-CDH2 variant as a likely pathogenic variant. Herein, it is demonstrated that genetic mutations in CDH2-encoded N-cadherin may represent a novel pathogenetic basis for ACM in humans. The prevalence of CDH2-mediated ACM in heretofore genetically elusive ACM remains to be determined. © 2017 Wiley Periodicals, Inc.

Identification and Characterization of Three Differentially Expressed Genes, Encoding S-Adenosylhomocysteine Hydrolase, Methionine Aminopeptidase, and a Histone-Like Protein, in the Toxic Dinoflagellate Alexandrium fundyense†

PubMed Central

Taroncher-Oldenburg, Gaspar; Anderson, Donald M.

2000-01-01

Genes showing differential expression related to the early G1 phase of the cell cycle during synchronized circadian growth of the toxic dinoflagellate Alexandrium fundyense were identified and characterized by differential display (DD). The determination in our previous work that toxin production in Alexandrium is relegated to a narrow time frame in early G1 led to the hypothesis that transcriptionally up- or downregulated genes during this subphase of the cell cycle might be related to toxin biosynthesis. Three genes, encoding S-adenosylhomocysteine hydrolase (Sahh), methionine aminopeptidase (Map), and a histone-like protein (HAf), were isolated. Sahh was downregulated, while Map and HAf were upregulated, during the early G1 phase of the cell cycle. Sahh and Map encoded amino acid sequences with about 90 and 70% similarity to those encoded by several eukaryotic and prokaryotic Sahh and Map genes, respectively. The partial Map sequence also contained three cobalt binding motifs characteristic of all Map genes. HAf encoded an amino acid sequence with 60% similarity to those of two histone-like proteins from the dinoflagellate Crypthecodinium cohnii Biecheler. This study documents the potential of applying DD to the identification of genes that are related to physiological processes or cell cycle events in phytoplankton under conditions where small sample volumes represent an experimental constraint. The identification of an additional 21 genes with various cell cycle-related DD patterns also provides evidence for the importance of pretranslational or transcriptional regulation in dinoflagellates, contrary to previous reports suggesting the possibility that translational mechanisms are the primary means of circadian regulation in this group of organisms. PMID:10788388

Analysis of expression of the argC and argD genes in the cyanobacterium Anabaena sp. strain PCC 7120.

PubMed Central

Floriano, B; Herrero, A; Flores, E

1994-01-01

A cloned DNA fragment from Anabaena sp. strain PCC 7120 that complements an arginine auxotrophic mutant from the same organism was found to include an open reading frame encoding a 427-residue polypeptide that is homologous to N-acetylornithine aminotransferase from Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. The gene encoding N-acetylornithine aminotransferase in bacteria has been named argD. The expression of Anabaena sp. strain PCC 7120 argD, as well as of argC, was analyzed at the mRNA level. Both genes were transcribed as monocistronic mRNAs, and their expression was not affected by exogenously added arginine. Primer extension analysis identified transcription start points for both genes which were preceded by sequences similar to that of the E. coli RNA polymerase sigma 70 consensus promoter. A second transcription start point for the argD gene that is not preceded by a sigma 70 consensus promoter was detected in dinitrogen-grown cultures. Images PMID:7929012

Functional Expression of Two Neuronal Nicotinic Acetylcholine Receptors from cDNA Clones Identifies a Gene Family

NASA Astrophysics Data System (ADS)

Boulter, Jim; Connolly, John; Deneris, Evan; Goldman, Dan; Heinemann, Steven; Patrick, Jim

1987-11-01

A family of genes coding for proteins homologous to the α subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by α -bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

Comparative and Evolutionary Analysis of Grass Pollen Allergens Using Brachypodium distachyon as a Model System

PubMed Central

Sharma, Akanksha; Sharma, Niharika; Bhalla, Prem; Singh, Mohan

2017-01-01

Comparative genomics have facilitated the mining of biological information from a genome sequence, through the detection of similarities and differences with genomes of closely or more distantly related species. By using such comparative approaches, knowledge can be transferred from the model to non-model organisms and insights can be gained in the structural and evolutionary patterns of specific genes. In the absence of sequenced genomes for allergenic grasses, this study was aimed at understanding the structure, organisation and expression profiles of grass pollen allergens using the genomic data from Brachypodium distachyon as it is phylogenetically related to the allergenic grasses. Combining genomic data with the anther RNA-Seq dataset revealed 24 pollen allergen genes belonging to eight allergen groups mapping on the five chromosomes in B. distachyon. High levels of anther-specific expression profiles were observed for the 24 identified putative allergen-encoding genes in Brachypodium. The genomic evidence suggests that gene encoding the group 5 allergen, the most potent trigger of hay fever and allergic asthma originated as a pollen specific orphan gene in a common grass ancestor of Brachypodium and Triticiae clades. Gene structure analysis showed that the putative allergen-encoding genes in Brachypodium either lack or contain reduced number of introns. Promoter analysis of the identified Brachypodium genes revealed the presence of specific cis-regulatory sequences likely responsible for high anther/pollen-specific expression. With the identification of putative allergen-encoding genes in Brachypodium, this study has also described some important plant gene families (e.g. expansin superfamily, EF-Hand family, profilins etc) for the first time in the model plant Brachypodium. Altogether, the present study provides new insights into structural characterization and evolution of pollen allergens and will further serve as a base for their functional characterization in related grass species. PMID:28103252

Expression and rearrangement of the ROS1 gene in human glioblastoma cells

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

Birchmeier, C.; Sharma, S.; Wigler, M.

1987-12-01

The human ROS1 gene, which possibly encodes a growth factor receptor, was found to be expressed in human tumor cell lines. In a survey of 45 different human cell lines, the authors found ROS1 to be expressed in glioblastoma-derived cell lines at high levels and not to be expressed at all, or expressed at very low levels, in the remaining cell lines. The ROS1 gene was present in normal copy numbers in all cell lines that expressed the gene. However, in one particular glioblastoma line, they detected a potentially activating mutation at the ROS1 locus.

Gene Duplication and Evolutionary Innovations in Hemoglobin-Oxygen Transport

PubMed Central

2016-01-01

During vertebrate evolution, duplicated hemoglobin (Hb) genes diverged with respect to functional properties as well as the developmental timing of expression. For example, the subfamilies of genes that encode the different subunit chains of Hb are ontogenetically regulated such that functionally distinct Hb isoforms are expressed during different developmental stages. In some vertebrate taxa, functional differentiation between co-expressed Hb isoforms may also contribute to physiologically important divisions of labor. PMID:27053736

DNA methylation of miRNA-encoding genes in non-small cell lung cancer patients.

PubMed

Heller, Gerwin; Altenberger, Corinna; Steiner, Irene; Topakian, Thais; Ziegler, Barbara; Tomasich, Erwin; Lang, György; End-Pfützenreuter, Adelheid; Zehetmayer, Sonja; Döme, Balazs; Arns, Britt-Madeleine; Klepetko, Walter; Zielinski, Christoph C; Zöchbauer-Müller, Sabine

2018-03-23

De-regulated DNA methylation leading to transcriptional inactivation of certain genes occurs frequently in non-small cell lung cancers (NSCLC). Besides protein-encoding genes also microRNA (miRNA)-encoding genes may be targets for methylation in NSCLCs, however, the number of known methylated miRNA genes is still small. Thus, we investigated methylation of miRNA genes in primary tumours (TU) and corresponding non-malignant lung tissue samples (NL) of 50 NSCLC patients using methylated DNA immunoprecipitation followed by custom designed tiling microarray analyses (MeDIP-chip) and 252 differentially methylated probes between TU and NL samples were identified. These probes were annotated which resulted in the identification of 34 miRNA-encoding genes with increased methylation in TU specimens. While some of these miRNA-encoding genes were already known to be methylated in NSCLCs (e.g. miR-9-3, miR-124), methylation of the vast majority of them was unknown so far. We selected six miRNA genes (miR-10b, miR-1179, miR-137, miR-572, miR-3150b and miR-129-2) for gene-specific methylation analyses in TU and corresponding NL samples of 104 NSCLC patients and observed a statistically significant increase of methylation of these miRNA genes in TU samples (p<0.0001, respectively). In silico target prediction of the six miRNAs identified several oncogenic/cell proliferation promoting factors (e.g. CCNE1 as miR-1179 target). To investigate if miR-1179 indeed targets CCNE1, we transfected miR-1179 mimics into CCNE1 expressing NSCLC cells and observed down-regulated CCNE1 mRNA expression in these cells compared to control cells. Similar effects on Cyclin E1 expression were seen in Western blot analyses. In addition, we found a statistically significant growth reduction of NSCLC cells transfected with miR-1179 mimics compared to control cells. In conclusion, we identified many methylated miRNA genes in NSCLC patients and found that miR-1179 is a potential tumour cell growth suppressor in NSCLCs. Overall, our findings emphasize the impact of miRNA gene methylation on the pathogenesis of NSCLCs. This article is protected by copyright. All rights reserved.

Cloning and expression analysis of FaPR-1 gene in strawberry

NASA Astrophysics Data System (ADS)

Mo, Fan; Luo, Ya; Ge, Cong; Mo, Qin; Ling, Yajie; Luo, Shu; Tang, Haoru

2018-04-01

The FaPR-1 gene was cloned by RT-PCR from `Benihoppe' strawberry and its bioinformatics analysis was conducted. The results showed that the open reading frame was 483 bp encoding encoding l60 amino acids which protein molecular weight and theoretical isoelectricity were 17854.17 and 8.72 respectively. Subcellular localization prediction shows that this gene is located extracellularly. By comparing strawberry FaPR-l and other plant Pathogenesis-related protein, homology and phylogenetic tree construction showed that the homology with grapes, peach is relatively close. In the treatments of ABA, sucrose and the mixture of the two, the expression of FaPR-1 in strawberry fruit were significantly increased.

Evidence for transceptor function of cellodextrin transporters in Neurospora crassa.

PubMed

Znameroski, Elizabeth A; Li, Xin; Tsai, Jordan C; Galazka, Jonathan M; Glass, N Louise; Cate, Jamie H D

2014-01-31

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as "transceptors" with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.

Evolution, functional differentiation, and co-expression of the RLK gene family revealed in Jilin ginseng, Panax ginseng C.A. Meyer.

PubMed

Lin, Yanping; Wang, Kangyu; Li, Xiangyu; Sun, Chunyu; Yin, Rui; Wang, Yanfang; Wang, Yi; Zhang, Meiping

2018-02-21

Most genes in a genome exist in the form of a gene family; therefore, it is necessary to have knowledge of how a gene family functions to comprehensively understand organismal biology. The receptor-like kinase (RLK)-encoding gene family is one of the most important gene families in plants. It plays important roles in biotic and abiotic stress tolerances, and growth and development. However, little is known about the functional differentiation and relationships among the gene members within a gene family in plants. This study has isolated 563 RLK genes (designated as PgRLK genes) expressed in Jilin ginseng (Panax ginseng C.A. Meyer), investigated their evolution, and deciphered their functional diversification and relationships. The PgRLK gene family is highly diverged and formed into eight types. The LRR type is the earliest and most prevalent, while only the Lec type originated after P. ginseng evolved. Furthermore, although the members of the PgRLK gene family all encode receptor-like protein kinases and share conservative domains, they are functionally very diverse, participating in numerous biological processes. The expressions of different members of the PgRLK gene family are extremely variable within a tissue, at a developmental stage and in the same cultivar, but most of the genes tend to express correlatively, forming a co-expression network. These results not only provide a deeper and comprehensive understanding of the evolution, functional differentiation and correlation of a gene family in plants, but also an RLK genic resource useful for enhanced ginseng genetic improvement.

Deregulation of Rab and Rab Effector Genes in Bladder Cancer

PubMed Central

Ho, Joel R.; Chapeaublanc, Elodie; Kirkwood, Lisa; Nicolle, Remy; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Southgate, Jennifer; Radvanyi, François; Goud, Bruno

2012-01-01

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer. PMID:22724020

Molecular and genomic basis of volatile-mediated indirect defense against insects in rice.

PubMed

Yuan, Joshua S; Köllner, Tobias G; Wiggins, Greg; Grant, Jerome; Degenhardt, Jörg; Chen, Feng

2008-08-01

Rice plants fed on by fall armyworm (Spodoptera frugiperda, FAW) caterpillars emit a blend of volatiles dominated by terpenoids. These volatiles were highly attractive to females of the parasitoid Cotesia marginiventris. Microarray analysis identified 196 rice genes whose expression was significantly upregulated by FAW feeding, 18 of which encode metabolic enzymes potentially involved in volatile biosynthesis. Significant induction of expression of seven of the 11 terpene synthase (TPS) genes identified through the microarray experiments was confirmd using real-time RT-PCR. Enzymes encoded by three TPS genes, Os02g02930, Os08g07100 and Os08g04500, were biochemically characterized. Os02g02930 was found to encode a monoterpene synthase producing the single product S-linalool, which is the most abundant volatile emitted from FAW-damaged rice plants. Both Os08g07100 and Os08g04500 were found to encode sesquiterpene synthases, each producing multiple products. These three enzymes are responsible for production of the majority of the terpenes released from FAW-damaged rice plants. In addition to TPS genes, several key genes in the upstream terpenoid pathways were also found to be upregulated by FAW feeding. This paper provides a comprehensive analysis of FAW-induced volatiles and the corresponding volatile biosynthetic genes potentially involved in indirect defense in rice. Evolution of the genetic basis governing volatile terpenoid biosynthesis for indirect defense is discussed.

Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression.

PubMed

Kidokoro, Satoshi; Watanabe, Keitaro; Ohori, Teppei; Moriwaki, Takashi; Maruyama, Kyonoshin; Mizoi, Junya; Myint Phyu Sin Htwe, Nang; Fujita, Yasunari; Sekita, Sachiko; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2015-02-01

Soybean (Glycine max) is a globally important crop, and its growth and yield are severely reduced by abiotic stresses, such as drought, heat, and cold. The cis-acting element DRE (dehydration-responsive element)/CRT plays an important role in activating gene expression in response to these stresses. The Arabidopsis DREB1/CBF genes that encode DRE-binding proteins function as transcriptional activators in the cold stress responsive gene expression. In this study, we identified 14 DREB1-type transcription factors (GmDREB1s) from a soybean genome database. The expression of most GmDREB1 genes in soybean was strongly induced by a variety of abiotic stresses, such as cold, drought, high salt, and heat. The GmDREB1 proteins activated transcription via DREs (dehydration-responsive element) in Arabidopsis and soybean protoplasts. Transcriptome analyses using transgenic Arabidopsis plants overexpressing GmDREB1s indicated that many of the downstream genes are cold-inducible and overlap with those of Arabidopsis DREB1A. We then comprehensively analyzed the downstream genes of GmDREB1B;1, which is closely related to DREB1A, using a transient expression system in soybean protoplasts. The expression of numerous genes induced by various abiotic stresses were increased by overexpressing GmDREB1B;1 in soybean, and DREs were the most conserved element in the promoters of these genes. The downstream genes of GmDREB1B;1 included numerous soybean-specific stress-inducible genes that encode an ABA receptor family protein, GmPYL21, and translation-related genes, such as ribosomal proteins. We confirmed that GmDREB1B;1 directly activates GmPYL21 expression and enhances ABRE-mediated gene expression in an ABA-independent manner. These results suggest that GmDREB1 proteins activate the expression of numerous soybean-specific stress-responsive genes under diverse abiotic stress conditions. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Functional characterization of MAT1-1-specific mating-type genes in the homothallic ascomycete Sordaria macrospora provides new insights into essential and nonessential sexual regulators.

PubMed

Klix, V; Nowrousian, M; Ringelberg, C; Loros, J J; Dunlap, J C; Pöggeler, S

2010-06-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the alpha domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes.

Functional Characterization of MAT1-1-Specific Mating-Type Genes in the Homothallic Ascomycete Sordaria macrospora Provides New Insights into Essential and Nonessential Sexual Regulators▿†

PubMed Central

Klix, V.; Nowrousian, M.; Ringelberg, C.; Loros, J. J.; Dunlap, J. C.; Pöggeler, S.

2010-01-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the α domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes. PMID:20435701

Gene duplication and fragment recombination drive functional diversification of a superfamily of cytoplasmic effectors in Phytophthora sojae.

PubMed

Shen, Danyu; Liu, Tingli; Ye, Wenwu; Liu, Li; Liu, Peihan; Wu, Yuren; Wang, Yuanchao; Dou, Daolong

2013-01-01

Phytophthora and other oomycetes secrete a large number of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling and necrosis inducing proteins (CRN), or Crinkler. Here, we first investigated the evolutionary patterns and mechanisms of CRN effectors in Phytophthora sojae and compared them to two other Phytophthora species. The genes encoding CRN effectors could be divided into 45 orthologous gene groups (OGG), and most OGGs unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. The 134 expanded genes in P. sojae encoded family proteins including 82 functional genes and expressed at higher levels while the other 68 genes encoding orphan proteins were less expressed and contained 50 pseudogenes. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination. Three different mechanisms that drove gene duplication or recombination were identified. Finally, the expanded CRN effectors exhibited varying pathogenic functions, including induction of programmed cell death (PCD) and suppression of PCD through PAMP-triggered immunity or/and effector-triggered immunity. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen.

Global analysis of gene expression in response to L-Cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica

PubMed Central

2011-01-01

Background Entamoeba histolytica, an enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. E. histolytica completely lacks glutathione metabolism but possesses L-cysteine as the principle low molecular weight thiol. L-Cysteine is essential for the structure, stability, and various protein functions, including catalysis, electron transfer, redox regulation, nitrogen fixation, and sensing for regulatory processes. Recently, we demonstrated that in E. histolytica, L-cysteine regulates various metabolic pathways including energy, amino acid, and phospholipid metabolism. Results In this study, employing custom-made Affymetrix microarrays, we performed time course (3, 6, 12, 24, and 48 h) gene expression analysis upon L-cysteine deprivation. We identified that out of 9,327 genes represented on the array, 290 genes encoding proteins with functions in metabolism, signalling, DNA/RNA regulation, electron transport, stress response, membrane transport, vesicular trafficking/secretion, and cytoskeleton were differentially expressed (≥3 fold) at one or more time points upon L-cysteine deprivation. Approximately 60% of these modulated genes encoded proteins of no known function and annotated as hypothetical proteins. We also attempted further functional analysis of some of the most highly modulated genes by L-cysteine depletion. Conclusions To our surprise, L-cysteine depletion caused only limited changes in the expression of genes involved in sulfur-containing amino acid metabolism and oxidative stress defense. In contrast, we observed significant changes in the expression of several genes encoding iron sulfur flavoproteins, a major facilitator super-family transporter, regulator of nonsense transcripts, NADPH-dependent oxido-reductase, short chain dehydrogenase, acetyltransferases, and various other genes involved in diverse cellular functions. This study represents the first genome-wide analysis of transcriptional changes induced by L-cysteine deprivation in protozoan parasites, and in eukaryotic organisms where L-cysteine represents the major intracellular thiol. PMID:21627801

A subset of replication-dependent histone mRNAs are expressed as polyadenylated RNAs in terminally differentiated tissues.

PubMed

Lyons, Shawn M; Cunningham, Clark H; Welch, Joshua D; Groh, Beezly; Guo, Andrew Y; Wei, Bruce; Whitfield, Michael L; Xiong, Yue; Marzluff, William F

2016-11-02

Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Effects of Caste on the Expression of Genes Associated with Septic Injury and Xenobiotic Exposure in the Formosan Subterranean Termite

PubMed Central

2014-01-01

As social insects, termites live in densely populated colonies with specialized castes under conditions conducive to microbial growth and transmission. Furthermore, termites are exposed to xenobiotics in soil and their lignocellulose diet. Therefore, termites are valuable models for studying gene expression involved in response to septic injury, immunity and detoxification in relation to caste membership. In this study, workers and soldiers of the Formosan subterranean termite, Coptotermes formosanus, were challenged by bacterial injection or by no-choice feeding with a sublethal concentration (0.5%) of phenobarbital. Constitutive and induced expression of six putative immune response genes (two encoding for lectin-like proteins, one for a ficolin-precursor, one for the Down syndrome cell adhesion molecule, one for a chitin binding protein, and one for the gram-negative binding protein 2) and four putative detoxification genes (two encoding for cytochrome P450s, one for glutathione S-transferase, and one for the multi antimicrobial extrusion protein), were measured via quantitative real time polymerase chain reaction and compared within and among 1) colonies, 2) treatment types and 3) castes via ANOVA. Eight genes were inducible by septic injury, feeding with phenobarbital or both. Colony origin had no effect on inducibility or differential gene expression. However, treatment type showed significant effects on the expression of the eight inducible genes. Caste effects on expression levels were significant in five of the eight inducible genes with constitutive and induced expression of most target genes being higher in workers than in soldiers. PMID:25141339

Isolation of pheromone precursor genes of Magnaporthe grisea.

PubMed

Shen, W C; Bobrowicz, P; Ebbole, D J

1999-01-01

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia. Copyright 1999 Academic Press.

Unbiased View of Synaptic and Neuronal Gene Complement in Ctenophores: Are There Pan-neuronal and Pan-synaptic Genes across Metazoa?

PubMed Central

Moroz, Leonid L.; Kohn, Andrea B.

2015-01-01

Hypotheses of origins and evolution of neurons and synapses are controversial, mostly due to limited comparative data. Here, we investigated the genome-wide distribution of the bilaterian “synaptic” and “neuronal” protein-coding genes in non-bilaterian basal metazoans (Ctenophora, Porifera, Placozoa, and Cnidaria). First, there are no recognized genes uniquely expressed in neurons across all metazoan lineages. None of the so-called pan-neuronal genes such as embryonic lethal abnormal vision (ELAV), Musashi, or Neuroglobin are expressed exclusively in neurons of the ctenophore Pleurobrachia. Second, our comparative analysis of about 200 genes encoding canonical presynaptic and postsynaptic proteins in bilaterians suggests that there are no true “pan-synaptic” genes or genes uniquely and specifically attributed to all classes of synapses. The majority of these genes encode receptive and secretory complexes in a broad spectrum of eukaryotes. Trichoplax (Placozoa) an organism without neurons and synapses has more orthologs of bilaterian synapse-related/neuron-related genes than do ctenophores—the group with well-developed neuronal and synaptic organization. Third, the majority of genes encoding ion channels and ionotropic receptors are broadly expressed in unicellular eukaryotes and non-neuronal tissues in metazoans. Therefore, they cannot be viewed as neuronal markers. Nevertheless, the co-expression of multiple types of ion channels and receptors does correlate with the presence of neural and synaptic organization. As an illustrative example, the ctenophore genomes encode a greater diversity of ion channels and ionotropic receptors compared with the genomes of the placozoan Trichoplax and the demosponge Amphimedon. Surprisingly, both placozoans and sponges have a similar number of orthologs of “synaptic” proteins as we identified in the genomes of two ctenophores. Ctenophores have a distinct synaptic organization compared with other animals. Our analysis of transcriptomes from 10 different ctenophores did not detect recognized orthologs of synthetic enzymes encoding several classical, low-molecular-weight (neuro)transmitters; glutamate signaling machinery is one of the few exceptions. Novel peptidergic signaling molecules were predicted for ctenophores, together with the diversity of putative receptors including SCNN1/amiloride-sensitive sodium channel-like channels, many of which could be examples of a lineage-specific expansion within this group. In summary, our analysis supports the hypothesis of independent evolution of neurons and, as corollary, a parallel evolution of synapses. We suggest that the formation of synaptic machinery might occur more than once over 600 million years of animal evolution. PMID:26454853

HEx: A heterologous expression platform for the discovery of fungal natural products

PubMed Central

Schlecht, Ulrich; Horecka, Joe; Lin, Hsiao-Ching; Naughton, Brian; Miranda, Molly; Li, Yong Fuga; Hennessy, James R.; Vandova, Gergana A.; Steinmetz, Lars M.; Sattely, Elizabeth; Khosla, Chaitan; Hillenmeyer, Maureen E.

2018-01-01

For decades, fungi have been a source of U.S. Food and Drug Administration–approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products. PMID:29651464

Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway.

PubMed Central

Hughes, E J; Bayly, R C; Skurray, R A

1984-01-01

Alcaligenes eutrophus wild-type strain 345 metabolizes m- and p-toluate via a catechol meta-cleavage pathway. DNA analysis, curing studies, and transfer of this phenotype by conjugation and transformation showed that the degradative genes are encoded on a self-transmissible 85-kilobase plasmid, pRA1000. HindIII and XhoI restriction endonuclease analysis of pRA1000 showed it to be similar to the archetypal TOL plasmid, pWWO, differing in the case of HindIII only by the absence of fragments B and D present in pWWO. In strain 345, the presence of pRA1000 prevented the expression of chromosomally encoded enzymes required for the degradation of p-cresol, whereas these enzymes were expressed in strains cured of pRA1000. On the basis of studies with an R68.45-pRA1000 cointegrate plasmid, pRA1001, we conclude that the gene(s) responsible for the effect of p-cresol degradation resides within or near the m- and p-toluate degradative region on pRA1000. Images PMID:6325399

Inactivation of DNA-Binding Response Regulator Sak189 Abrogates β-Antigen Expression and Affects Virulence of Streptococcus agalactiae

PubMed Central

Rozhdestvenskaya, Anastasia S.; Totolian, Artem A.; Dmitriev, Alexander V.

2010-01-01

Background Streptococcus agalactiae is able to colonize numerous tissues employing different mechanisms of gene regulation, particularly via two-component regulatory systems. These systems sense the environmental stimuli and regulate expression of the genes including virulence genes. Recently, the novel two-component regulatory system Sak188/Sak189 was identified. In S. agalactiae genome, it was adjacent to the bac gene encoding for β-antigen, an important virulence factor. Methodology/Principal Findings In this study, the sak188 and sak189 genes were inactivated, and the functional role of Sak188/Sak189 two-component system in regulation of the β-antigen expression was investigated. It was demonstrated that both transcription of bac gene and expression of encoded β-antigen were controlled by Sak189 response regulator, but not Sak188 histidine kinase. It was also found that the regulation occurred at transcriptional level. Finally, insertional inactivation of sak189 gene, but not sak188 gene, significantly affected virulent properties of S. agalactiae. Conclusions/Significance Sak189 response regulator is necessary for activation of bac gene transcription. It also controls the virulent properties of S. agalactiae. Given that the primary functional role of Sak188/Sak189 two-component systems is a control of bac gene transcription, this system can be annotated as BgrR/S (bac gene regulatory system). PMID:20419089

Sequence and Expression Analyses of Ethylene Response Factors Highly Expressed in Latex Cells from Hevea brasiliensis

PubMed Central

Piyatrakul, Piyanuch; Yang, Meng; Putranto, Riza-Arief; Pirrello, Julien; Dessailly, Florence; Hu, Songnian; Summo, Marilyne; Theeravatanasuk, Kannikar; Leclercq, Julie; Kuswanhadi; Montoro, Pascal

2014-01-01

The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors. PMID:24971876

Sequence and expression analyses of ethylene response factors highly expressed in latex cells from Hevea brasiliensis.

PubMed

Piyatrakul, Piyanuch; Yang, Meng; Putranto, Riza-Arief; Pirrello, Julien; Dessailly, Florence; Hu, Songnian; Summo, Marilyne; Theeravatanasuk, Kannikar; Leclercq, Julie; Kuswanhadi; Montoro, Pascal

2014-01-01

The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors.

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

An Alternative Nested Reading Frame May Participate in the Stress-Dependent Expression of a Plant Gene

PubMed Central

Sheshukova, Ekaterina V.; Komarova, Tatiana V.; Ershova, Natalia M.; Shindyapina, Anastasia V.; Dorokhov, Yuri L.

2017-01-01

Although plants as sessile organisms are affected by a variety of stressors in the field, the stress factors for the above-ground and underground parts of the plant and their gene expression profiles are not the same. Here, we investigated NbKPILP, a gene encoding a new member of the ubiquitous, pathogenesis-related Kunitz peptidase inhibitor (KPI)-like protein family, that we discovered in the genome of Nicotiana benthamiana and other representatives of the Solanaceae family. The NbKPILP gene encodes a protein that has all the structural elements characteristic of KPI but in contrast to the proven A. thaliana KPI (AtKPI), it does not inhibit serine peptidases. Unlike roots, NbKPILP mRNA and its corresponding protein were not detected in intact leaves, but abiotic and biotic stressors drastically affected NbKPILP mRNA accumulation. In search of the causes of suppressed NbKPILP mRNA accumulation in leaves, we found that the NbKPILP gene is “matryoshka,” containing an alternative nested reading frame (ANRF) encoding a 53-amino acid (aa) polypeptide (53aa-ANRF) which has an amphipathic helix (AH). We confirmed ANRF expression experimentally. A vector containing a GFP-encoding sequence was inserted into the NbKPILP gene in frame with 53aa-ANRF, resulting in a 53aa-GFP fused protein that localized in the membrane fraction of cells. Using the 5′-RACE approach, we have shown that the expression of ANRF was not explained by the existence of a cryptic promoter within the NbKPILP gene but was controlled by the maternal NbKPILP mRNA. We found that insertion of mutations destroying the 53aa-ANRF AH resulted in more than a two-fold increase of the NbKPILP mRNA level. The NbKPILP gene represents the first example of ANRF functioning as a repressor of a maternal gene in an intact plant. We proposed a model where the stress influencing the translation initiation promotes the accumulation of NbKPILP and its mRNA in leaves. PMID:29312392

An Alternative Nested Reading Frame May Participate in the Stress-Dependent Expression of a Plant Gene.

PubMed

Sheshukova, Ekaterina V; Komarova, Tatiana V; Ershova, Natalia M; Shindyapina, Anastasia V; Dorokhov, Yuri L

2017-01-01

Although plants as sessile organisms are affected by a variety of stressors in the field, the stress factors for the above-ground and underground parts of the plant and their gene expression profiles are not the same. Here, we investigated NbKPILP , a gene encoding a new member of the ubiquitous, pathogenesis-related Kunitz peptidase inhibitor (KPI)-like protein family, that we discovered in the genome of Nicotiana benthamiana and other representatives of the Solanaceae family. The NbKPILP gene encodes a protein that has all the structural elements characteristic of KPI but in contrast to the proven A. thaliana KPI (AtKPI), it does not inhibit serine peptidases. Unlike roots, NbKPILP mRNA and its corresponding protein were not detected in intact leaves, but abiotic and biotic stressors drastically affected NbKPILP mRNA accumulation. In search of the causes of suppressed NbKPILP mRNA accumulation in leaves, we found that the NbKPILP gene is "matryoshka," containing an alternative nested reading frame (ANRF) encoding a 53-amino acid (aa) polypeptide (53aa-ANRF) which has an amphipathic helix (AH). We confirmed ANRF expression experimentally. A vector containing a GFP-encoding sequence was inserted into the NbKPILP gene in frame with 53aa-ANRF, resulting in a 53aa-GFP fused protein that localized in the membrane fraction of cells. Using the 5'-RACE approach, we have shown that the expression of ANRF was not explained by the existence of a cryptic promoter within the NbKPILP gene but was controlled by the maternal NbKPILP mRNA. We found that insertion of mutations destroying the 53aa-ANRF AH resulted in more than a two-fold increase of the NbKPILP mRNA level. The NbKPILP gene represents the first example of ANRF functioning as a repressor of a maternal gene in an intact plant. We proposed a model where the stress influencing the translation initiation promotes the accumulation of NbKPILP and its mRNA in leaves.

Transgenic tobacco expressing a modified spider peptide inhibits the growth of plant pathogens and insect larvae

USDA-ARS?s Scientific Manuscript database

The gene encoding lycotoxin I, an amphipathic pore-forming peptide, was modified to increase oral toxicity to insects. One of the most active modified genes was then constitutively expressed in tobacco (Nicotiana tabacum) and transformants were evaluated for insect and disease resistance. Pathogenic...

Acetylcholinesterase of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: Gene identification, expression, and biochemical properties of recombinant proteins

USDA-ARS?s Scientific Manuscript database

Rhipicephalus (Boophilus) microplus (Bm) ticks are vectors of bovine babesiosis and anaplasmosis. Tick resistance to organophosphate (OP) acaricide involves acetylcholinesterase (AChE) insensitivity to OP and metabolic detoxification. Sequencing and in vitro expression of Bm genes encoding AChE allo...

Acetylcholinesterases of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: Gene identification, expression and biochemical properties of recombinant proteins

USDA-ARS?s Scientific Manuscript database

Rhipicephalus (Boophilus) microplus (Bm) is a vector of bovine babesiosis and anaplasmosis. Tick resistance to organophosphate (OP) acaricide involves acetylcholinesterase (AChE) insensitivity to OP and metabolic detoxification. In vitro expression of Bm genes encoding AChE allowed biochemical chara...

Features of cues and processes during chloroplast-mediated retrograde signaling in the alga Chlamydomonas

USDA-ARS?s Scientific Manuscript database

Retrograde signalling is a selective process defined by cues generated in chloroplast/mitochondria which traverse membranes and end up regulating nuclear gene expression and protein synthesis. The coding and encoding of organellar message(s) that alter nuclear gene expression and/or cellular metabo...

The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1

USDA-ARS?s Scientific Manuscript database

The gene encoding SnTox1, a necrotrophic effector from Stagonospora nodorum that causes necrosis of wheat lines expressing Snn1, has been verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid cysteine rich protein with the first 17 amino acids predicted as a signal ...

Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components.

PubMed

Condino-Neto, A; Whitney, C; Newburger, P E

1998-11-01

We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-gamma and TNF-alpha, alone or in combination, for up to 7 days. IFN-gamma and TNF-alpha, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-gamma and TNF-alpha for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-gamma and TNF-alpha during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.

Dextransucrase Expression Is Concomitant with that of Replication and Maintenance Functions of the pMN1 Plasmid in Lactobacillus sakei MN1

PubMed Central

Nácher-Vázquez, Montserrat; Ruiz-Masó, José A.; Mohedano, María L.; del Solar, Gloria; Aznar, Rosa; López, Paloma

2017-01-01

The exopolysaccharide synthesized by Lactobacillus sakei MN1 is a dextran with antiviral and immunomodulatory properties of potential utility in aquaculture. In this work we have investigated the genetic basis of dextran production by this bacterium. Southern blot hybridization experiments demonstrated the plasmidic location of the dsrLS gene, which encodes the dextransucrase involved in dextran synthesis. DNA sequencing of the 11,126 kbp plasmid (pMN1) revealed that it belongs to a family which replicates by the theta mechanism, whose prototype is pUCL287. The plasmid comprises the origin of replication, repA, repB, and dsrLS genes, as well as seven open reading frames of uncharacterized function. Lb. sakei MN1 produces dextran when sucrose, but not glucose, is present in the growth medium. Therefore, plasmid copy number and stability, as well as dsrLS expression, were investigated in cultures grown in the presence of either sucrose or glucose. The results revealed that pMN1 is a stable low-copy-number plasmid in both conditions. Gene expression studies showed that dsrLS is constitutively expressed, irrespective of the carbon source present in the medium. Moreover, dsrLS is expressed from a monocistronic transcript as well as from a polycistronic repA-repB-orf1-dsrLS mRNA. To our knowledge, this is the first report of a plasmid-borne dextransucrase-encoding gene, as well as the first time that co-transcription of genes involved in plasmid maintenance and replication with a gene encoding an enzyme has been established. PMID:29209293

Mutations in nuclear genes alter post-transcriptional regulation of mitochondrial genes.

USDA-ARS?s Scientific Manuscript database

Nuclear gene products are required for the expression of mitochondrial genes and elaboration of functional mitochondrial protein complexes. To better understand the roles of these nuclear genes, we exploited the mitochondrial encoded S-type of cytoplasmic male sterility (CMS-S) and developed a nove...

Differential Gene Expression by Lactobacillus plantarum WCFS1 in Response to Phenolic Compounds Reveals New Genes Involved in Tannin Degradation.

PubMed

Reverón, Inés; Jiménez, Natalia; Curiel, José Antonio; Peñas, Elena; López de Felipe, Félix; de Las Rivas, Blanca; Muñoz, Rosario

2017-04-01

Lactobacillus plantarum is a lactic acid bacterium that can degrade food tannins by the successive action of tannase and gallate decarboxylase enzymes. In the L. plantarum genome, the gene encoding the catalytic subunit of gallate decarboxylase ( lpdC , or lp_2945 ) is only 6.5 kb distant from the gene encoding inducible tannase ( L. plantarum tanB [ tanB Lp ], or lp_2956 ). This genomic context suggests concomitant activity and regulation of both enzymatic activities. Reverse transcription analysis revealed that subunits B ( lpdB , or lp_0271 ) and D ( lpdD , or lp_0272 ) of the gallate decarboxylase are cotranscribed, whereas subunit C ( lpdC , or lp_2945 ) is cotranscribed with a gene encoding a transport protein ( gacP , or lp_2943 ). In contrast, the tannase gene is transcribed as a monocistronic mRNA. Investigation of knockout mutations of genes located in this chromosomal region indicated that only mutants of the gallate decarboxylase (subunits B and C), tannase, GacP transport protein, and TanR transcriptional regulator ( lp_2942 ) genes exhibited altered tannin metabolism. The expression profile of genes involved in tannin metabolism was also analyzed in these mutants in the presence of methyl gallate and gallic acid. It is noteworthy that inactivation of tanR suppresses the induction of all genes overexpressed in the presence of methyl gallate and gallic acid. This transcriptional regulator was also induced in the presence of other phenolic compounds, such as kaempferol and myricetin. This study complements the catalog of L. plantarum expression profiles responsive to phenolic compounds, which enable this bacterium to adapt to a plant food environment. IMPORTANCE Lactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables when tannins are present. L. plantarum strains degrade tannins to the less-toxic pyrogallol by the successive action of tannase and gallate decarboxylase enzymes. The genes encoding these enzymes are located close to each other in the chromosome, suggesting concomitant regulation. Proteins involved in tannin metabolism and regulation, such GacP (gallic acid permease) and TanR (tannin transcriptional regulator), were identified by differential gene expression in knockout mutants with mutations in genes from this region. This study provides insights into the highly coordinated mechanisms that enable L. plantarum to adapt to plant food fermentations. Copyright © 2017 American Society for Microbiology.

Differential Gene Expression by Lactobacillus plantarum WCFS1 in Response to Phenolic Compounds Reveals New Genes Involved in Tannin Degradation

PubMed Central

Reverón, Inés; Jiménez, Natalia; Curiel, José Antonio; Peñas, Elena; López de Felipe, Félix; de las Rivas, Blanca

2017-01-01

ABSTRACT Lactobacillus plantarum is a lactic acid bacterium that can degrade food tannins by the successive action of tannase and gallate decarboxylase enzymes. In the L. plantarum genome, the gene encoding the catalytic subunit of gallate decarboxylase (lpdC, or lp_2945) is only 6.5 kb distant from the gene encoding inducible tannase (L. plantarum tanB [tanBLp], or lp_2956). This genomic context suggests concomitant activity and regulation of both enzymatic activities. Reverse transcription analysis revealed that subunits B (lpdB, or lp_0271) and D (lpdD, or lp_0272) of the gallate decarboxylase are cotranscribed, whereas subunit C (lpdC, or lp_2945) is cotranscribed with a gene encoding a transport protein (gacP, or lp_2943). In contrast, the tannase gene is transcribed as a monocistronic mRNA. Investigation of knockout mutations of genes located in this chromosomal region indicated that only mutants of the gallate decarboxylase (subunits B and C), tannase, GacP transport protein, and TanR transcriptional regulator (lp_2942) genes exhibited altered tannin metabolism. The expression profile of genes involved in tannin metabolism was also analyzed in these mutants in the presence of methyl gallate and gallic acid. It is noteworthy that inactivation of tanR suppresses the induction of all genes overexpressed in the presence of methyl gallate and gallic acid. This transcriptional regulator was also induced in the presence of other phenolic compounds, such as kaempferol and myricetin. This study complements the catalog of L. plantarum expression profiles responsive to phenolic compounds, which enable this bacterium to adapt to a plant food environment. IMPORTANCE Lactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables when tannins are present. L. plantarum strains degrade tannins to the less-toxic pyrogallol by the successive action of tannase and gallate decarboxylase enzymes. The genes encoding these enzymes are located close to each other in the chromosome, suggesting concomitant regulation. Proteins involved in tannin metabolism and regulation, such GacP (gallic acid permease) and TanR (tannin transcriptional regulator), were identified by differential gene expression in knockout mutants with mutations in genes from this region. This study provides insights into the highly coordinated mechanisms that enable L. plantarum to adapt to plant food fermentations. PMID:28115379

LAMP-1-chimeric DNA vaccines enhance the antibody response in Japanese flounder, Paralichthys olivaceus.

PubMed

Rondón-Barragán, Iang; Nozaki, Reiko; Hirono, Ikuo; Kondo, Hidehiro

2017-08-01

DNA vaccination is one method to protect farmed fish from viral and bacterial diseases. Chimeric antigens encoded by DNA vaccines have been shown to increase the resistance to viral diseases. Here, we sequenced the gene encoding lysosome-associated membrane protein-1 from Japanese flounder, Paralichthys olivaceus, (JfLAMP-1) and assessed its use in a chimeric DNA vaccine fused with the major capsule protein (MCP) from red seabream iridovirus (RSIV). JfLAMP-1 cDNA has a length of 1248 bp encoding 415 aa, which contains transmembrane and cytoplasmic domains. JfLAMP-1 is constitutively expressed in several tissues and its expression in spleen was upregulated following injection of formalin-killed cells (FKC) of Edwardsiella tarda. Immunofluorescence analysis showed that JfLAMP-1 is distributed in the small and large granules in the cytoplasm and groups close to the nucleus. The DNA encoding the luminal domain of JfLAMP-1 was replaced with the gene for the RSIV MCP, and the construct was cloned in an expression vector (pCIneo). Fish vaccinated with pCLAMP-MCP had significantly higher antibody levels than fish vaccinated with pCIneo vector harboring the MCP gene (p < 0.05) at day 30 post-vaccination. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-wide analysis of WRKY gene family in Cucumis sativus

PubMed Central

2011-01-01

Background WRKY proteins are a large family of transcriptional regulators in higher plant. They are involved in many biological processes, such as plant development, metabolism, and responses to biotic and abiotic stresses. Prior to the present study, only one full-length cucumber WRKY protein had been reported. The recent publication of the draft genome sequence of cucumber allowed us to conduct a genome-wide search for cucumber WRKY proteins, and to compare these positively identified proteins with their homologs in model plants, such as Arabidopsis. Results We identified a total of 55 WRKY genes in the cucumber genome. According to structural features of their encoded proteins, the cucumber WRKY (CsWRKY) genes were classified into three groups (group 1-3). Analysis of expression profiles of CsWRKY genes indicated that 48 WRKY genes display differential expression either in their transcript abundance or in their expression patterns under normal growth conditions, and 23 WRKY genes were differentially expressed in response to at least one abiotic stresses (cold, drought or salinity). The expression profile of stress-inducible CsWRKY genes were correlated with those of their putative Arabidopsis WRKY (AtWRKY) orthologs, except for the group 3 WRKY genes. Interestingly, duplicated group 3 AtWRKY genes appear to have been under positive selection pressure during evolution. In contrast, there was no evidence of recent gene duplication or positive selection pressure among CsWRKY group 3 genes, which may have led to the expressional divergence of group 3 orthologs. Conclusions Fifty-five WRKY genes were identified in cucumber and the structure of their encoded proteins, their expression, and their evolution were examined. Considering that there has been extensive expansion of group 3 WRKY genes in angiosperms, the occurrence of different evolutionary events could explain the functional divergence of these genes. PMID:21955985

Genome-wide analysis of WRKY gene family in Cucumis sativus.

PubMed

Ling, Jian; Jiang, Weijie; Zhang, Ying; Yu, Hongjun; Mao, Zhenchuan; Gu, Xingfang; Huang, Sanwen; Xie, Bingyan

2011-09-28

WRKY proteins are a large family of transcriptional regulators in higher plant. They are involved in many biological processes, such as plant development, metabolism, and responses to biotic and abiotic stresses. Prior to the present study, only one full-length cucumber WRKY protein had been reported. The recent publication of the draft genome sequence of cucumber allowed us to conduct a genome-wide search for cucumber WRKY proteins, and to compare these positively identified proteins with their homologs in model plants, such as Arabidopsis. We identified a total of 55 WRKY genes in the cucumber genome. According to structural features of their encoded proteins, the cucumber WRKY (CsWRKY) genes were classified into three groups (group 1-3). Analysis of expression profiles of CsWRKY genes indicated that 48 WRKY genes display differential expression either in their transcript abundance or in their expression patterns under normal growth conditions, and 23 WRKY genes were differentially expressed in response to at least one abiotic stresses (cold, drought or salinity). The expression profile of stress-inducible CsWRKY genes were correlated with those of their putative Arabidopsis WRKY (AtWRKY) orthologs, except for the group 3 WRKY genes. Interestingly, duplicated group 3 AtWRKY genes appear to have been under positive selection pressure during evolution. In contrast, there was no evidence of recent gene duplication or positive selection pressure among CsWRKY group 3 genes, which may have led to the expressional divergence of group 3 orthologs. Fifty-five WRKY genes were identified in cucumber and the structure of their encoded proteins, their expression, and their evolution were examined. Considering that there has been extensive expansion of group 3 WRKY genes in angiosperms, the occurrence of different evolutionary events could explain the functional divergence of these genes.

Shifts in Host Mucosal Innate Immune Function Are Associated with Ruminal Microbial Succession in Supplemental Feeding and Grazing Goats at Different Ages

PubMed Central

Jiao, Jinzhen; Zhou, Chuanshe; Guan, L. L.; McSweeney, C. S.; Tang, Shaoxun; Wang, Min; Tan, Zhiliang

2017-01-01

Gastrointestinal microbiota may play an important role in regulating host mucosal innate immune function. This study was conducted to test the hypothesis that age (non-rumination, transition and rumination) and feeding type [Supplemental feeding (S) vs. Grazing (G)] could alter ruminal microbial diversity and maturation of host mucosal innate immune system in goat kids. MiSeq sequencing was applied to investigate ruminal microbial composition and diversity, and RT-PCR was used to test expression of immune-related genes in ruminal mucosa. Results showed that higher (P < 0.05) relative abundances of Prevotella, Butyrivibrio, Pseudobutyrivibrio, Methanobrevibacter.gottschalkii, Neocallimastix, Anoplodinium–Diplodinium, and Polyplastron, and lower relative abundance of Methanosphaera (P = 0.042) were detected in the rumen of S kids when compared to those in G kids. The expression of genes encoding TLRs, IL1α, IL1β and TICAM2 was down-regulated (P < 0.01), while expression of genes encoding tight junction proteins was up-regulated (P < 0.05) in the ruminal mucosa of S kids when compared to that in G kids. Moreover, irrespective of feeding type, relative abundances of ruminal Prevotella, Fibrobacter, Ruminococcus, Butyrivibrio, Methanobrevibacter, Neocallimastix, and Entodinium increased with age. The expression of most genes encoding TLRs and cytokines increased (P < 0.05) from day 0 to 7, while expression of genes encoding tight junction proteins declined with age (P < 0.05). This study revealed that the composition of each microbial domain changed as animals grew, and these changes might be associated with variations in host mucosal innate immune function. Moreover, supplementing goat kids with concentrate could modulate ruminal microbial composition, enhance barrier function and decrease local inflammation. The findings provide useful information in interpreting microbiota and host interactions, and developing nutritional strategies to improve the productivity and health of rumen during early life. PMID:28912767

Molecular evolution and expression profile of the chemerine encoding gene RARRES2 in baboon and chimpanzee.

PubMed

González-Alvarez, Rafael; Garza-Rodríguez, María de Lourdes; Delgado-Enciso, Iván; Treviño-Alvarado, Víctor Manuel; Canales-Del-Castillo, Ricardo; Martínez-De-Villarreal, Laura Elia; Lugo-Trampe, Ángel; Tejero, María Elizabeth; Schlabritz-Loutsevitch, Natalia E; Rocha-Pizaña, María Del Refugio; Cole, Shelley A; Reséndez-Pérez, Diana; Moises-Alvarez, Mario; Comuzzie, Anthony G; Barrera-Saldaña, Hugo Alberto; Garza-Guajardo, Raquel; Barboza-Quintana, Oralia; Rodríguez-Sánchez, Irám Pablo

2015-06-12

Chemerin, encoded by the retinoic acid receptor responder 2 (RARRES2) gene is an adipocytesecreted protein with autocrine/paracrine functions in adipose tissue, metabolism and inflammation with a recently described function in vascular tone regulation, liver, steatosis, etc. This molecule is believed to represent a critical endocrine signal linking obesity to diabetes. There are no data available regarding evolution of RARRES2 in non-human primates and great apes. Expression profile and orthology in RARRES2 genes are unknown aspects in the biology of this multigene family in primates. Thus; we attempt to describe expression profile and phylogenetic relationship as complementary knowledge in the function of this gene in primates. To do that, we performed A RT-PCR from different tissues obtained during necropsies. Also we tested the hypotheses of positive evolution, purifying selection, and neutrality. And finally a phylogenetic analysis was made between primates RARRES2 protein. RARRES2 transcripts were present in liver, lung, adipose tissue, ovary, pancreas, heart, hypothalamus and pituitary tissues. Expression in kidney and leukocytes were not detectable in either species. It was determined that the studied genes are orthologous. RARRES2 evolution fits the hypothesis of purifying selection. Expression profiles of the RARRES2 gene are similar in baboons and chimpanzees and are also phylogenetically related.

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

Mitochondrial genes are altered in blood early in Alzheimer's disease.

PubMed

Lunnon, Katie; Keohane, Aoife; Pidsley, Ruth; Newhouse, Stephen; Riddoch-Contreras, Joanna; Thubron, Elisabeth B; Devall, Matthew; Soininen, Hikka; Kłoszewska, Iwona; Mecocci, Patrizia; Tsolaki, Magda; Vellas, Bruno; Schalkwyk, Leonard; Dobson, Richard; Malik, Afshan N; Powell, John; Lovestone, Simon; Hodges, Angela

2017-05-01

Although mitochondrial dysfunction is a consistent feature of Alzheimer's disease in the brain and blood, the molecular mechanisms behind these phenomena are unknown. Here we have replicated our previous findings demonstrating reduced expression of nuclear-encoded oxidative phosphorylation (OXPHOS) subunits and subunits required for the translation of mitochondrial-encoded OXPHOS genes in blood from people with Alzheimer's disease and mild cognitive impairment. Interestingly this was accompanied by increased expression of some mitochondrial-encoded OXPHOS genes, namely those residing closest to the transcription start site of the polycistronic heavy chain mitochondrial transcript (MT-ND1, MT-ND2, MT-ATP6, MT-CO1, MT-CO2, MT-C03) and MT-ND6 transcribed from the light chain. Further we show that mitochondrial DNA copy number was unchanged suggesting no change in steady-state numbers of mitochondria. We suggest that an imbalance in nuclear and mitochondrial genome-encoded OXPHOS transcripts may drive a negative feedback loop reducing mitochondrial translation and compromising OXPHOS efficiency, which is likely to generate damaging reactive oxygen species. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum

PubMed Central

Weber, Barbara; Lindell, Kristoffer; El Qaidi, Samir; Hjerde, Erik; Willassen, Nils-Peder

2011-01-01

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ54 and the σ54-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment. PMID:21948044

The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum.

PubMed

Weber, Barbara; Lindell, Kristoffer; El Qaidi, Samir; Hjerde, Erik; Willassen, Nils-Peder; Milton, Debra L

2011-12-01

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ(54) and the σ(54)-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

Genomics Analysis of Genes Expressed in Maize Endosperm Identifies Novel Seed Proteins and Clarifies Patterns of Zein Gene Expression

PubMed Central

Woo, Young-Min; Hu, David Wang-Nan; Larkins, Brian A.; Jung, Rudolf

2001-01-01

We analyzed cDNA libraries from developing endosperm of the B73 maize inbred line to evaluate the expression of storage protein genes. This study showed that zeins are by far the most highly expressed genes in the endosperm, but we found an inverse relationship between the number of zein genes and the relative amount of specific mRNAs. Although α-zeins are encoded by large multigene families, only a few of these genes are transcribed at high or detectable levels. In contrast, relatively small gene families encode the γ- and δ-zeins, and members of these gene families, especially the γ-zeins, are highly expressed. Knowledge of expressed storage protein genes allowed the development of DNA and antibody probes that distinguish between closely related gene family members. Using in situ hybridization, we found differences in the temporal and spatial expression of the α-, γ-, and δ-zein gene families, which provides evidence that γ-zeins are synthesized throughout the endosperm before α- and δ-zeins. This observation is consistent with earlier studies that suggested that γ-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD γ-zein, an 18-kD α-globulin, and a legumin-related protein. Immunolocalization of the 50-kD γ-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other γ-zeins. The 18-kD α-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm. PMID:11595803

Transcriptome Analysis Reveals Genes Commonly Induced by Botrytis cinerea Infection, Cold, Drought and Oxidative Stresses in Arabidopsis

PubMed Central

Al-Ameri, Salma; Al-Mahmoud, Bassam; Awwad, Falah; Al-Rawashdeh, Ahmed; Iratni, Rabah; AbuQamar, Synan

2014-01-01

Signaling pathways controlling biotic and abiotic stress responses may interact synergistically or antagonistically. To identify the similarities and differences among responses to diverse stresses, we analyzed previously published microarray data on the transcriptomic responses of Arabidopsis to infection with Botrytis cinerea (a biotic stress), and to cold, drought, and oxidative stresses (abiotic stresses). Our analyses showed that at early stages after B. cinerea inoculation, 1498 genes were up-regulated (B. cinerea up-regulated genes; BUGs) and 1138 genes were down-regulated (B. cinerea down-regulated genes; BDGs). We showed a unique program of gene expression was activated in response each biotic and abiotic stress, but that some genes were similarly induced or repressed by all of the tested stresses. Of the identified BUGs, 25%, 6% and 12% were also induced by cold, drought and oxidative stress, respectively; whereas 33%, 7% and 5.5% of the BDGs were also down-regulated by the same abiotic stresses. Coexpression and protein-protein interaction network analyses revealed a dynamic range in the expression levels of genes encoding regulatory proteins. Analysis of gene expression in response to electrophilic oxylipins suggested that these compounds are involved in mediating responses to B. cinerea infection and abiotic stress through TGA transcription factors. Our results suggest an overlap among genes involved in the responses to biotic and abiotic stresses in Arabidopsis. Changes in the transcript levels of genes encoding components of the cyclopentenone signaling pathway in response to biotic and abiotic stresses suggest that the oxylipin signal transduction pathway plays a role in plant defense. Identifying genes that are commonly expressed in response to environmental stresses, and further analyzing the functions of their encoded products, will increase our understanding of the plant stress response. This information could identify targets for genetic modification to improve plant resistance to multiple stresses. PMID:25422934

Cloning and expression of calmodulin gene in Scoparia dulcis.

PubMed

Saitoh, Daisuke; Asakura, Yuki; Nkembo, Marguerite Kasidimoko; Shite, Masato; Sugiyama, Ryuji; Lee, Jung-Bum; Hayashi, Toshimitsu; Kurosaki, Fumiya

2007-06-01

A homology-based cloning strategy yielded a cDNA clone, designated Sd-cam, encoding calmodulin protein from Scoparia dulcis. The restriction digests of genomic DNA of S. dulcis showed a single hybridized signal when probed with the fragment of this gene in Southern blot analyses, suggesting that Sd-cam occurs as a sole gene encoding calmodulin in the plant. The reverse-transcription polymerase chain reaction analysis revealed that Sd-cam was appreciably expressed in leaf, root and stem tissues. It appeared that transcription of this gene increased transiently when the leaf cultures of S. dulcis were treated with methyl jasmonate and calcium ionophore A23187. These results suggest that transcriptional activation of Sd-cam is one of the early cellular events of the methyl jasmonate-induced responses of S. dulcis.

Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation

PubMed Central

Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Madhav, Sheshu M.; Kirti, P. B.

2016-01-01

Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2–3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in rice and other crops, which may be achieved by overexpressing and raising independent transgenic plants carrying the genes that became up-regulated significantly and instantaneously. PMID:27605933

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions.

PubMed

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity.

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions

PubMed Central

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity. PMID:25191551

prtH2, Not prtH, Is the Ubiquitous Cell Wall Proteinase Gene in Lactobacillus helveticus▿

PubMed Central

Genay, M.; Sadat, L.; Gagnaire, V.; Lortal, S.

2009-01-01

Lactobacillus helveticus strains possess an efficient proteolytic system that releases peptides which are essential for lactobacillus growth in various fermented dairy products and also affect textural properties or biological activities. Cell envelope proteinases (CEPs) are bacterial enzymes that hydrolyze milk proteins. In the case of L. helveticus, two CEPs with low percentages of amino acid identity have been described, i.e., PrtH and PrtH2. However, the distribution of the genes that encode CEPs still remains unclear, rendering it difficult to further control the formation of particular peptides. This study evaluated the diversity of genes that encode CEPs in a collection of strains of L. helveticus isolated from various biotopes, both in terms of the presence or absence of these genes and in terms of nucleotide sequence, and studied their transcription in dairy matrices. After defining three sets of primers for both the prtH and prtH2 genes, we studied the distribution of the genes by using PCR and Southern blotting experiments. The prtH2 gene was ubiquitous in the 29 strains of L. helveticus studied, whereas only 18 of them also exhibited the prtH gene. Sequencing of a 350-bp internal fragment of these genes revealed the existence of intraspecific diversity. Finally, expression of these two CEP-encoding genes was followed during the growth in dairy matrices of two strains, ITG LH77 and CNRZ32, which possess one and two CEP-encoding genes, respectively. Both genes were shown to be expressed by L. helveticus at each stage of growth in milk and at different stages of mini-Swiss-type cheese making and ripening. PMID:19286786

Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)

PubMed Central

2010-01-01

Background Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here. PMID:20089196

Distinct Calcium Signaling Pathways Regulate Calmodulin Gene Expression in Tobacco1

PubMed Central

van der Luit, Arnold H.; Olivari, Claudio; Haley, Ann; Knight, Marc R.; Trewavas, Anthony J.

1999-01-01

Cold shock and wind stimuli initiate Ca2+ transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca2+ pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca2+ transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca2+ signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca2+ dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca2+ signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm. PMID:10557218

aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

PubMed Central

de Vries, Ronald P.; Poulsen, Charlotte H.; Madrid, Susan; Visser, Jaap

1998-01-01

An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose. PMID:9440512

aguA, the gene encoding an extracellular alpha-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid.

PubMed

de Vries, R P; Poulsen, C H; Madrid, S; Visser, J

1998-01-01

An extracellular alpha-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70 degrees C. The alpha-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and beta-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this alpha-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and alpha-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.

Selection and validation of reference genes for RT-qPCR indicates that juice of sugarcane varieties modulate the expression of C metabolism genes in the endophytic diazotrophic Herbaspirillum rubrisubalbicans strain HCC103.

PubMed

Polese, Valéria; de Paula Soares, Cleiton; da Silva, Paula Renata Alves; Simões-Araújo, Jean Luiz; Baldani, José Ivo; Vidal, Marcia Soares

2017-12-01

Quantitative reverse transcription PCR (RT-qPCR) is an important tool for evaluating gene expression. However, this technique requires that specific internal normalizing genes be identified for different experimental conditions. To date, no internal normalizing genes are available for validation of data analyses for Herbaspirillum rubrisubalbicans strain HCC103, an endophyte that is part of the sugarcane consortium inoculant. This work seeks to identify and evaluate suitable reference genes for gene expression studies in HCC103 grown until middle log phase in sugarcane juice obtained from four sugarcane varieties or media with three different carbon sources. The mRNA levels of five candidate genes (rpoA, gyrA, dnaG, recA and gmK) and seven target genes involved in carbon metabolism (acnA, fbp, galE, suhB, wcaA, ORF_0127.0101 and _0127.0123) were quantified by RT-qPCR. Analysis of expression stability of these genes was carried out using geNorm and Normfinder software. The results indicated that the HCC103 dnaG and gyrA genes are the most stable and showed adequate relative expression level changes among the different sugarcane juices. The highest expression level was seen for ORF_0127.0101, which encodes a sugar transporter, in juice from sugarcane variety RB867515 and glucose as the carbon source. The suhB gene, encoding SuhB inositol monophosphatase, had a higher relative expression level on 0.5% glucose, 100% sugarcane juice from variety RB867515 and 0.5% aconitate. Together the results suggest that dnaG and gyrA genes are suitable as reference genes for RT-qPCR analysis of strain HCC103 and that juice from different sugarcane varieties modulates the expression of key genes involved in carbon metabolism.

A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system.

PubMed

Sudomoina, Marina; Latypova, Ekaterina; Favorova, Olga O; Golemis, Erica A; Serebriiskii, Ilya G

2004-04-29

Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo.

Thermo-Regulation of Genes Mediating Motility and Plant Interactions in Pseudomonas syringae

PubMed Central

Hockett, Kevin L.; Burch, Adrien Y.; Lindow, Steven E.

2013-01-01

Pseudomonas syringae is an important phyllosphere colonist that utilizes flagellum-mediated motility both as a means to explore leaf surfaces, as well as to invade into leaf interiors, where it survives as a pathogen. We found that multiple forms of flagellum-mediated motility are thermo-suppressed, including swarming and swimming motility. Suppression of swarming motility occurs between 28° and 30°C, which coincides with the optimal growth temperature of P. syringae. Both fliC (encoding flagellin) and syfA (encoding a non-ribosomal peptide synthetase involved in syringafactin biosynthesis) were suppressed with increasing temperature. RNA-seq revealed 1440 genes of the P. syringae genome are temperature sensitive in expression. Genes involved in polysaccharide synthesis and regulation, phage and IS elements, type VI secretion, chemosensing and chemotaxis, translation, flagellar synthesis and motility, and phytotoxin synthesis and transport were generally repressed at 30°C, while genes involved in transcriptional regulation, quaternary ammonium compound metabolism and transport, chaperone/heat shock proteins, and hypothetical genes were generally induced at 30°C. Deletion of flgM, a key regulator in the transition from class III to class IV gene expression, led to elevated and constitutive expression of fliC regardless of temperature, but did not affect thermo-regulation of syfA. This work highlights the importance of temperature in the biology of P. syringae, as many genes encoding traits important for plant-microbe interactions were thermo-regulated. PMID:23527276

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

PubMed Central

Park, Jong-Won; Beyene, Getu; Buenrostro-Nava, Marco T.; Molina, Joe; Wang, Xiaofeng; Ciomperlik, Jessica J.; Manabayeva, Shuga A.; Alvarado, Veria Y.; Rathore, Keerti S.; Scholthof, Herman B.; Mirkov, T. Erik

2013-01-01

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane. PMID:23799071

Effects of TCDD on the Expression of Nuclear Encoded Mitochondrial Genes

PubMed Central

Forgacs, Agnes L.; Burgoon, Lyle D.; Lynn, Scott G.; LaPres, John J.; Zacharewski, Timothy

2014-01-01

Generation of mitochondrial reactive oxygen species (ROS) can be perturbed following exposure to environmental chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Reports indicate that the aryl hydrocarbon receptor (AhR) mediates TCDD-induced sustained hepatic oxidative stress by decreasing hepatic ATP levels and through hyperpolarization of the inner mitochondrial membrane. To further elucidate the effects of TCDD on the mitochondria, high-throughput quantitative real-time PCR (HTP-QRTPCR) was used to evaluate the expression of 90 genes encoding mitochondrial proteins involved in electron transport, oxidative phosphorylation, uncoupling, and associated chaperones. HTP-QRTPCR analysis of time course (30 μg/kg TCDD at 2, 4, 8, 12, 18, 24, 72, and 168 hrs) liver samples obtained from orally gavaged immature, ovariectomized C57BL/6 mice identified 54 differentially expressed genes (|fold change|>1.5 and P-value <0.1). Of these, 8 exhibited a dose response (0.03 to 300 μg/kg TCDD) at 4, 24 or 72 hrs. Dose responsive genes encoded proteins associated with electron transport chain (ETC) complex I (NADH dehydrogenase), III (cytochrome c reductase), IV (cytochrome c oxidase), and V (ATP synthase) and could be generally categorized as having proton gradient, ATP synthesis, and chaperone activities. In contrast, transcript levels of ETC complex II, succinate dehydrogenase, remained unchanged. Putative dioxin response elements were computationally found in the promoter regions of the 8 dose-responsive genes. This high-throughput approach suggests that TCDD alters the expression of genes associated with mitochondrial function which may contribute to TCDD-elicited mitochondrial toxicity. PMID:20399798

Novel Variants in ZNF34 and Other Brain-Expressed Transcription Factors are Shared Among Early-Onset MDD Relatives

PubMed Central

Subaran, Ryan L.; Odgerel, Zagaa; Swaminathan, Rajeswari; Glatt, Charles E.; Weissman, Myrna M.

2018-01-01

There are no known genetic variants with large effects on susceptibility to major depressive disorder (MDD). Although one proposed study approach is to increase sensitivity by increasing sample sizes, another is to focus on families with multiple affected individuals to identify genes with rare or novel variants with strong effects. Choosing the family-based approach, we performed whole-exome analysis on affected individuals (n = 12) across five MDD families, each with at least five affected individuals, early onset, and prepubertal diagnoses. We identified 67 genes where novel deleterious variants were shared among affected relatives. Gene ontology analysis shows that of these 67 genes, 18 encode transcriptional regulators, eight of which are expressed in the human brain, including four KRAB-A box-containing Zn2+ finger repressors. One of these, ZNF34, has been reported as being associated with bipolar disorder and as differentially expressed in bipolar disorder patients compared to healthy controls. We found a novel variant—encoding a non-conservative P17R substitution in the conserved repressor domain of ZNF34 protein—segregating completely with MDD in all available individuals in the family in which it was discovered. Further analysis showed a common ZNF34 coding indel segregating with MDD in a separate family, possibly indicating the presence of an unobserved, linked, rare variant in that particular family. Our results indicate that genes encoding transcription factors expressed in the brain might be an important group of MDD candidate genes and that rare variants in ZNF34 might contribute to susceptibility to MDD and perhaps other affective disorders. PMID:26823146

Phylogenetic analysis of fungal heterotrimeric G protein-encoding genes and their expression during dimorphism in Mucor circinelloides.

PubMed

Valle-Maldonado, Marco Iván; Jácome-Galarza, Irvin Eduardo; Díaz-Pérez, Alma Laura; Martínez-Cadena, Guadalupe; Campos-García, Jesús; Ramírez-Díaz, Martha Isela; Reyes-De la Cruz, Homero; Riveros-Rosas, Héctor; Díaz-Pérez, César; Meza-Carmen, Víctor

2015-12-01

In fungi, heterotrimeric G proteins are key regulators of biological processes such as mating, virulence, morphology, among others. Mucor circinelloides is a model organism for many biological processes, and its genome contains the largest known repertoire of genes that encode putative heterotrimeric G protein subunits in the fungal kingdom: twelve Gα (McGpa1-12), three Gβ (McGpb1-3), and three Gγ (McGpg1-3). Phylogenetic analysis of fungal Gα showed that they are divided into four distinct groups as reported previously. Fungal Gβ and Gγ are also divided into four phylogenetic groups, and to our understanding this is the first report of a phylogenetic classification for fungal Gβ and Gγ subunits. Almost all genes that encode putative heterotrimeric G subunits in M. circinelloides are differentially expressed during dimorphic growth, except for McGpg1 (Gγ) that showed very low mRNA levels at all developmental stages. Moreover, several of the subunits are expressed in a similar pattern and at the same level, suggesting that they constitute discrete complexes. For example, McGpb3 (Gβ), and McGpg2 (Gγ), are co-expressed during mycelium growth, and McGpa1, McGpb2, and McGpg2, are co-expressed during yeast development. These findings provide the conceptual framework to study the biological role of these genes during M. circinelloides morphogenesis. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Pnrc2 regulates 3'UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation.

PubMed

Gallagher, Thomas L; Tietz, Kiel T; Morrow, Zachary T; McCammon, Jasmine M; Goldrich, Michael L; Derr, Nicolas L; Amacher, Sharon L

2017-09-01

Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and Characterization of a Cis Antisense RNA of the rpoH Gene of Salmonella enterica Serovar Typhi.

PubMed

Xiong, Changyan; Li, Xuejiao; Liu, Juanli; Zhao, Xin; Xu, Shungao; Huang, Xinxiang

2018-01-01

Antisense RNAs from complementary strands of protein coding genes regulate the expression of genes involved in many cellular processes. Using deep sequencing analysis of the Salmonella enterica serovar Typhi ( S. Typhi) transcriptome, a novel antisense RNA encoded on the strand complementary to the rpoH gene was revealed. In this study, the molecular features of this antisense RNA were assessed using northern blotting and rapid amplification of cDNA ends. The 3,508 nt sequence of RNA was identified as the antisense RNA of the rpoH gene and was named ArpH. ArpH was found to attenuate the invasion of HeLa cells by S. Typhi by regulating the expression of SPI-1 genes. In an rpoH mutant strain, the invasive capacity of S. Typhi was increased, whereas overexpression of ArpH positively regulates rpoH mRNA levels. Results of this study suggest that the cis -encoded antisense RNA ArpH is likely to affect the invasive capacity of S. Typhi by regulating the expression of rpoH .

Physiological impact of transposable elements encoding DDE transposases in the environmental adaptation of Streptococcus agalactiae.

PubMed

Fléchard, Maud; Gilot, Philippe

2014-07-01

We have referenced and described Streptococcus agalactiae transposable elements encoding DDE transposases. These elements belonged to nine families of insertion sequences (ISs) and to a family of conjugative transposons (TnGBSs). An overview of the physiological impact of the insertion of all these elements is provided. DDE-transposable elements affect S. agalactiae in a number of aspects of its capability to adapt to various environments and modulate the expression of several virulence genes, the scpB-lmB genomic region and the genes involved in capsule expression and haemolysin transport being the targets of several different mobile elements. The referenced mobile elements modify S. agalactiae behaviour by transferring new gene(s) to its genome, by modifying the expression of neighbouring genes at the integration site or by promoting genomic rearrangements. Transposition of some of these elements occurs in vivo, suggesting that by dynamically regulating some adaptation and/or virulence genes, they improve the ability of S. agalactiae to reach different niches within its host and ensure the 'success' of the infectious process. © 2014 The Authors.

Inventory of high-abundance mRNAs in skeletal muscle of normal men.

PubMed

Welle, S; Bhatt, K; Thornton, C A

1999-05-01

G42875rial analysis of gene expression (SAGE) method was used to generate a catalog of 53,875 short (14 base) expressed sequence tags from polyadenylated RNA obtained from vastus lateralis muscle of healthy young men. Over 12,000 unique tags were detected. The frequency of occurrence of each tag reflects the relative abundance of the corresponding mRNA. The mRNA species that were detected 10 or more times, each comprising >/=0.02% of the mRNA population, accounted for 64% of the mRNA mass but <10% of the total number of mRNA species detected. Almost all of the abundant tags matched mRNA or EST sequences cataloged in GenBank. Mitochondrial transcripts accounted for approximately 20% of the polyadenylated RNA. Transcripts encoding proteins of the myofibrils were the most abundant nuclear-encoded mRNAs. Transcripts encoding ribosomal proteins, and those encoding proteins involved in energy metabolism, also were very abundant. The database can be used as a reference for investigations of alterations in gene expression associated with conditions that influence muscle function, such as muscular dystrophies, aging, and exercise.

Positive selection on human gamete-recognition genes

PubMed Central

Stover, Daryn A.; Guerra, Vanessa; Mozaffari, Sahar V.; Ober, Carole; Mugal, Carina F.; Kaj, Ingemar

2018-01-01

Coevolution of genes that encode interacting proteins expressed on the surfaces of sperm and eggs can lead to variation in reproductive compatibility between mates and reproductive isolation between members of different species. Previous studies in mice and other mammals have focused in particular on evidence for positive or diversifying selection that shapes the evolution of genes that encode sperm-binding proteins expressed in the egg coat or zona pellucida (ZP). By fitting phylogenetic models of codon evolution to data from the 1000 Genomes Project, we identified candidate sites evolving under diversifying selection in the human genes ZP3 and ZP2. We also identified one candidate site under positive selection in C4BPA, which encodes a repetitive protein similar to the mouse protein ZP3R that is expressed in the sperm head and binds to the ZP at fertilization. Results from several additional analyses that applied population genetic models to the same data were consistent with the hypothesis of selection on those candidate sites leading to coevolution of sperm- and egg-expressed genes. By contrast, we found no candidate sites under selection in a fourth gene (ZP1) that encodes an egg coat structural protein not directly involved in sperm binding. Finally, we found that two of the candidate sites (in C4BPA and ZP2) were correlated with variation in family size and birth rate among Hutterite couples, and those two candidate sites were also in linkage disequilibrium in the same Hutterite study population. All of these lines of evidence are consistent with predictions from a previously proposed hypothesis of balancing selection on epistatic interactions between C4BPA and ZP3 at fertilization that lead to the evolution of co-adapted allele pairs. Such patterns also suggest specific molecular traits that may be associated with both natural reproductive variation and clinical infertility. PMID:29340252

Alcoholism and alternative splicing of candidate genes.

PubMed

Sasabe, Toshikazu; Ishiura, Shoichi

2010-04-01

Gene expression studies have shown that expression patterns of several genes have changed during the development of alcoholism. Gene expression is regulated not only at the level of transcription but also through alternative splicing of pre-mRNA. In this review, we discuss some of the evidence suggesting that alternative splicing of candidate genes such as DRD2 (encoding dopamine D2 receptor) may form the basis of the mechanisms underlying the pathophysiology of alcoholism. These reports suggest that aberrant expression of splice variants affects alcohol sensitivities, and alcohol consumption also regulates alternative splicing. Thus, investigations of alternative splicing are essential for understanding the molecular events underlying the development of alcoholism.

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

PubMed Central

Lopez, I; Anthony, R G; Maciver, S K; Jiang, C J; Khan, S; Weeds, A G; Hussey, P J

1996-01-01

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8693008

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites.

PubMed

Lempereur, Laetitia; Larcombe, Stephen D; Durrani, Zeeshan; Karagenc, Tulin; Bilgic, Huseyin Bilgin; Bakirci, Serkan; Hacilarlioglu, Selin; Kinnaird, Jane; Thompson, Joanne; Weir, William; Shiels, Brian

2017-06-05

Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a 'One Health' approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans.

[Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].

PubMed

Yang, Xian-Xian; Zhang, Mei; Yan, Zhao-Wen; Zhang, Ru-Hong; Mu, Xiong-Zheng

2008-01-01

To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.

Epigenetic control of effector gene expression in the plant pathogenic fungus Leptosphaeria maculans.

PubMed

Soyer, Jessica L; El Ghalid, Mennat; Glaser, Nicolas; Ollivier, Bénédicte; Linglin, Juliette; Grandaubert, Jonathan; Balesdent, Marie-Hélène; Connolly, Lanelle R; Freitag, Michael; Rouxel, Thierry; Fudal, Isabelle

2014-03-01

Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions.

Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

PubMed

Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

1997-02-01

Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

Odors regulate Arc expression in neuronal ensembles engaged in odor processing.

PubMed

Guthrie, K; Rayhanabad, J; Kuhl, D; Gall, C

2000-06-26

Synaptic activity is critical to developmental and plastic processes that produce long-term changes in neuronal connectivity and function. Genes expressed by neurons in an activity-dependent fashion are of particular interest since the proteins they encode may mediate neuronal plasticity. One such gene encodes the activity-regulated cytoskeleton-associated protein, Arc. The present study evaluated the effects of odor stimulation on Arc expression in rat olfactory bulb. Arc mRNA was rapidly increased in functionally linked cohorts of neurons topographically activated by odor stimuli. These included neurons surrounding individual glomeruli, mitral cells and transynaptically activated granule cells. Dendritic Arc immunoreactivity was also increased in odor-activated glomeruli. Our results suggest that odor regulation of Arc expression may contribute to activity-dependent structural changes associated with olfactory experience.

Central leptin regulates heart lipid content by selectively increasing PPAR β/δ expression.

PubMed

Mora, Cristina; Pintado, Cristina; Rubio, Blanca; Mazuecos, Lorena; López, Virginia; Fernández, Alejandro; Salamanca, Aurora; Bárcena, Brenda; Fernández-Agulló, Teresa; Arribas, Carmen; Gallardo, Nilda; Andrés, Antonio

2018-01-01

The role of central leptin in regulating the heart from lipid accumulation in lean leptin-sensitive animals has not been fully elucidated. Herein, we investigated the effects of central leptin infusion on the expression of genes involved in cardiac metabolism and its role in the control of myocardial triacylglyceride (TAG) accumulation in adult Wistar rats. Intracerebroventricular (icv) leptin infusion (0.2 µg/day) for 7 days markedly decreased TAG levels in cardiac tissue. Remarkably, the cardiac anti-steatotic effects of central leptin were associated with the selective upregulation of gene and protein expression of peroxisome proliferator-activated receptor β/δ (PPARβ/δ, encoded by Pparb/d ) and their target genes, adipose triglyceride lipase (encoded by Pnpla2 , herefater referred to as Atgl ), hormone sensitive lipase (encoded by Lipe , herefater referred to as Hsl ), pyruvate dehydrogenase kinase 4 ( Pdk4 ) and acyl CoA oxidase 1 ( Acox1 ), involved in myocardial intracellular lipolysis and mitochondrial/peroxisomal fatty acid utilization. Besides, central leptin decreased the expression of stearoyl-CoA deaturase 1 ( Scd1 ) and diacylglycerol acyltransferase 1 ( Dgat1 ) involved in TAG synthesis and increased the CPT-1 independent palmitate oxidation, as an index of peroxisomal β-oxidation. Finally, the pharmacological inhibition of PPARβ/δ decreased the effects on gene expression and cardiac TAG content induced by leptin. These results indicate that leptin, acting at central level, regulates selectively the cardiac expression of PPARβ/δ, contributing in this way to regulate the cardiac TAG accumulation in rats, independently of its effects on body weight. © 2018 Society for Endocrinology.

Stable zymomonas mobilis xylose and arabinose fermenting strains

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Taipei, TW

2008-04-08

The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided.The transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

PubMed Central

Weigel, B J; Burgett, S G; Chen, V J; Skatrud, P L; Frolik, C A; Queener, S W; Ingolia, T D

1988-01-01

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution. Images PMID:3045077

Functional relevance of three proopiomelanocortin (POMC) genes in darkening camouflage, blind-side hypermelanosis, and appetite of Paralichthys olivaceus.

PubMed

Kang, Duk-Young; Kim, Hyo-Chan

2015-01-01

To determine whether proopiomelanocortin (POMC) genes are involved in darkening color camouflage, blind-side hypermelanosis, and appetite in flatfish, we isolated and cloned three POMC genes from the pituitary of the olive flounder (Paralichthys olivaceus) and compared their amino acid (aa) structures to those of POMC genes from other animals. Next, we examined the relationship of these pituitary POMC genes to camouflage color change, blind-side hypermelanosis, and appetite by quantifying mRNA expression. Olive flounder (of)-POMC1, 2, and 3 cDNAs consisted of 648-bp, 582-bp, and 693-bp open reading frames (ORF) encoding 216 aa, 194 aa, and 231 aa residues, respectively. Structurally, the three of-POMC cDNAs consisted of seven peptides (signal peptide, N-POMC, α-MSH, CLIP, N-β-LPH, β-MSH and β-END [or END-like peptide]) that are similar to those of other fish POMC cDNAs. α-MSH encoded a protein composed of 13 aa and β-MSH encoded a protein composed of 17 aa. The three POMC genes were predominantly expressed in the pituitary gland, but they were also expressed in a variety of tissues, including brain, eye, kidney, heart, testis, and skin. of-POMC2 exhibited the highest expression, while of-POMC3 displayed the lowest expression. The relative levels of of-POMC1 and 3 mRNAs were not influenced by background color and feeding (or fasting), but the relative level of of-POMC2 mRNA significantly increased in response to a dark background and fasting. The relative levels of of-POMC1 and 2 mRNAs were significantly higher in hypermelanic fish; however, we did not determine a direct anorexigenic or orexigenic relationship for the three POMC genes. These results indicate that pituitary POMC genes are related to darkening color change and the differentiation of pigment cells, but they are not directly related to appetite. Copyright © 2014 Elsevier Inc. All rights reserved.

β-Lactamase Genes of the Penicillin-Susceptible Bacillus anthracis Sterne Strain

PubMed Central

Chen, Yahua; Succi, Janice; Tenover, Fred C.; Koehler, Theresa M.

2003-01-01

Susceptibility to penicillin and other β-lactam-containing compounds is a common trait of Bacillus anthracis. β-lactam agents, particularly penicillin, have been used worldwide to treat anthrax in humans. Nonetheless, surveys of clinical and soil-derived strains reveal penicillin G resistance in 2 to 16% of isolates tested. Bacterial resistance to β-lactam agents is often mediated by production of one or more types of β-lactamases that hydrolyze the β-lactam ring, inactivating the antimicrobial agent. Here, we report the presence of two β-lactamase (bla) genes in the penicillin-susceptible Sterne strain of B. anthracis. We identified bla1 by functional cloning with Escherichia coli. bla1 is a 927-nucleotide (nt) gene predicted to encode a protein with 93.8% identity to the type I β-lactamase gene of Bacillus cereus. A second gene, bla2, was identified by searching the unfinished B. anthracis chromosome sequence database of The Institute for Genome Research for open reading frames (ORFs) predicted to encode β-lactamases. We found a partial ORF predicted to encode a protein with significant similarity to the carboxy-terminal end of the type II β-lactamase of B. cereus. DNA adjacent to the 5′ end of the partial ORF was cloned using inverse PCR. bla2 is a 768-nt gene predicted to encode a protein with 92% identity to the B. cereus type II enzyme. The bla1 and bla2 genes confer ampicillin resistance to E. coli and Bacillus subtilis when cloned individually in these species. The MICs of various antimicrobial agents for the E. coli clones indicate that the two β-lactamase genes confer different susceptibility profiles to E. coli; bla1 is a penicillinase, while bla2 appears to be a cephalosporinase. The β-galactosidase activities of B. cereus group species harboring bla promoter-lacZ transcriptional fusions indicate that bla1 is poorly transcribed in B. anthracis, B. cereus, and B. thuringiensis. The bla2 gene is strongly expressed in B. cereus and B. thuringiensis and weakly expressed in B. anthracis. Taken together, these data indicate that the bla1 and bla2 genes of the B. anthracis Sterne strain encode functional β-lactamases of different types, but gene expression is usually not sufficient to confer resistance to β-lactam agents. PMID:12533457

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai

PubMed Central

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-01-01

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%–3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones. PMID:26593905

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai.

PubMed

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-11-18

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%-3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones.

The bromodomain protein LEX-1 acts with TAM-1 to modulate gene expression in C. elegans.

PubMed

Tseng, Rong-Jeng; Armstrong, Kristin R; Wang, Xiaodong; Chamberlin, Helen M

2007-11-01

In many organisms, repetitive DNA serves as a trigger for gene silencing. However, some gene expression is observed from repetitive genomic regions such as heterochromatin, suggesting mechanisms exist to modulate the silencing effects. From a genetic screen in C. elegans, we have identified mutations in two genes important for expression of repetitive sequences: lex-1 and tam-1. Here we show that lex-1 encodes a protein containing an ATPase domain and a bromodomain. LEX-1 is similar to the yeast Yta7 protein, which maintains boundaries between silenced and active chromatin. tam-1 has previously been shown to encode a RING finger/B-box protein that modulates gene expression from repetitive DNA. We find that lex-1, like tam-1, acts as a class B synthetic multivulva (synMuv) gene. However, since lex-1 and tam-1 mutants have normal P granule localization, it suggests they act through a mechanism distinct from other class B synMuvs. We observe intragenic (interallelic) complementation with lex-1 and a genetic interaction between lex-1 and tam-1, data consistent with the idea that the gene products function in the same biological process, perhaps as part of a protein complex. We propose that LEX-1 and TAM-1 function together to influence chromatin structure and to promote expression from repetitive sequences.

Insights into the transcriptional and translational mechanisms of linear organellar chromosomes in the box jellyfish Alatina alata (Cnidaria: Medusozoa: Cubozoa).

PubMed

Kayal, Ehsan; Bentlage, Bastian; Collins, Allen G

2016-09-01

In most animals, the mitochondrial genome is characterized by its small size, organization into a single circular molecule, and a relative conservation of the number of encoded genes. In box jellyfish (Cubozoa, Cnidaria), the mitochondrial genome is organized into 8 linear mito-chromosomes harboring between one and 4 genes each, including 2 extra protein-coding genes: mt-polB and orf314. Such an organization challenges the traditional view of mitochondrial DNA (mtDNA) expression in animals. In this study, we investigate the pattern of mitochondrial gene expression in the box jellyfish Alatina alata, as well as several key nuclear-encoded molecular pathways involved in the processing of mitochondrial gene transcription. Read coverage of DNA-seq data is relatively uniform for all 8 mito-chromosomes, suggesting that each mito-chromosome is present in equimolar proportion in the mitochondrion. Comparison of DNA and RNA-seq based assemblies indicates that mito-chromosomes are transcribed into individual transcripts in which the beginning and ending are highly conserved. Expression levels for mt-polB and orf314 are similar to those of other mitochondrial-encoded genes, which provides further evidence for them having functional roles in the mitochondrion. Survey of the transcriptome suggests recognition of the mitochondrial tRNA-Met by the cytoplasmic aminoacyl-tRNA synthetase counterpart and C-to-U editing of the cytoplasmic tRNA-Trp after import into the mitochondrion. Moreover, several mitochondrial ribosomal proteins appear to be lost. This study represents the first survey of mitochondrial gene expression of the linear multi-chromosomal mtDNA in box jellyfish (Cubozoa). Future exploration of small RNAs and the proteome of the mitochondrion will test the hypotheses presented herein.

A novel gene encoding a TIG multiple domain protein is a positional candidate for autosomal recessive polycystic kidney disease.

PubMed

Xiong, Huaqi; Chen, Yongxiong; Yi, Yajun; Tsuchiya, Karen; Moeckel, Gilbert; Cheung, Joseph; Liang, Dan; Tham, Kyi; Xu, Xiaohu; Chen, Xing-Zhen; Pei, York; Zhao, Zhizhuang Jeo; Wu, Guanqing

2002-07-01

Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-p12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. We recently identified a novel gene in this genetic interval from kidney cDNA, using cloning strategies. The gene PKHD1 (PKHD1-tentative) encodes a novel 3396-amino-acid protein with no apparent homology with any known proteins. We named its gene product "tigmin" because it contains multiple TIG domains, which usually are seen in proteins containing immunoglobulin-like folds. PKHD1 encodes an 11.6-kb transcript and is composed of 61 exons spanning an approximately 365-kb genomic region on chromosome 6p12-p11.2 adjacent to the marker D6S1714. Northern blot analyses demonstrated that the gene has discrete bands with one peak signal at approximately 11 kb, indicating that PKHD1 is likely to have multiple alternative transcripts. PKHD1 is highly expressed in adult and infant kidneys and weakly expressed in liver in northern blot analysis. This expression pattern parallels the tissue involvement observed in ARPKD. In situ hybridization analysis further revealed that the expression of PKHD1 in the kidney is mainly localized to the epithelial cells of the collecting duct, the specific tubular segment involved in cyst formation in ARPKD. These features of PKHD1 make it a strong positional candidate gene for ARPKD.

LEFPS1, a Tomato Farnesyl Pyrophosphate Gene Highly Expressed during Early Fruit Development1

PubMed Central

Gaffe, Joel; Bru, Jean-Philippe; Causse, Mathilde; Vidal, Alain; Stamitti-Bert, Linda; Carde, Jean-Pierre; Gallusci, Philippe

2000-01-01

Farnesyl pyrophosphate synthase (FPS) catalyzes the synthesis of farnesyl pyrophosphate, a key intermediate in sterol and sesquiterpene biosynthesis. Using a polymerase chain reaction-based approach, we have characterized LeFPS1, a tomato (Lycoperscion esculentum cv Wva 106) fruit cDNA, which encodes a functional FPS. We demonstrate that tomato FPSs are encoded by a small multigenic family with genes located on chromosomes 10 and 12. Consistent with farnesyl pyrophosphate requirement in sterol biosynthesis, FPS genes are ubiquitously expressed in tomato plants. Using an LeFPS1 specific probe, we show that the corresponding gene can account for most of FPS mRNA in most plant organs, but not during young seedling development, indicating a differential regulation of FPS genes in tomato. FPS gene expression is also under strict developmental control: FPS mRNA was mainly abundant in young organs and decreased as organs matured with the exception of fruits that presented a biphasic accumulation pattern. In this latter case in situ hybridization studies have shown that FPS mRNA is similarly abundant in all tissues of young fruit. Taken together our results suggest that several FPS isoforms are involved in tomato farnesyl pyrophosphate metabolism and that FPS genes are mostly expressed in relation to cell division and enlargement. PMID:10938353

SOA genes encode proteins controlling lipase expression in response to triacylglycerol utilization in the yeast Yarrowia lipolytica.

PubMed

Desfougères, Thomas; Haddouche, Ramdane; Fudalej, Franck; Neuvéglise, Cécile; Nicaud, Jean-Marc

2010-02-01

The oleaginous yeast Yarrowia lipolytica efficiently metabolizes hydrophobic substrates such as alkanes, fatty acids or triacylglycerol. This yeast has been identified in oil-polluted water and in lipid-rich food. The enzymes involved in lipid breakdown, for use as a carbon source, are known, but the molecular mechanisms controlling the expression of the genes encoding these enzymes are still poorly understood. The study of mRNAs obtained from cells grown on oleic acid identified a new group of genes called SOA genes (specific for oleic acid). SOA1 and SOA2 are two small genes coding for proteins with no known homologs. Single- and double-disrupted strains were constructed. Wild-type and mutant strains were grown on dextrose, oleic acid and triacylglycerols. The double mutant presents a clear phenotype consisting of a growth defect on tributyrin and triolein, but not on dextrose or oleic acid media. Lipase activity was 50-fold lower in this mutant than in the wild-type strain. The impact of SOA deletion on the expression of the main extracellular lipase gene (LIP2) was monitored using a LIP2-beta-galactosidase promoter fusion protein. These data suggest that Soa proteins are components of a molecular mechanism controlling lipase gene expression in response to extracellular triacylglycerol.

Expression of mouse Tla region class I genes in tissues enriched for gamma delta cells.

PubMed

Eghtesady, P; Brorson, K A; Cheroutre, H; Tigelaar, R E; Hood, L; Kronenberg, M

1992-01-01

The Tla region of the BALB/c mouse major histocompatibility complex contains at least 20 class I genes. The function of the products of these genes is unknown, but recent evidence demonstrates that some Tla region gene products could be involved in presentation of antigens to gamma delta T cells. We have generated a set of polymerase chain reaction (PCR) oligonucleotide primers and hybridization probes that permit us to specifically amplify and detect expression of 11 of the 20 BALB/c Tla region genes. cDNA prepared from 12 adult and fetal tissues and from seven cell lines was analyzed. In some cases, northern blot analysis or staining with monoclonal antibodies specific for the Tla-encoded thymus leukemia (TL) antigen were used to confirm the expression pattern of several of the genes as determined by PCR. Some Tla region genes, such as T24d and the members of the T10d/T22d gene pair, are expressed in a wide variety of tissues in a manner similar to the class I transplantation antigens. The members of the TL antigen encoding gene pair, T3d/T18d, are expressed in only a limited number of organs, including several sites enriched for gamma delta T cells. Other Tla region genes, including T1d, T2d, T16d, and T17d, are transcriptionally silent and transcripts from the T8d/T20d gene pair do not undergo proper splicing. In general, sites that contain gamma delta T lymphocytes have Tla region transcripts. The newly identified pattern of expression of the genes analyzed in sites containing gamma delta T cells further extends the list of potential candidates for antigen presentation to gamma delta T cells.

Functional Analysis of Mating Type Genes and Transcriptome Analysis during Fruiting Body Development of Botrytis cinerea

PubMed Central

2018-01-01

ABSTRACT Botrytis cinerea is a plant-pathogenic fungus producing apothecia as sexual fruiting bodies. To study the function of mating type (MAT) genes, single-gene deletion mutants were generated in both genes of the MAT1-1 locus and both genes of the MAT1-2 locus. Deletion mutants in two MAT genes were entirely sterile, while mutants in the other two MAT genes were able to develop stipes but never formed an apothecial disk. Little was known about the reprogramming of gene expression during apothecium development. We analyzed transcriptomes of sclerotia, three stages of apothecium development (primordia, stipes, and apothecial disks), and ascospores by RNA sequencing. Ten secondary metabolite gene clusters were upregulated at the onset of sexual development and downregulated in ascospores released from apothecia. Notably, more than 3,900 genes were differentially expressed in ascospores compared to mature apothecial disks. Among the genes that were upregulated in ascospores were numerous genes encoding virulence factors, which reveals that ascospores are transcriptionally primed for infection prior to their arrival on a host plant. Strikingly, the massive transcriptional changes at the initiation and completion of the sexual cycle often affected clusters of genes, rather than randomly dispersed genes. Thirty-five clusters of genes were jointly upregulated during the onset of sexual reproduction, while 99 clusters of genes (comprising >900 genes) were jointly downregulated in ascospores. These transcriptional changes coincided with changes in expression of genes encoding enzymes participating in chromatin organization, hinting at the occurrence of massive epigenetic regulation of gene expression during sexual reproduction. PMID:29440571

Expression of the insect metalloproteinase inhibitor IMPI in the fat body of Galleria mellonella exposed to infection with Beauveria bassiana.

PubMed

Vertyporokh, Lidiia; Wojda, Iwona

2017-01-01

The inducible metalloproteinase inhibitor (IMPI) discovered in Galleria mellonella is currently the only specific inhibitor of metalloproteinases found in animals. Its role is to inhibit the activity of metalloproteinases secreted by pathogenic organisms as virulence factors to degrade immune-relevant polypeptides of the infected host. This is a good example of an evolutionary arms race between the insect hosts and their natural pathogens. In this report, we analyze the expression of a gene encoding an inducible metalloproteinase inhibitor (IMPI) in fat bodies of the greater wax moth larvae Galleria mellonella infected with an entomopathogenic fungus Beauveria bassiana. We have used a natural infection, i.e. covering larval integument with fungal aerospores, as well as injection of fungal blastospores directly into the larval hemocel. We compare the expression of IMPI with the expression of genes encoding proteins with fungicidal activity, gallerimycin and galiomycin, whose expression reflects the stimulation of Galleria mellonella defense mechanisms. Also, gene expression is analyzed in the light of survival of animals after spore injection.

Saponin Biosynthesis in Saponaria vaccaria. cDNAs Encoding β-Amyrin Synthase and a Triterpene Carboxylic Acid Glucosyltransferase1[OA

PubMed Central

Meesapyodsuk, Dauenpen; Balsevich, John; Reed, Darwin W.; Covello, Patrick S.

2007-01-01

Saponaria vaccaria (Caryophyllaceae), a soapwort, known in western Canada as cowcockle, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). To improve our understanding of the biosynthesis of these saponins, a combined polymerase chain reaction and expressed sequence tag approach was taken to identify the genes involved. A cDNA encoding a β-amyrin synthase (SvBS) was isolated by reverse transcription-polymerase chain reaction and characterized by expression in yeast (Saccharomyces cerevisiae). The SvBS gene is predominantly expressed in leaves. A S. vaccaria developing seed expressed sequence tag collection was developed and used for the isolation of a full-length cDNA bearing sequence similarity to ester-forming glycosyltransferases. The gene product of the cDNA, classified as UGT74M1, was expressed in Escherichia coli, purified, and identified as a triterpene carboxylic acid glucosyltransferase. UGT74M1 is expressed in roots and leaves and appears to be involved in monodesmoside biosynthesis in S. vaccaria. PMID:17172290

Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

PubMed

Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

2012-04-15

Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall thickening after cessation of phellogen cell division. Published by Elsevier GmbH.

[Characteristics of extracellular invertase of Saccharomyces cerevisiae in Heterologous expression of the suc2 gene in Solarium Tuberosum plants].

PubMed

Deriabin, A N; Berdichevets, I N; Burakhanova, E A; Trunova, T I

2014-01-01

Some properties and activity of extracellular invertase in the Saccharomyces cerevisiae yeasts encoded by the suc2 gene in heterologous expression were described. It was shown that the target suc2 gene is actively expressed in the genome of the transformed potato plants and S. cerevisiae invertase synthesized by this gene is transported into the apoplast due to the signal peptide of the proteinase II inhibitor. This enzyme is present in the apoplast in a soluble form and absorbed into the cell wall.

Influence of human lactoferrin expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco.

PubMed

Kumar, Vinay; Gill, Tejpal; Grover, Sunita; Ahuja, Paramvir Singh; Yadav, Sudesh Kumar

2013-02-01

This study was aimed at to check the influence of human lactoferrin (hLF) expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco. Transgenic tobacco expressing hLF cDNA under the control of a CaMV 35S promoter was produced. The iron content as well as chlorophyll content of transgenic tobacco was lower compared to mock and untransformed wild plants. Interestingly, hLF transgenic tobacco showed higher level of transcript expression for genes related to iron content regulation like iron transporter and metal transporter. While expression of genes related to iron storage such as ferritin 1 and ferritin 2 was downregulated. The transcript expression of genes encoding antioxidant enzymes such as glutathione reductase, glutathione-S-transferase, ascorbate peroxidase, and catalase was downregulated in hLF transgenic tobacco compared to controls. Further, the transcript expression of two important genes encoding dihydroflavonol reductase (DFR) and phenylalanine ammonia lyase regulatory enzymes of flavonoid biosynthesis pathway was analyzed. The expression of DFR was found to be downregulated, while PAL expression was upregulated in hLF transgenic tobacco compared to mock and untransformed wild plant. Total phenolics, flavonoids, and proanthocyanidins contents were found to be higher in hLF transgenic tobacco than the mock and untransformed wild plant. Results suggest that hLF expression in transgenic tobacco leads to iron deficiency, downregulation of antioxidant enzymes, and increase in total flavonoids.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

PubMed

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.

Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene.

PubMed

Shimizu, Masami; Kimura, Tetsuya; Koyama, Takayoshi; Suzuki, Katsuhisa; Ogawa, Naoto; Miyashita, Kiyotaka; Sakka, Kazuo; Ohmiya, Kunio

2002-08-01

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.

Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival.

PubMed

Echenique-Rivera, Hebert; Muzzi, Alessandro; Del Tordello, Elena; Seib, Kate L; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

2011-05-01

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism.

Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

PubMed Central

Del Tordello, Elena; Seib, Kate L.; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

2011-01-01

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism. PMID:21589640

Bacterial infection as assessed by in vivo gene expression

PubMed Central

Heithoff, Douglas M.; Conner, Christopher P.; Hanna, Philip C.; Julio, Steven M.; Hentschel, Ute; Mahan, Michael J.

1997-01-01

In vivo expression technology (IVET) has been used to identify >100 Salmonella typhimurium genes that are specifically expressed during infection of BALB/c mice and/or murine cultured macrophages. Induction of these genes is shown to be required for survival in the animal under conditions of the IVET selection. One class of in vivo induced (ivi) genes, iviVI-A and iviVI-B, constitute an operon that resides in a region of the Salmonella genome with low G+C content and presumably has been acquired by horizontal transfer. These ivi genes encode predicted proteins that are similar to adhesins and invasins from prokaryotic and eukaryotic pathogens (Escherichia coli [tia], Plasmodium falciparum [PfEMP1]) and have coopted the PhoPQ regulatory circuitry of Salmonella virulence genes. Examination of the in vivo induction profile indicates (i) many ivi genes encode regulatory functions (e.g., phoPQ and pmrAB) that serve to enhance the sensitivity and amplitude of virulence gene expression (e.g., spvB); (ii) the biochemical function of many metabolic genes may not represent their sole contribution to virulence; (iii) the host ecology can be inferred from the biochemical functions of ivi genes; and (iv) nutrient limitation plays a dual signaling role in pathogenesis: to induce metabolic functions that complement host nutritional deficiencies and to induce virulence functions required for immediate survival and spread to subsequent host sites. PMID:9023360

Differential expression of copper-zinc superoxide dismutase gene of Polygonum sibiricum leaves, stems and underground stems, subjected to high-salt stress.

PubMed

Qu, Chun-Pu; Xu, Zhi-Ru; Liu, Guan-Jun; Liu, Chun; Li, Yang; Wei, Zhi-Gang; Liu, Gui-Feng

2010-01-01

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5' untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3' untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1-16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO(3). The different mRNA levels' expression of PS-CuZnSOD show the gene's different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.

Expression of an Arabidopsis sodium/proton antiporter gene (AtNHX1)in peanut to improve salt tolerance

USDA-ARS?s Scientific Manuscript database

Salinity is a major environmental stress that affects agricultural productivity worldwide. One approach to improving salt tolerance in crops is through high expression of the Arabidopsis gene AtNHX1, which encodes a vacuolar sodium/proton antiporter that sequesters excess sodium ion into the large i...

Extensive diversification of IgD-, IgY-, and truncated IgY(δFc)-encoding genes in the red-eared turtle (Trachemys scripta elegans).

PubMed

Li, Lingxiao; Wang, Tao; Sun, Yi; Cheng, Gang; Yang, Hui; Wei, Zhiguo; Wang, Ping; Hu, Xiaoxiang; Ren, Liming; Meng, Qingyong; Zhang, Ran; Guo, Ying; Hammarström, Lennart; Li, Ning; Zhao, Yaofeng

2012-10-15

IgY(ΔFc), containing only CH1 and CH2 domains, is expressed in the serum of some birds and reptiles, such as ducks and turtles. The duck IgY(ΔFc) is produced by the same υ gene that expresses the intact IgY form (CH1-4) using different transcriptional termination sites. In this study, we show that intact IgY and IgY(ΔFc) are encoded by distinct genes in the red-eared turtle (Trachemys scripta elegans). At least eight IgY and five IgY(ΔFc) transcripts were found in a single turtle. Together with Southern blotting, our data suggest that multiple genes encoding both IgY forms are present in the turtle genome. Both of the IgY forms were detected in the serum using rabbit polyclonal Abs. In addition, we show that multiple copies of the turtle δ gene are present in the genome and that alternative splicing is extensively involved in the generation of both the secretory and membrane-bound forms of the IgD H chain transcripts. Although a single μ gene was identified, the α gene was not identified in this species.

Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

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

Altabella, T.; Chrispeels, M.J.

Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{submore » r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.« less

Streptomyces coelicolor encodes a urate-responsive transcriptional regulator with homology to PecS from plant pathogens.

PubMed

Huang, Hao; Mackel, Brian J; Grove, Anne

2013-11-01

Many transcriptional regulators control gene activity by responding to specific ligands. Members of the multiple-antibiotic resistance regulator (MarR) family of transcriptional regulators feature prominently in this regard, and they frequently function as repressors in the absence of their cognate ligands. Plant pathogens such as Dickeya dadantii encode a MarR homolog named PecS that controls expression of a gene encoding the efflux pump PecM in addition to other virulence genes. We report here that the soil bacterium Streptomyces coelicolor also encodes a PecS homolog (SCO2647) that regulates a pecM gene (SCO2646). S. coelicolor PecS, which exists as a homodimer, binds the intergenic region between pecS and pecM genes with high affinity. Several potential PecS binding sites were found in this intergenic region. The binding of PecS to its target DNA can be efficiently attenuated by the ligand urate, which also quenches the intrinsic fluorescence of PecS, indicating a direct interaction between urate and PecS. In vivo measurement of gene expression showed that activity of pecS and pecM genes is significantly elevated after exposure of S. coelicolor cultures to urate. These results indicate that S. coelicolor PecS responds to the ligand urate by attenuated DNA binding in vitro and upregulation of gene activity in vivo. Since production of urate is associated with generation of reactive oxygen species by xanthine dehydrogenase, we propose that PecS functions under conditions of oxidative stress.

Streptomyces coelicolor Encodes a Urate-Responsive Transcriptional Regulator with Homology to PecS from Plant Pathogens

PubMed Central

Huang, Hao; Mackel, Brian J.

2013-01-01

Many transcriptional regulators control gene activity by responding to specific ligands. Members of the multiple-antibiotic resistance regulator (MarR) family of transcriptional regulators feature prominently in this regard, and they frequently function as repressors in the absence of their cognate ligands. Plant pathogens such as Dickeya dadantii encode a MarR homolog named PecS that controls expression of a gene encoding the efflux pump PecM in addition to other virulence genes. We report here that the soil bacterium Streptomyces coelicolor also encodes a PecS homolog (SCO2647) that regulates a pecM gene (SCO2646). S. coelicolor PecS, which exists as a homodimer, binds the intergenic region between pecS and pecM genes with high affinity. Several potential PecS binding sites were found in this intergenic region. The binding of PecS to its target DNA can be efficiently attenuated by the ligand urate, which also quenches the intrinsic fluorescence of PecS, indicating a direct interaction between urate and PecS. In vivo measurement of gene expression showed that activity of pecS and pecM genes is significantly elevated after exposure of S. coelicolor cultures to urate. These results indicate that S. coelicolor PecS responds to the ligand urate by attenuated DNA binding in vitro and upregulation of gene activity in vivo. Since production of urate is associated with generation of reactive oxygen species by xanthine dehydrogenase, we propose that PecS functions under conditions of oxidative stress. PMID:23995633

Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening.

PubMed

Schallmey, Marcus; Ly, Anh; Wang, Chunxia; Meglei, Gabriela; Voget, Sonja; Streit, Wolfgang R; Driscoll, Brian T; Charles, Trevor C

2011-08-01

We previously reported the construction of metagenomic libraries in the IncP cosmid vector pRK7813, enabling heterologous expression of these broad-host-range libraries in multiple bacterial hosts. Expressing these libraries in Sinorhizobium meliloti, we have successfully complemented associated phenotypes of polyhydroxyalkanoate synthesis mutants. DNA sequence analysis of three clones indicates that the complementing genes are homologous to, but substantially different from, known polyhydroxyalkanaote synthase-encoding genes. Thus we have demonstrated the ability to isolate diverse genes for polyhydroxyalkanaote synthesis by functional complementation of defined mutants. Such genes might be of use in the engineering of more efficient systems for the industrial production of bioplastics. The use of functional complementation will also provide a vehicle to probe the genetics of polyhydroxyalkanaote metabolism and its relation to carbon availability in complex microbial assemblages. 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The GMD1 and GMD2 genes of Arabidopsis encode isoforms of GDP-D-mannose 4,6-dehydratase with cell type-specific expression patterns.

PubMed

Bonin, Christopher P; Freshour, Glenn; Hahn, Michael G; Vanzin, Gary F; Reiter, Wolf-Dieter

2003-06-01

l-Fucose (l-Fuc) is a monosaccharide constituent of plant cell wall polysaccharides and glycoproteins. The committing step in the de novo synthesis of l-Fuc is catalyzed by GDP-d-mannose 4,6-dehydratase, which, in Arabidopsis, is encoded by the GMD1 and GMD2 (MUR1) genes. To determine the functional significance of this genetic redundancy, the expression patterns of both genes were investigated via promoter-beta-glucuronidase fusions and immunolocalization of a Fuc-containing epitope. GMD2 is expressed in most cell types of the root, with the notable exception of the root tip where strong expression of GMD1 is observed. Within shoot organs, GMD1::GUS expression is confined to stipules and pollen grains leading to fucosylation of the walls of these cell types in the mur1 mutant. These results suggest that GMD2 represents the major housekeeping gene for the de novo synthesis of GDP-l-Fuc, whereas GMD1 expression is limited to a number of specialized cell types. We conclude that the synthesis of GDP-l-Fuc is controlled in a cell-autonomous manner by differential expression of two isoforms of the same enzyme.

CD30 expression defines a novel subgroup of diffuse large B-cell lymphoma with favorable prognosis and distinct gene expression signature: a report from the International DLBCL Rituximab-CHOP Consortium Program Study

PubMed Central

Hu, Shimin; Xu-Monette, Zijun Y.; Balasubramanyam, Aarthi; Manyam, Ganiraju C.; Visco, Carlo; Tzankov, Alexander; Liu, Wei-min; Miranda, Roberto N.; Zhang, Li; Montes-Moreno, Santiago; Dybkær, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W. L.; Han van Krieken, J.; Huang, Qin; Huh, Jooryung; Ai, Weiyun; Ponzoni, Maurilio; Ferreri, Andrés J. M.; Zhao, Xiaoying; Winter, Jane N.; Zhang, Mingzhi; Li, Ling; Møller, Michael B.; Piris, Miguel A.; Li, Yong; Go, Ronald S.; Wu, Lin; Medeiros, L. Jeffrey; Young, Ken H.

2013-01-01

CD30, originally identified as a cell-surface marker of Reed-Sternberg and Hodgkin cells of classical Hodgkin lymphoma, is also expressed by several types of non-Hodgkin lymphoma, including a subset of diffuse large B-cell lymphoma (DLBCL). However, the prognostic and biological importance of CD30 expression in DLBCL is unknown. Here we report that CD30 expression is a favorable prognostic factor in a cohort of 903 de novo DLBCL patients. CD30 was expressed in ∼14% of DLBCL patients. Patients with CD30+ DLBCL had superior 5-year overall survival (CD30+, 79% vs CD30–, 59%; P = .001) and progression-free survival (P = .003). The favorable outcome of CD30 expression was maintained in both the germinal center B-cell and activated B-cell subtypes. Gene expression profiling revealed the upregulation of genes encoding negative regulators of nuclear factor κB activation and lymphocyte survival, and downregulation of genes encoding B-cell receptor signaling and proliferation, as well as prominent cytokine and stromal signatures in CD30+ DLBCL patients, suggesting a distinct molecular basis for its favorable outcome. Given the superior prognostic value, unique gene expression signature, and significant value of CD30 as a therapeutic target for brentuximab vedotin in ongoing successful clinical trials, it seems appropriate to consider CD30+ DLBCL as a distinct subgroup of DLBCL. PMID:23343832

Expression of genes encoding IGFBPs, SNARK, CD36, and PECAM1 in the liver of mice treated with chromium disilicide and titanium nitride nanoparticles.

PubMed

Minchenko, Dmytro O; Tsymbal, D O; Yavorovsky, O P; Solokha, N V; Minchenko, O H

2017-04-25

The aim of the present study was to examine the effect of chromium disilicide and titanium nitride nanoparticles on the expression level of genes encoding important regulatory factors (IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, SNARK/NUAK2, CD36, and PECAM1/CD31) in mouse liver for evaluation of possible toxic effects of these nanoparticles. Male mice received 20 mg chromium disilicide nanoparticles (45 nm) and titanium nitride nanoparticles (20 nm) with food every working day for 2 months. The expression of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, SNARK, CD36, and PECAM1 genes in mouse liver was studied by quantitative polymerase chain reaction. Treatment of mice with chromium disilicide nanoparticles led to down-regulation of the expression of IGFBP2, IGFBP5, PECAM1, and SNARK genes in the liver in comparison with control mice, with more prominent changes for SNARK gene. At the same time, the expression of IGFBP3 and CD36 genes was increased in mouse liver upon treatment with chromium disilicide nanoparticles. We have also shown that treatment with titanium nitride nanoparticles resulted in down-regulation of the expression of IGFBP2 and SNARK genes in the liver with more prominent changes for SNARK gene. At the same time, the expression of IGFBP3, IGFBP4, and CD36 genes was increased in the liver of mice treated with titanium nitride nanoparticles. Furthermore, the effect of chromium disilicide nanoparticles on IGFBP2 and CD36 genes expression was significantly stronger as compared to titanium nitride nanoparticles. The results of this study demonstrate that chromium disilicide and titanium nitride nanoparticles have variable effects on the expression of IGFBP2, IGFBP3, IGFBP4, IGFBP5, SNARK, CD36, and PECAM1 genes in mouse liver, which may reflect the genotoxic activities of the studied nanoparticles.

Identification of drought-response genes and a study of their expression during sucrose accumulation and water deficit in sugarcane culms.

PubMed

Iskandar, Hayati M; Casu, Rosanne E; Fletcher, Andrew T; Schmidt, Susanne; Xu, Jingsheng; Maclean, Donald J; Manners, John M; Bonnett, Graham D

2011-01-13

The ability of sugarcane to accumulate high concentrations of sucrose in its culm requires adaptation to maintain cellular function under the high solute load. We have investigated the expression of 51 genes implicated in abiotic stress to determine their expression in the context of sucrose accumulation by studying mature and immature culm internodes of a high sucrose accumulating sugarcane cultivar. Using a sub-set of eight genes, expression was examined in mature internode tissues of sugarcane cultivars as well as ancestral and more widely related species with a range of sucrose contents. Expression of these genes was also analysed in internode tissue from a high sucrose cultivar undergoing water deficit stress to compare effects of sucrose accumulation and water deficit. A sub-set of stress-related genes that are potentially associated with sucrose accumulation in sugarcane culms was identified through correlation analysis, and these included genes encoding enzymes involved in amino acid metabolism, a sugar transporter and a transcription factor. Subsequent analysis of the expression of these stress-response genes in sugarcane plants that were under water deficit stress revealed a different transcriptional profile to that which correlated with sucrose accumulation. For example, genes with homology to late embryogenesis abundant-related proteins and dehydrin were strongly induced under water deficit but this did not correlate with sucrose content. The expression of genes encoding proline biosynthesis was associated with both sucrose accumulation and water deficit, but amino acid analysis indicated that proline was negatively correlated with sucrose concentration, and whilst total amino acid concentrations increased about seven-fold under water deficit, the relatively low concentration of proline suggested that it had no osmoprotectant role in sugarcane culms. The results show that while there was a change in stress-related gene expression associated with sucrose accumulation, different mechanisms are responding to the stress induced by water deficit, because different genes had altered expression under water deficit.

Identification of drought-response genes and a study of their expression during sucrose accumulation and water deficit in sugarcane culms

PubMed Central

2011-01-01

Background The ability of sugarcane to accumulate high concentrations of sucrose in its culm requires adaptation to maintain cellular function under the high solute load. We have investigated the expression of 51 genes implicated in abiotic stress to determine their expression in the context of sucrose accumulation by studying mature and immature culm internodes of a high sucrose accumulating sugarcane cultivar. Using a sub-set of eight genes, expression was examined in mature internode tissues of sugarcane cultivars as well as ancestral and more widely related species with a range of sucrose contents. Expression of these genes was also analysed in internode tissue from a high sucrose cultivar undergoing water deficit stress to compare effects of sucrose accumulation and water deficit. Results A sub-set of stress-related genes that are potentially associated with sucrose accumulation in sugarcane culms was identified through correlation analysis, and these included genes encoding enzymes involved in amino acid metabolism, a sugar transporter and a transcription factor. Subsequent analysis of the expression of these stress-response genes in sugarcane plants that were under water deficit stress revealed a different transcriptional profile to that which correlated with sucrose accumulation. For example, genes with homology to late embryogenesis abundant-related proteins and dehydrin were strongly induced under water deficit but this did not correlate with sucrose content. The expression of genes encoding proline biosynthesis was associated with both sucrose accumulation and water deficit, but amino acid analysis indicated that proline was negatively correlated with sucrose concentration, and whilst total amino acid concentrations increased about seven-fold under water deficit, the relatively low concentration of proline suggested that it had no osmoprotectant role in sugarcane culms. Conclusions The results show that while there was a change in stress-related gene expression associated with sucrose accumulation, different mechanisms are responding to the stress induced by water deficit, because different genes had altered expression under water deficit. PMID:21226964

Isolation and expression of a Bacillus cereus gene encoding benzil reductase.

PubMed

Maruyama, R; Nishizawa, M; Itoi, Y; Ito, S; Inoue, M

2001-12-20

Benzil was reduced stereospecifically to (S)-benzoin by Bacillus cereus strain Tim-r01. To isolate the gene responsible for asymmetric reduction, we constructed a library consisting of Escherichia coli clones that harbored plasmids expressing Bacillus cereus genes. The library was screened using the halo formation assay, and one clone showed benzil reduction to (S)-benzoin. Thus, this clone seemed to carry a plasmid encoding a Bacillus cereus benzil reductase. The deduced amino acid sequence had marked homologies to the Bacillus subtilis yueD protein (41% identity), the yeast open reading frame YIR036C protein (31%), and the mammalian sepiapterin reductases (28% to 30%), suggesting that benzil reductase is a novel short-chain de-hydrogenases/ reductase. Copyright 2001 John Wiley & Sons, Inc.

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.

Induction of virulence gene expression in Staphylococcus aureus by pulmonary surfactant.

PubMed

Ishii, Kenichi; Adachi, Tatsuo; Yasukawa, Jyunichiro; Suzuki, Yutaka; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-04-01

We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

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

Characterization and functional analysis of the MAL and MPH Loci for maltose utilization in some ale and lager yeast strains.

PubMed

Vidgren, Virve; Ruohonen, Laura; Londesborough, John

2005-12-01

Maltose and maltotriose are the major sugars in brewer's wort. Brewer's yeasts contain multiple genes for maltose transporters. It is not known which of these express functional transporters. We correlated maltose transport kinetics with the genotypes of some ale and lager yeasts. Maltose transport by two ale strains was strongly inhibited by other alpha-glucosides, suggesting the use of broad substrate specificity transporters, such as Agt1p. Maltose transport by three lager strains was weakly inhibited by other alpha-glucosides, suggesting the use of narrow substrate specificity transporters. Hybridization studies showed that all five strains contained complete MAL1, MAL2, MAL3, and MAL4 loci, except for one ale strain, which lacked a MAL2 locus. All five strains also contained both AGT1 (coding a broad specificity alpha-glucoside transporter) and MAL11 alleles. MPH genes (maltose permease homologues) were present in the lager but not in the ale strains. During growth on maltose, the lager strains expressed AGT1 at low levels and MALx1 genes at high levels, whereas the ale strains expressed AGT1 at high levels and MALx1 genes at low levels. MPHx expression was negligible in all strains. The AGT1 sequences from the ale strains encoded full-length (616 amino acid) polypeptides, but those from both sequenced lager strains encoded truncated (394 amino acid) polypeptides that are unlikely to be functional transporters. Thus, despite the apparently similar genotypes of these ale and lager strains revealed by hybridization, maltose is predominantly carried by AGT1-encoded transporters in the ale strains and by MALx1-encoded transporters in the lager strains.

In vitro expression of erythropoietin by transfected human mesenchymal stromal cells.

PubMed

Mok, P-L; Cheong, S-K; Leong, C-F; Othman, A

2008-01-01

Mesenchymal stromal cells (MSC) are pluripotent progenitor cells that can be found in human bone marrow (BM). These cells have low immunogenicity and could suppress alloreactive T-cell responses. In the current study, MSC were tested for their capacity to carry and deliver the erythropoietin (EPO) gene in vitro. Expanded BM MSC was transfected with EPO-encoded plasmid pMCV1.2 and EPO-encoded MIDGE (minimalistic immunologically defined gene expression) vector by electroporation. The expressed EPO was used to induce hematopoietic stem cells (HSC) into erythroid colonies. The results showed that the MIDGE vector was more effective and stable than the plasmid (pMCV1.2) in delivering EPO gene into MSC. The supernatants containing EPO obtained from the transfected cell culture were able to induce the differentiation of HSC into erythroid colonies. MSC hold promise as a cell factory for the production of biologic molecules, and MIDGE vector is more effective and stable than the plasmid in nucleofection involving the EPO gene.

Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

PubMed Central

Sundaram, Vasavi; Choudhary, Mayank N. K.; Pehrsson, Erica; Xing, Xiaoyun; Fiore, Christopher; Pandey, Manishi; Maricque, Brett; Udawatta, Methma; Ngo, Duc; Chen, Yujie; Paguntalan, Asia; Ray, Tammy; Hughes, Ava; Cohen, Barak A.; Wang, Ting

2017-01-01

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome. PMID:28348391

Final technical report for award NO. DE-FG02-95ER20206

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

James P. Shapleigh

2010-02-23

ABSTRACT Initial work focused on the regulation of nitrite reductase, the defining reaction of denitrification as well as nitric oxide (NO) reductase. Expression of the genes encoding both proteins was controlled by NnrR. This regulator was shown to be responsive to NO. More recent work has shown NnrR function is also likely inhibited by oxygen. Therefore, it is this protein that sets the oxygen level at which nitrate respiration takes over from aerobic respiration. The gene encoding NO reductase appears to only require NnrR for expression. Expression of the gene encoding nitrite reductase is more complex. In addition to NnrR,more » a two component sensor regulator complex termed PrrA and PrrB is also required for expression. These proteins are global regulators and serve to link denitrification with other bioenergetic processes in the cell. They also provide an additional layer of oxygen dependent regulation. The sequencing of the R. sphaeroides 2.4.3 genome allowed us to identify several other genes regulated by NnrR. Surprisingly, most of the genes were not essential for denitrification. Their high level of conservation in related denitrifiers suggests they do provide a selectable benefit to the bacterium, however. We also examined the role of nitrate reductase in contributing to denitrification in R. sphaeroides. Strain 2.4.3 is unusual in having two distinct, but related clusters of genes encoding nitrate reductase. One of these genes clusters is expressed under high oxygen conditions but is repressed, likely by PrrB-PrrA, under low oxygen conditions. The other cluster is expressed only under low oxygen conditions. This cluster expresses the nitrate reductase used during denitrification. The high oxygen expressed cluster encodes a protein used for redox homeostasis. Surprisingly, both clusters are fully expressed even in the absence of nitrate. During the course of this work we found that the type strain of R. sphaeroides, 2.4.1, is a partial denitrifier because it has the nitrate and NO reductases but lacks nitrite reductase. Like 2.4.3 it uses NnrR to regulate NO reductase. This unexpected arrangement suggested that it may use NO reductase to detoxify NO produced in its environment. Using a green fluorescent protein based reporter system we were able to demonstrate that NO produced by a denitrifier such as 2.4.3 can induce expression of NO reductase in 2.4.1. We then went on to show that the NO produced by denitrifiers can induce a stress response in other non-denitrifying bacteria. This suggests that the NO produced during denitrification will have a significant impact on the non-denitrifiers present in the surrounding environment. We also expanded our studies to include the denitrifier Agrobacterium tumefaciens. We demonstrated that the expression of the nitrite and NO reductase genes in this bacterium follows the same general scheme as in R. sphaeroides. We also were able to show that this bacterium would induce NO reductase in response to the NO produced by plants. Importantly, we were able to demonstrate that A. tumefaciens had difficulty transitioning from aerobic respiration to denitrification if the transition was sudden. This difficulty manifested as an accumulation of NO. In some conditions cells were slowly able to switch modes of respiration but in other cases NO accumulations seemed to kill the cells. The difficulty in transition appears to be due to an inability to produce enough energy once the oxygen has been completely consumed.« less

Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae*

PubMed Central

Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko

2016-01-01

Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes. PMID:26755723

Identification of a core set of rhizobial infection genes using data from single cell-types.

PubMed

Chen, Da-Song; Liu, Cheng-Wu; Roy, Sonali; Cousins, Donna; Stacey, Nicola; Murray, Jeremy D

2015-01-01

Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia (NP). More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4',4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

Structure and expression of genes for a class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair follicles

PubMed Central

1990-01-01

The major histological components of the hair follicle are the hair cortex and cuticle. The hair cuticle cells encase and protect the cortex and undergo a different developmental program to that of the cortex. We report the molecular characterization of a set of evolutionarily conserved hair genes which are transcribed in the hair cuticle late in follicle development. Two genes were isolated and characterized, one expressed in the human follicle and one in the sheep follicle. Each gene encodes a small protein of 16 kD, containing greater than 50 cysteine residues, ranging from 31 to 36 mol% cysteine. Their high cysteine content and in vitro expression data identify them as ultra-high-sulfur (UHS) keratin proteins. The predicted proteins are composed almost entirely of cysteine-rich and glycine-rich repeats. Genomic blots reveal that the UHS keratin proteins are encoded by related multigene families in both the human and sheep genomes. Tissue in situ hybridization demonstrates that the expression of both genes is localized to the hair fiber cuticle and occurs at a late stage in fiber morphogenesis. PMID:1703541

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

Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence.

PubMed

Leroch, Michaela; Mernke, Dennis; Koppenhoefer, Dieter; Schneider, Prisca; Mosbach, Andreas; Doehlemann, Gunther; Hahn, Matthias

2011-05-01

The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungus Botrytis cinerea because of low fluorescence intensity. The codon usage of B. cinerea genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of the B. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression in B. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfp was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfp fusions for quantitative evaluation of various toxic compounds as inducers of the atrB gene encoding an ABC-type drug efflux transporter of B. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in B. cinerea.

Two Closely Related Genes of Arabidopsis Encode Plastidial Cytidinediphosphate Diacylglycerol Synthases Essential for Photoautotrophic Growth1[C

PubMed Central

Haselier, André; Akbari, Hana; Weth, Agnes; Baumgartner, Werner; Frentzen, Margrit

2010-01-01

Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the formation of cytidinediphosphate diacylglycerol, an essential precursor of anionic phosphoglycerolipids like phosphatidylglycerol or -inositol. In plant cells, CDS isozymes are located in plastids, mitochondria, and microsomes. Here, we show that these isozymes are encoded by five genes in Arabidopsis (Arabidopsis thaliana). Alternative translation initiation or alternative splicing of CDS2 and CDS4 transcripts can result in up to 10 isoforms. Most of the cDNAs encoding the various plant isoforms were functionally expressed in yeast and rescued the nonviable phenotype of the mutant strain lacking CDS activity. The closely related genes CDS4 and CDS5 were found to encode plastidial isozymes with similar catalytic properties. Inactivation of both genes was required to obtain Arabidopsis mutant lines with a visible phenotype, suggesting that the genes have redundant functions. Analysis of these Arabidopsis mutants provided further independent evidence for the importance of plastidial phosphatidylglycerol for structure and function of thylakoid membranes and, hence, for photoautotrophic growth. PMID:20442275

A comprehensive strategy for identifying long-distance mobile peptides in xylem sap.

PubMed

Okamoto, Satoru; Suzuki, Takamasa; Kawaguchi, Masayoshi; Higashiyama, Tetsuya; Matsubayashi, Yoshikatsu

2015-11-01

There is a growing awareness that secreted pemediate organ-to-organ communication in higher plants. Xylem sap peptidomics is an effective but challenging approach for identifying long-distance mobile peptides. In this study we developed a simple, gel-free purification system that combines o-chlorophenol extraction with HPLC separation. Using this system, we successfully identified seven oligopeptides from soybean xylem sap exudate that had one or more post-transcriptional modifications: glycosylation, sulfation and/or hydroxylation. RNA sequencing and quantitative PCR analyses showed that the peptide-encoding genes are expressed in multiple tissues. We further analyzed the long-distance translocation of four of the seven peptides using gene-encoding peptides with single amino acid substitutions, and identified these four peptides as potential root-to-shoot mobile oligopeptides. Promoter-GUS analysis showed that all four peptide-encoding genes were expressed in the inner tissues of the root endodermis. Moreover, we found that some of these peptide-encoding genes responded to biotic and/or abiotic factors. These results indicate that our purification system provides a comprehensive approach for effectively identifying endogenous small peptides and reinforce the concept that higher plants employ various peptides in root-to-shoot signaling. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae.

PubMed Central

Colicelli, J; Nicolette, C; Birchmeier, C; Rodgers, L; Riggs, M; Wigler, M

1991-01-01

Saccharomyces cerevisiae strains expressing the activated RAS2Val19 gene or lacking both cAMP phosphodiesterase genes, PDE1 and PDE2, have impaired growth control and display an acute sensitivity to heat shock. We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of RAS2Val19 strain. Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pde1- pde2- strains and encode cAMP phosphodiesterases. Members of the second class fail to suppress the phenotype of pde1- pde2- strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins. We report the nucleotide sequence of three members of this class. Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes. Images PMID:1849280

Methylomics of gene expression in human monocytes

PubMed Central

Liu, Yongmei; Ding, Jingzhong; Reynolds, Lindsay M.; Lohman, Kurt; Register, Thomas C.; De La Fuente, Alberto; Howard, Timothy D.; Hawkins, Greg A.; Cui, Wei; Morris, Jessica; Smith, Shelly G.; Barr, R. Graham; Kaufman, Joel D.; Burke, Gregory L.; Post, Wendy; Shea, Steven; Mccall, Charles E.; Siscovick, David; Jacobs, David R.; Tracy, Russell P.; Herrington, David M.; Hoeschele, Ina

2013-01-01

DNA methylation is one of several epigenetic mechanisms that contribute to the regulation of gene expression; however, the extent to which methylation of CpG dinucleotides correlates with gene expression at the genome-wide level is still largely unknown. Using purified primary monocytes from subjects in a large community-based cohort (n = 1264), we characterized methylation (>485 000 CpG sites) and mRNA expression (>48K transcripts) and carried out genome-wide association analyses of 8370 expression phenotypes. We identified 11 203 potential cis-acting CpG loci whose degree of methylation was associated with gene expression (eMS) at a false discovery rate threshold of 0.001. Most of the associations were consistent in effect size and direction of effect across sex and three ethnicities. Contrary to expectation, these eMS were not predominately enriched in promoter regions, or CpG islands, but rather in the 3′ UTR, gene bodies, CpG shores or ‘offshore’ sites, and both positive and negative correlations between methylation and expression were observed across all locations. eMS were enriched for regions predicted to be regulatory by ENCODE (Encyclopedia of DNA Elements) data in multiple cell types, particularly enhancers. One of the strongest association signals detected (P < 2.2 × 10−308) was a methylation probe (cg17005068) in the promoter/enhancer region of the glutathione S-transferase theta 1 gene (GSTT1, encoding the detoxification enzyme) with GSTT1 mRNA expression. Our study provides a detailed description of the epigenetic architecture in human monocytes and its relationship to gene expression. These data may help prioritize interrogation of biologically relevant methylation loci and provide new insights into the epigenetic basis of human health and diseases. PMID:23900078

Proglucagons in vertebrates: Expression and processing of multiple genes in a bony fish.

PubMed

Busby, Ellen R; Mommsen, Thomas P

2016-09-01

In contrast to mammals, where a single proglucagon (PG) gene encodes three peptides: glucagon, glucagon-like peptide 1 and glucagon-like peptide 2 (GLP-1; GLP-2), many non-mammalian vertebrates carry multiple PG genes. Here, we investigate proglucagon mRNA sequences, their tissue expression and processing in a diploid bony fish. Copper rockfish (Sebastes caurinus) express two independent genes coding for distinct proglucagon sequences (PG I, PG II), with PG II lacking the GLP-2 sequence. These genes are differentially transcribed in the endocrine pancreas, the brain, and the gastrointestinal tract. Alternative splicing identified in rockfish is only one part of this complex regulation of the PG transcripts: the system has the potential to produce two glucagons, four GLP-1s and a single GLP-2, or any combination of these peptides. Mass spectrometric analysis of partially purified PG-derived peptides in endocrine pancreas confirms translation of both PG transcripts and differential processing of the resulting peptides. The complex differential regulation of the two PG genes and their continued presence in this extant teleostean fish strongly suggests unique and, as yet largely unidentified, roles for the peptide products encoded in each gene. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of barosensitive mechanisms in yeast for Pressure Regulated Fermentation

NASA Astrophysics Data System (ADS)

Nomura, Kazuki; Iwahashi, Hitoshi; Iguchi, Akinori; Shigematsu, Toru

2013-06-01

Introduction: We are intending to develop a novel food processing technology, Pressure Regulated Fermentation (PReF), using pressure sensitive (barosensitive) fermentation microorganisms. Objectives of our study are to clarify barosensitive mechanisms for application to PReF technology. We isolated Saccharomyces cerevisiae barosensitive mutant a924E1 that was derived from the parent KA31a. Methods: Gene expression levels were analyzed by DNA microarray. The altered genes of expression levels were classified according to the gene function. Mutated genes were estimated by mating and producing diploid strains and confirmed by PCR of mitochondrial DNA (mtDNA). Results and Discussion: Gene expression profiles showed that genes of `Energy' function and that of encoding protein localized in ``Mitochondria'' were significantly down regulated in the mutant. These results suggest the respiratory deficiency and relationship between barosensitivity and respiratory deficiency. Since the respiratory functions of diploids showed non Mendelian inheritance, the respiratory deficiency was indicated to be due to mtDNA mutation. PCR analysis showed that the region of COX1 locus was deleted. COX1 gene encodes the subunit 1 of cytochrome c oxidase. For this reason, barosensitivity is strongly correlated with mitochondrial functions.

Lignin-degrading peroxidases in white-rot fungus Trametes hirsuta 072. Absolute expression quantification of full multigene family

PubMed Central

Vasina, Daria V.; Moiseenko, Konstantin V.; Fedorova, Tatiana V.; Tyazhelova, Tatiana V.

2017-01-01

Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle. PMID:28301519

Quantum changes in Helicobacter pylori gene expression accompany host-adaptation

PubMed Central

Wise, Michael J.; Khosravi, Yalda; Seow, Shih-Wee; Amoyo, Arlaine A.; Pettersson, Sven; Peters, Fanny; Tay, Chin-Yen; Perkins, Timothy T.; Loke, Mun-Fai; Marshall, Barry J.; Vadivelu, Jamuna

2017-01-01

Abstract Helicobacter pylori is a highly successful gastric pathogen. High genomic plasticity allows its adaptation to changing host environments. Complete genomes of H. pylori clinical isolate UM032 and its mice-adapted serial derivatives 298 and 299, generated using both PacBio RS and Illumina MiSeq sequencing technologies, were compared to identify novel elements responsible for host-adaptation. The acquisition of a jhp0562-like allele, which encodes for a galactosyltransferase, was identified in the mice-adapted strains. Our analysis implies a new β-1,4-galactosyltransferase role for this enzyme, essential for Ley antigen expression. Intragenomic recombination between babA and babB genes was also observed. Further, we expanded on the list of candidate genes whose expression patterns have been mediated by upstream homopolymer-length alterations to facilitate host adaption. Importantly, greater than four-fold reduction of mRNA levels was demonstrated in five genes. Among the down-regulated genes, three encode for outer membrane proteins, including BabA, BabB and HopD. As expected, a substantial reduction in BabA protein abundance was detected in mice-adapted strains 298 and 299 via Western analysis. Our results suggest that the expression of Ley antigen and reduced outer membrane protein expressions may facilitate H. pylori colonisation of mouse gastric epithelium. PMID:27803027

The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis.

PubMed

Zhao, D; Yu, Q; Chen, M; Ma, H

2001-07-01

The Arabidopsis floral regulatory genes APETALA3 (AP3) and PISTILLATA (PI) are required for the B function according to the ABC model for floral organ identity. AP3 and PI expression are positively regulated by the LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) genes. UFO encodes an F-box protein, and we have shown previously that UFO genetically interacts with the ASK1 gene encoding a SKP1 homologue; both the F-box containing protein and SKP1 are subunits of ubiquitin ligases. We show here that the ask1-1 mutation can enhance the floral phenotypes of weak lfy and ap3 mutants; therefore, like UFO, ASK1 also interacts with LFY and AP3 genetically. Furthermore, our results from RNA in situ hybridizations indicate that ASK1 regulates early AP3 and PI expression. These results support the idea that UFO and ASK1 together positively regulate AP3 and PI expression. We propose that the UFO and ASK1 proteins are components of a ubiquitin ligase that mediates the proteolysis of a repressor of AP3 and PI expression. Our genetic studies also indicate that ASK1 and UFO play a role in regulating the number of floral organ primordia, and we discuss possible mechanisms for such a regulation.

Parasitization by Scleroderma guani influences expression of superoxide dismutase genes in Tenebrio molitor.

PubMed

Zhu, Jia-Ying; Ze, Sang-Zi; Stanley, David W; Yang, Bin

2014-09-01

Superoxide dismutase (SOD) is an antioxidant enzyme involved in detoxifying reactive oxygen species. In this study, we identified genes encoding the extracellular and intracellular copper-zinc SODs (ecCuZnSOD and icCuZnSOD) and a manganese SOD (MnSOD) in the yellow mealworm beetle, Tenebrio molitor. The cDNAs for ecCuZnSOD, icCuZnSOD, and MnSOD, respectively, encode 24.55, 15.81, and 23.14 kDa polypeptides, which possess structural features typical of other insect SODs. They showed 20-94% identity to other known SOD sequences from Bombyx mori, Musca domestica, Nasonia vitripennis, Pediculus humanus corporis, and Tribolium castaneum. Expression of these genes was analyzed in selected tissues and developmental stages, and following exposure to Escherichia coli and parasitization by Scleroderma guani. We recorded expression of all three SODs in cuticle, fat body, and hemocytes and in the major developmental stages. Relatively higher expressions were detected in late-instar larvae and pupae, compared to other developmental stages. Transcriptional levels were upregulated following bacterial infection. Analysis of pupae parasitized by S. guani revealed that expression of T. molitor SOD genes was significantly induced following parasitization. We infer that these genes act in immune response and in host-parasitoid interactions. © 2014 Wiley Periodicals, Inc.

Woodpecker drumming behavior is linked to the elevated expression of genes that encode calcium handling proteins in the neck musculature.

PubMed

Schuppe, Eric R; Petersen, John O; Fuxjager, Matthew J

2018-05-31

Many animals perform elaborate physical displays for social communication. Identifying molecular mechanisms that co-evolve with these complex behavioral signals can therefore help reveal how forces of selection shape animal design. To study this issue, we examine gene expression profiles in select skeletal muscles that actuate woodpecker drum displays. This remarkable whole-body signal is produced when individuals rapidly hammer their bill against trees. We find that, compared to muscles that play no part in producing this behavior, the main muscle used to drum abundantly expresses two genes that encode proteins that support myocytic calcium (Ca 2+ ) handling dynamics-namely parvalbumin (PV) and sarcoplasmic reticulum Ca 2+ ATPase (SERCA1). Meanwhile, we find no such difference in the expression of another gene similarly vital to Ca 2+ handling, the ryanodine receptor (RYR1). These differences are not present in a non-woodpecker species, which readily produce much slower drum-like movements for foraging (but not social signaling). Our data therefore point to an association between the fast drum displays of woodpeckers and muscle-specific expression of genes whose protein products enhance select aspects of myocytic Ca 2+ handling. © 2018. Published by The Company of Biologists Ltd.

A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

NASA Technical Reports Server (NTRS)

Wang, W.; Takezawa, D.; Poovaiah, B. W.

1996-01-01

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

Functional and evolutionary implications from the molecular characterization of five spermatophore CHH/MIH/GIH genes in the shrimp Fenneropenaeus merguiensis.

PubMed

Shi, LiLi; Li, Bin; Zhou, Ting Ting; Wang, Wei; Chan, Siuming F

2018-01-01

The recent use of RNA-Seq to study the transcriptomes of different species has helped identify a large number of new genes from different non-model organisms. In this study, five distinctive transcripts encoding for neuropeptide members of the CHH/MIH/GIH family have been identified from the spermatophore transcriptome of the shrimp Fenneropenaeus merguiensis. The size of these transcripts ranged from 531 bp to 1771 bp. Four transcripts encoded different CHH-family subtype I members, and one transcript encoded a subtype II member. RT-PCR and RACE approaches have confirmed the expression of these genes in males. The low degree of amino acid sequence identity among these neuropeptides suggests that they may have different specific function(s). Results from a phylogenetic tree analysis indicated that these neuropeptides were likely derived from a common ancestor gene resulting from mutation and gene duplication. These CHH-family members could be grouped into distinct clusters, indicating a strong structural/functional relationship among these neuropeptides. Eyestalk removal caused a significant increase in the expression of transcript 32710 but decreases in expression for transcript 28020. These findings suggest the possible regulation of these genes by eyestalk factor(s). In summary, the results of this study would justify a re-evaluation of the more generalized and pleiotropic functions of these neuropeptides. This study also represents the first report on the cloning/identification of five CHH family neuropeptides in a non-neuronal tissue from a single crustacean species.

The alpha glycerophosphate cycle in Drosophila melanogaster VI. structure and evolution of enzyme paralogs in the genus Drosophila.

PubMed

Carmon, Amber; MacIntyre, Ross

2010-01-01

The genome sequences of 12 Drosophila species contain 3 paralogs for alpha glycerophosphate dehydrogenase (GPDH) and for the mitochondrial alpha glycerophosphate oxidase (GPO). These 2 enzymes participate in the alpha glycerophosphate cycle in the adult thoracic flight muscles. The flight muscle enzymes are encoded by gpdh-1 at 26A2 and gpo-1 at 52C8. In this paper, we show that the GPDH paralogs share the same evolutionarily conserved functional domains and most intron positions, whereas the GPO paralogs share only some of the functional domains of mitochondrial oxidoreductases. The GPO paralogs not expressed in the flight muscles essentially lack introns. GPDH paralogs encoded by gpdh-2 and gpdh-3 and the GPO paralogs encoded by gpo-2 and gpo-3 are expressed only in the testes. Gene trees for the GPDH and GPO paralogs indicate that the genes expressed in the flight muscles are evolving very slowly presumably under strong purifying selection whereas the paralogs expressed in the testes are evolving more rapidly. The concordance between species and gene trees, d(N)/d(S) ratios, phylogenetic analysis by maximum likelihood-based tests, and analyses of radical and conservative substitutions all indicate that the additional GPDH and GPO paralogs are also evolving under purifying selection.

Molecular cloning and functional analysis of ESGP, an embryonic stem cell and germ cell specific protein.

PubMed

Chen, Yan-Mei; Du, Zhong-Wei; Yao, Zhen

2005-12-01

Several putative Oct-4 downstream genes from mouse embryonic stem (ES) cells have been identified using the suppression-subtractive hybridization method. In this study, one of the novel genes encoding an ES cell and germ cell specific protein (ESGP) was cloned by rapid amplification of cDNA ends. ESGP contains 801 bp encoding an 84 amino acid small protein and has no significant homology to any known genes. There is a signal peptide at the N-terminal of ESGP protein as predicted by SeqWeb (GCG) (SeqWeb version 2.0.2, http://gcg.biosino.org:8080/). The result of immunofluorescence assay suggested that ESGP might encode a secretory protein. The expression pattern of ESGP is consistent with the expression of Oct-4 during embryonic development. ESGP protein was detected in fertilized oocyte, from 3.5 day postcoital (dpc) blastocyst to 17.5 dpc embryo, and was only detected in testis and ovary tissues in adult. In vitro, ESGP was only expressed in pluripotent cell lines, such as embryonic stem cells, embryonic caoma cells and embryonic germ cells, but not in their differentiated progenies. Despite its specific expression, forced expression of ESGP is not indispensable for the effect of Oct-4 on ES cell self-renewal, and does not affect the differentiation to three germ layers.

A distinct subgroup of cardiomyopathy patients characterized by transcriptionally active cardiotropic erythrovirus and altered cardiac gene expression.

PubMed

Kuhl, U; Lassner, D; Dorner, A; Rohde, M; Escher, F; Seeberg, B; Hertel, E; Tschope, C; Skurk, C; Gross, U M; Schultheiss, H-P; Poller, W

2013-09-01

Recent studies have detected erythrovirus genomes in the hearts of cardiomyopathy and cardiac transplant patients. Assessment of the functional status of viruses may provide clinically important information beyond detection of the viral genomes. Here, we report transcriptional activation of cardiotropic erythrovirus to be associated with strongly altered myocardial gene expression in a distinct subgroup of cardiomyopathy patients. Endomyocardial biopsies (EMBs) from 415 consecutive cardiac erythrovirus (B19V)-positive patients with clinically suspected cardiomyopathy were screened for virus-encoded VP1/VP2 mRNA indicating transcriptional activation of the virus, and correlated with cardiac host gene expression patterns in transcriptionally active versus latent infections, and in virus-free control hearts. Transcriptional activity was detected in baseline biopsies of only 66/415 patients (15.9 %) harbouring erythrovirus. At the molecular level, significant differences between cardiac B19V-positive patients with transcriptionally active versus latent virus were revealed by expression profiling of EMBs. Importantly, latent B19V infection was indistinguishable from controls. Genes involved encode proteins of antiviral immune response, B19V receptor complex, and mitochondrial energy metabolism. Thus, functional mapping of erythrovirus allows definition of a subgroup of B19V-infected cardiomyopathy patients characterized by virus-encoded VP1/VP2 transcripts and anomalous host myocardial transcriptomes. Cardiac B19V reactivation from latency, as reported here for the first time, is a key factor required for erythrovirus to induce altered cardiac gene expression in a subgroup of cardiomyopathy patients. Virus genome detection is insufficient to assess pathogenic potential, but additional transcriptional mapping should be incorporated into future pathogenetic and therapeutic studies both in cardiology and transplantation medicine.

The euryhaline yeast Debaryomyces hansenii has two catalase genes encoding enzymes with differential activity profile.

PubMed

Segal-Kischinevzky, Claudia; Rodarte-Murguía, Beatriz; Valdés-López, Victor; Mendoza-Hernández, Guillermo; González, Alicia; Alba-Lois, Luisa

2011-03-01

Debaryomyces hansenii is a spoilage yeast able to grow in a variety of ecological niches, from seawater to dairy products. Results presented in this article show that (i) D. hansenii has an inherent resistance to H2O2 which could be attributed to the fact that this yeast has a basal catalase activity which is several-fold higher than that observed in Saccharomyces cerevisiae under the same culture conditions, (ii) D. hansenii has two genes (DhCTA1 and DhCTT1) encoding two catalase isozymes with a differential enzymatic activity profile which is not strictly correlated with a differential expression profile of the encoding genes.

Expression of a synthetic gene encoding human insulin-like growth factor I in cultured mouse fibroblasts

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

Bayne, M.L.; Cascieri, M.A.; Kelder, B.

1987-05-01

A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fibroblasts, directs synthesis of the fusion protein, subsequent proteolytic removal of the bovine growth hormone signal peptide, and secretion of hIGF-I into the culture medium. Conditioned medium frommore » transfected cells inhibits binding of /sup 125/I-labeled IGF-I to type I IGF receptors on human placental membranes and to acid-stable human serum carrier proteins. The recombinant hIGF-I produced is biologically active, as monitored by the stimulation of DNA synthesis in vascular smooth muscle cells.« less

Natural variation in the VELVET gene bcvel1 affects virulence and light-dependent differentiation in Botrytis cinerea.

PubMed

Schumacher, Julia; Pradier, Jean-Marc; Simon, Adeline; Traeger, Stefanie; Moraga, Javier; Collado, Isidro González; Viaud, Muriel; Tudzynski, Bettina

2012-01-01

Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the full-length protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Δbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Δbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Δbcvel1 mutants to nutrient starvation conditions.

Natural Variation in the VELVET Gene bcvel1 Affects Virulence and Light-Dependent Differentiation in Botrytis cinerea

PubMed Central

Schumacher, Julia; Pradier, Jean-Marc; Simon, Adeline; Traeger, Stefanie; Moraga, Javier; Collado, Isidro González; Viaud, Muriel; Tudzynski, Bettina

2012-01-01

Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the full-length protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Δbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Δbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Δbcvel1 mutants to nutrient starvation conditions. PMID:23118899

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.

Characterization of the aes gene of Escherichia coli encoding an enzyme with esterase activity.

PubMed Central

Peist, R; Koch, A; Bolek, P; Sewitz, S; Kolbus, T; Boos, W

1997-01-01

malQ mutants of Escherichia coli lacking amylomaltase cannot grow on maltose. They express the maltose system constitutively and are sensitive to maltose when grown on another carbon source. In an attempt to isolate a multicopy suppressor that would result in growth on maltose, we transformed a malQ mutant with a gene bank of E. coli DNA which had been digested with Sau3a and cloned in pBR322. We screened the transformants on MacConkey maltose plates. A colony was isolated that appeared to be resistant to maltose and was pink on these plates, but it was still unable to grow on minimal medium with maltose as the carbon source. The plasmid was isolated, and the gene causing this phenotype was characterized. The deduced amino acid sequence of the encoded protein shows homology to that of lipases and esterases. We termed the gene aes, for acetyl esterase. Extracts of cells harboring plasmid-encoded aes under its own promoter exhibit a fivefold higher capacity to hydrolyze p-nitrophenyl acetate than do extracts of cells of plasmid-free strains. Similarly, strains harboring plasmid-encoded aes are able to grow on triacetyl glycerol (triacetin) whereas the plasmid-free strains are not. The expression of plasmid-encoded aes resulted in strong repression of the maltose transport genes in malT+ strains (10-fold reduction), but not in a malT(Con) strain which is independent of the inducer. Also, overproduction of MalT counteracted the Aes-dependent repression, indicating a direct interaction between MalT and Aes. PMID:9401025

In situ hybridization analysis of the expression of futsch, tau, and MESK2 homologues in the brain of the European honeybee (Apis mellifera L.).

PubMed

Kaneko, Kumi; Hori, Sayaka; Morimoto, Mai M; Nakaoka, Takayoshi; Paul, Rajib Kumar; Fujiyuki, Tomoko; Shirai, Kenichi; Wakamoto, Akiko; Tsuboko, Satomi; Takeuchi, Hideaki; Kubo, Takeo

2010-02-16

The importance of visual sense in Hymenopteran social behavior is suggested by the existence of a Hymenopteran insect-specific neural circuit related to visual processing and the fact that worker honeybee brain changes morphologically according to its foraging experience. To analyze molecular and neural bases that underlie the visual abilities of the honeybees, we used a cDNA microarray to search for gene(s) expressed in a neural cell-type preferential manner in a visual center of the honeybee brain, the optic lobes (OLs). Expression analysis of candidate genes using in situ hybridization revealed two genes expressed in a neural cell-type preferential manner in the OLs. One is a homologue of Drosophila futsch, which encodes a microtubule-associated protein and is preferentially expressed in the monopolar cells in the lamina of the OLs. The gene for another microtubule-associated protein, tau, which functionally overlaps with futsch, was also preferentially expressed in the monopolar cells, strongly suggesting the functional importance of these two microtubule-associated proteins in monopolar cells. The other gene encoded a homologue of Misexpression Suppressor of Dominant-negative Kinase Suppressor of Ras 2 (MESK2), which might activate Ras/MAPK-signaling in Drosophila. MESK2 was expressed preferentially in a subclass of neurons located in the ventral region between the lamina and medulla neuropil in the OLs, suggesting that this subclass is a novel OL neuron type characterized by MESK2-expression. These three genes exhibited similar expression patterns in the worker, drone, and queen brains, suggesting that they function similarly irrespective of the honeybee sex or caste. Here we identified genes that are expressed in a monopolar cell (Amfutsch and Amtau) or ventral medulla-preferential manner (AmMESK2) in insect OLs. These genes may aid in visualizing neurites of monopolar cells and ventral medulla cells, as well as in analyzing the function of these neurons.

Control of Flagellar Gene Regulation in Legionella pneumophila and Its Relation to Growth Phase▿ †

PubMed Central

Albert-Weissenberger, Christiane; Sahr, Tobias; Sismeiro, Odile; Hacker, Jörg; Heuner, Klaus; Buchrieser, Carmen

2010-01-01

The bacterial pathogen Legionella pneumophila responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of fliA encoding sigma 28. Expression analysis of a fliA mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene fliA is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum. PMID:19915024

Expression of the tachykinin receptor mRNAs in healthy human colon.

PubMed

Jaafari, Nadia; Hua, Guoqiang; Adélaïde, José; Julé, Yvon; Imbert, Jean

2008-12-03

Tachykinins are a family of neuropeptides, involved in a variety of physiological and pathological processes occurring in the gastrointestinal tract. They act via three distinct types of receptors, tachykinin NK(1), NK(2), and NK(3) receptors, which belong to the family of G protein-coupled receptors. The aim of the present study was to characterize, for the first time in the healthy human colon, the TACR(1), TACR(2) and TACR(3) mRNAs encoding the three different tachykinin receptors and to measure their relative expression by quantitative reverse transcription-PCR assay. Our results confirm the broad distribution of the tachykinin receptors but evidenced significant differences in the expression level of their respective mRNAs. A higher expression level of the TACR2 mRNA alpha isoform, the gene encoding the functional tachykinin NK(2) receptor, was observed in comparison to TACR1 and TACR3 mRNAs genes encoding for NK(1) and NK(3) receptors respectively. The prevalence of the TACR2 mRNA alpha isoform strongly suggests a major involvement of tachykinin NK(2) receptor in the regulation of human colonic functions.

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

Neurotactin functions in concert with other identified CAMs in growth cone guidance in Drosophila.

PubMed

Speicher, S; García-Alonso, L; Carmena, A; Martín-Bermudo, M D; de la Escalera, S; Jiménez, F

1998-02-01

We have isolated and characterized mutations in Drosophila neurotactin, a gene that encodes a cell adhesion protein widely expressed during neural development. Analysis of both loss and gain of gene function conditions during embryonic and postembryonic development revealed specific requirements for neurotactin during axon outgrowth, fasciculation, and guidance. Furthermore, embryos of some double mutant combinations of neurotactin and other genes encoding adhesion/signaling molecules, including neuroglian, derailed, and kekkon1, displayed phenotypic synergy. This result provides evidence for functional cooperativity in vivo between the adhesion and signaling pathways controlled by neurotactin and the other three genes.

Analysis of expressed sequence tags (ESTs) from avocado seed (Persea americana var. drymifolia) reveals abundant expression of the gene encoding the antimicrobial peptide snakin.

PubMed

Guzmán-Rodríguez, Jaquelina J; Ibarra-Laclette, Enrique; Herrera-Estrella, Luis; Ochoa-Zarzosa, Alejandra; Suárez-Rodríguez, Luis María; Rodríguez-Zapata, Luis C; Salgado-Garciglia, Rafael; Jimenez-Moraila, Beatriz; López-Meza, Joel E; López-Gómez, Rodolfo

2013-09-01

Avocado is one of the most important fruits in the world. Avocado "native mexicano" (Persea americana var. drymifolia) seeds are widely used in the propagation of this plant and are the primary source of rootstocks globally for a variety of avocado cultivars, such as the Hass avocado. Here, we report the isolation of 5005 ESTs from the 5' ends of P. americana var. drymifolia seed cDNA clones representing 1584 possible unigenes. These avocado seed ESTs were compared with the avocado flower EST library, and we detected several genes that are expressed either in both tissues or only in the seed. The snakin gene, which encodes an element of the innate immune response in plants, was one of those most frequently found among the seed ESTs, and this suggests that it is abundantly expressed in the avocado seed. We expressed the snakin gene in a heterologous system, namely the bovine endothelial cell line BVE-E6E7. Conditioned media from transfected BVE-E6E7 cells showed antimicrobial activity against strains of Escherichia coli and Staphylococcus aureus. This is the first study of the function of the snakin gene in plant seed tissue, and our observations suggest that this gene might play a protective role in the avocado seed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Comparison of the Structure and Expression of Odd-Skipped and Two Related Genes That Encode a New Family of Zinc Finger Proteins in Drosophila

PubMed Central

Hart, M. C.; Wang, L.; Coulter, D. E.

1996-01-01

The odd-skipped (odd) gene, which was identified on the basis of a pair-rule segmentation phenotype in mutant embryos, is initially expressed in the Drosophila embryo in seven pair-rule stripes, but later exhibits a segment polarity-like pattern for which no phenotypic correlate is apparent. We have molecularly characterized two embryonically expressed odd-cognate genes, sob and bowel (bowl), that encode proteins with highly conserved C(2)H(2) zinc fingers. While the Sob and Bowl proteins each contain five tandem fingers, the Odd protein lacks a fifth (C-terminal) finger and is also less conserved among the four common fingers. Reminiscent of many segmentation gene paralogues, the closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns; in contrast, the less-tightly linked bowl gene is expressed in a distinctly different pattern at the termini of the early embryo. Although our results indicate that odd and sob are more likely than bowl to share overlapping developmental roles, some functional divergence between the Odd and Sob proteins is suggested by the absence of homology outside the zinc fingers, and also by amino acid substitutions in the Odd zinc fingers at positions that appear to be constrained in Sob and Bowl. PMID:8878683

Expression of inflammation-related genes is altered in gastric tissue of patients with advanced stages of NAFLD.

PubMed

Mehta, Rohini; Birerdinc, Aybike; Neupane, Arpan; Shamsaddini, Amirhossein; Afendy, Arian; Elariny, Hazem; Chandhoke, Vikas; Baranova, Ancha; Younossi, Zobair M

2013-01-01

Obesity is associated with chronic low-grade inflammation perpetuated by visceral adipose. Other organs, particularly stomach and intestine, may also overproduce proinflammatory molecules. We examined the gene expression patterns in gastric tissue of morbidly obese patients with nonalcoholic fatty liver disease (NAFLD) and compared the changes in gene expression in different histological forms of NAFLD. Stomach tissue samples from 20 morbidly obese NAFLD patients who were undergoing sleeve gastrectomy were profiled using qPCR for 84 genes encoding inflammatory cytokines, chemokines, their receptors, and other components of inflammatory cascades. Interleukin 8 receptor-beta (IL8RB) gene overexpression in gastric tissue was correlated with the presence of hepatic steatosis, hepatic fibrosis, and histologic diagnosis of nonalcoholic steatohepatitis (NASH). Expression levels of soluble interleukin 1 receptor antagonist (IL1RN) were correlated with the presence of NASH and hepatic fibrosis. mRNA levels of interleukin 8 (IL8), chemokine (C-C motif) ligand 4 (CCL4), and its receptor chemokine (C-C motif) receptor type 5 (CCR5) showed a significant increase in patients with advanced hepatic inflammation and were correlated with the severity of the hepatic inflammation. The results of our study suggest that changes in expression patterns for inflammatory molecule encoding genes within gastric tissue may contribute to the pathogenesis of obesity-related NAFLD.

Expression of Inflammation-Related Genes Is Altered in Gastric Tissue of Patients with Advanced Stages of NAFLD

PubMed Central

Mehta, Rohini; Birerdinc, Aybike; Neupane, Arpan; Shamsaddini, Amirhossein; Afendy, Arian; Elariny, Hazem; Chandhoke, Vikas; Baranova, Ancha; Younossi, Zobair M.

2013-01-01

Obesity is associated with chronic low-grade inflammation perpetuated by visceral adipose. Other organs, particularly stomach and intestine, may also overproduce proinflammatory molecules. We examined the gene expression patterns in gastric tissue of morbidly obese patients with nonalcoholic fatty liver disease (NAFLD) and compared the changes in gene expression in different histological forms of NAFLD. Stomach tissue samples from 20 morbidly obese NAFLD patients who were undergoing sleeve gastrectomy were profiled using qPCR for 84 genes encoding inflammatory cytokines, chemokines, their receptors, and other components of inflammatory cascades. Interleukin 8 receptor-beta (IL8RB) gene overexpression in gastric tissue was correlated with the presence of hepatic steatosis, hepatic fibrosis, and histologic diagnosis of nonalcoholic steatohepatitis (NASH). Expression levels of soluble interleukin 1 receptor antagonist (IL1RN) were correlated with the presence of NASH and hepatic fibrosis. mRNA levels of interleukin 8 (IL8), chemokine (C-C motif) ligand 4 (CCL4), and its receptor chemokine (C-C motif) receptor type 5 (CCR5) showed a significant increase in patients with advanced hepatic inflammation and were correlated with the severity of the hepatic inflammation. The results of our study suggest that changes in expression patterns for inflammatory molecule encoding genes within gastric tissue may contribute to the pathogenesis of obesity-related NAFLD. PMID:23661906

Two human homeobox genes, c1 and c8: structure analysis and expression in embryonic development.

PubMed

Simeone, A; Mavilio, F; Acampora, D; Giampaolo, A; Faiella, A; Zappavigna, V; D'Esposito, M; Pannese, M; Russo, G; Boncinelli, E

1987-07-01

Two human cDNA clones (HHO.c1.95 and HHO.c8.5111) containing a homeobox region have been characterized, and the respective genomic regions have been partially analyzed. Expression of the corresponding genes, termed c1 and c8, was evaluated in different organs and body parts during human embryonic/fetal development. HHO.c1.95 apparently encodes a 217-amino acid protein containing a class I homeodomain that shares 60 out of 61 amino acid residues with the Antennapedia homeodomain of Drosophila melanogaster. HHO.c8.5111 encodes a 153-amino acid protein containing a homeodomain identical to that of the frog AC1 gene. Clones HHO.c1 and HHO.c8 detect by blot-hydridization one and two specific polyadenylylated transcripts, respectively. These are differentially expressed in spinal cord, backbone rudiments, limb buds (or limbs), heart, and skin of human embryos and early fetuses in the 5- to 9-week postfertilization period, thus suggesting that the c1 and c8 genes play a key role in a variety of developmental processes. Together, the results of the embryonic/fetal expression of c1 and c8 and those of two previously analyzed genes (c10 and c13) indicate a coherent pattern of expression of these genes in early human ontogeny.

IGF-1 modulates gene expression of proteins involved in inflammation, cytoskeleton, and liver architecture.

PubMed

Lara-Diaz, V J; Castilla-Cortazar, I; Martín-Estal, I; García-Magariño, M; Aguirre, G A; Puche, J E; de la Garza, R G; Morales, L A; Muñoz, U

2017-05-01

Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1's effects on liver by comparing wild-type controls, heterozygous igf1 +/- , and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.

Cloning and Expression of the Erwinia carotovora subsp. carotovora Gene Encoding the Low-Molecular-Weight Bacteriocin Carocin S1▿

PubMed Central

Chuang, Duen-yau; Chien, Yung-chei; Wu, Huang-Pin

2007-01-01

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (−312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity. PMID:17071754

Transcriptome analysis of Gossypium hirsutum flower buds infested by cotton boll weevil (Anthonomus grandis) larvae.

PubMed

Artico, Sinara; Ribeiro-Alves, Marcelo; Oliveira-Neto, Osmundo Brilhante; de Macedo, Leonardo Lima Pepino; Silveira, Sylvia; Grossi-de-Sa, Maria Fátima; Martinelli, Adriana Pinheiro; Alves-Ferreira, Marcio

2014-10-04

Cotton is a major fibre crop grown worldwide that suffers extensive damage from chewing insects, including the cotton boll weevil larvae (Anthonomus grandis). Transcriptome analysis was performed to understand the molecular interactions between Gossypium hirsutum L. and cotton boll weevil larvae. The Illumina HiSeq 2000 platform was used to sequence the transcriptome of cotton flower buds infested with boll weevil larvae. The analysis generated a total of 327,489,418 sequence reads that were aligned to the G. hirsutum reference transcriptome. The total number of expressed genes was over 21,697 per sample with an average length of 1,063 bp. The DEGseq analysis identified 443 differentially expressed genes (DEG) in cotton flower buds infected with boll weevil larvae. Among them, 402 (90.7%) were up-regulated, 41 (9.3%) were down-regulated and 432 (97.5%) were identified as orthologues of A. thaliana genes using Blastx. Mapman analysis of DEG indicated that many genes were involved in the biotic stress response spanning a range of functions, from a gene encoding a receptor-like kinase to genes involved in triggering defensive responses such as MAPK, transcription factors (WRKY and ERF) and signalling by ethylene (ET) and jasmonic acid (JA) hormones. Furthermore, the spatial expression pattern of 32 of the genes responsive to boll weevil larvae feeding was determined by "in situ" qPCR analysis from RNA isolated from two flower structures, the stamen and the carpel, by laser microdissection (LMD). A large number of cotton transcripts were significantly altered upon infestation by larvae. Among the changes in gene expression, we highlighted the transcription of receptors/sensors that recognise chitin or insect oral secretions; the altered regulation of transcripts encoding enzymes related to kinase cascades, transcription factors, Ca2+ influxes, and reactive oxygen species; and the modulation of transcripts encoding enzymes from phytohormone signalling pathways. These data will aid in the selection of target genes to genetically engineer cotton to control the cotton boll weevil.

Association of breast cancer risk with genetic variants showing differential allelic expression: Identification of a novel breast cancer susceptibility locus at 4q21

PubMed Central

Adoue, Véronique; Michailidou, Kyriaki; Canisius, Sander; Lemaçon, Audrey; Droit, Arnaud; Andrulis, Irene L; Anton-Culver, Hoda; Arndt, Volker; Baynes, Caroline; Blomqvist, Carl; Bogdanova, Natalia V.; Bojesen, Stig E.; Bolla, Manjeet K.; Bonanni, Bernardo; Borresen-Dale, Anne-Lise; Brand, Judith S.; Brauch, Hiltrud; Brenner, Hermann; Broeks, Annegien; Burwinkel, Barbara; Chang-Claude, Jenny; Couch, Fergus J.; Cox, Angela; Cross, Simon S.; Czene, Kamila; Darabi, Hatef; Dennis, Joe; Devilee, Peter; Dörk, Thilo; Dos-Santos-Silva, Isabel; Eriksson, Mikael; Fasching, Peter A.; Figueroa, Jonine; Flyger, Henrik; García-Closas, Montserrat; Giles, Graham G.; Goldberg, Mark S.; González-Neira, Anna; Grenaker-Alnæs, Grethe; Guénel, Pascal; Haeberle, Lothar; Haiman, Christopher A.; Hamann, Ute; Hallberg, Emily; Hooning, Maartje J.; Hopper, John L.; Jakubowska, Anna; Jones, Michael; Kabisch, Maria; Kataja, Vesa; Lambrechts, Diether; Marchand, Loic Le; Lindblom, Annika; Lubinski, Jan; Mannermaa, Arto; Maranian, Mel; Margolin, Sara; Marme, Frederik; Milne, Roger L.; Neuhausen, Susan L.; Nevanlinna, Heli; Neven, Patrick; Olswold, Curtis; Peto, Julian; Plaseska-Karanfilska, Dijana; Pylkäs, Katri; Radice, Paolo; Rudolph, Anja; Sawyer, Elinor J.; Schmidt, Marjanka K.; Shu, Xiao-Ou; Southey, Melissa C.; Swerdlow, Anthony; Tollenaar, Rob A.E.M.; Tomlinson, Ian; Torres, Diana; Truong, Thérèse; Vachon, Celine; Van Den Ouweland, Ans M. W.; Wang, Qin; Winqvist, Robert; Investigators, kConFab/AOCS; Zheng, Wei; Benitez, Javier; Chenevix-Trench, Georgia; Dunning, Alison M.; Pharoah, Paul D. P.; Kristensen, Vessela; Hall, Per; Easton, Douglas F.; Pastinen, Tomi; Nord, Silje; Simard, Jacques

2016-01-01

There are significant inter-individual differences in the levels of gene expression. Through modulation of gene expression, cis-acting variants represent an important source of phenotypic variation. Consequently, cis-regulatory SNPs associated with differential allelic expression are functional candidates for further investigation as disease-causing variants. To investigate whether common variants associated with differential allelic expression were involved in breast cancer susceptibility, a list of genes was established on the basis of their involvement in cancer related pathways and/or mechanisms. Thereafter, using data from a genome-wide map of allelic expression associated SNPs, 313 genetic variants were selected and their association with breast cancer risk was then evaluated in 46,451 breast cancer cases and 42,599 controls of European ancestry ascertained from 41 studies participating in the Breast Cancer Association Consortium. The associations were evaluated with overall breast cancer risk and with estrogen receptor negative and positive disease. One novel breast cancer susceptibility locus on 4q21 (rs11099601) was identified (OR = 1.05, P = 5.6x10-6). rs11099601 lies in a 135 kb linkage disequilibrium block containing several genes, including, HELQ, encoding the protein HEL308 a DNA dependant ATPase and DNA Helicase involved in DNA repair, MRPS18C encoding the Mitochondrial Ribosomal Protein S18C and FAM175A (ABRAXAS), encoding a BRCA1 BRCT domain-interacting protein involved in DNA damage response and double-strand break (DSB) repair. Expression QTL analysis in breast cancer tissue showed rs11099601 to be associated with HELQ (P = 8.28x10-14), MRPS18C (P = 1.94x10-27) and FAM175A (P = 3.83x10-3), explaining about 20%, 14% and 1%, respectively of the variance inexpression of these genes in breast carcinomas. PMID:27792995

Association of breast cancer risk with genetic variants showing differential allelic expression: Identification of a novel breast cancer susceptibility locus at 4q21.

PubMed

Hamdi, Yosr; Soucy, Penny; Adoue, Véronique; Michailidou, Kyriaki; Canisius, Sander; Lemaçon, Audrey; Droit, Arnaud; Andrulis, Irene L; Anton-Culver, Hoda; Arndt, Volker; Baynes, Caroline; Blomqvist, Carl; Bogdanova, Natalia V; Bojesen, Stig E; Bolla, Manjeet K; Bonanni, Bernardo; Borresen-Dale, Anne-Lise; Brand, Judith S; Brauch, Hiltrud; Brenner, Hermann; Broeks, Annegien; Burwinkel, Barbara; Chang-Claude, Jenny; Couch, Fergus J; Cox, Angela; Cross, Simon S; Czene, Kamila; Darabi, Hatef; Dennis, Joe; Devilee, Peter; Dörk, Thilo; Dos-Santos-Silva, Isabel; Eriksson, Mikael; Fasching, Peter A; Figueroa, Jonine; Flyger, Henrik; García-Closas, Montserrat; Giles, Graham G; Goldberg, Mark S; González-Neira, Anna; Grenaker-Alnæs, Grethe; Guénel, Pascal; Haeberle, Lothar; Haiman, Christopher A; Hamann, Ute; Hallberg, Emily; Hooning, Maartje J; Hopper, John L; Jakubowska, Anna; Jones, Michael; Kabisch, Maria; Kataja, Vesa; Lambrechts, Diether; Le Marchand, Loic; Lindblom, Annika; Lubinski, Jan; Mannermaa, Arto; Maranian, Mel; Margolin, Sara; Marme, Frederik; Milne, Roger L; Neuhausen, Susan L; Nevanlinna, Heli; Neven, Patrick; Olswold, Curtis; Peto, Julian; Plaseska-Karanfilska, Dijana; Pylkäs, Katri; Radice, Paolo; Rudolph, Anja; Sawyer, Elinor J; Schmidt, Marjanka K; Shu, Xiao-Ou; Southey, Melissa C; Swerdlow, Anthony; Tollenaar, Rob A E M; Tomlinson, Ian; Torres, Diana; Truong, Thérèse; Vachon, Celine; Van Den Ouweland, Ans M W; Wang, Qin; Winqvist, Robert; Zheng, Wei; Benitez, Javier; Chenevix-Trench, Georgia; Dunning, Alison M; Pharoah, Paul D P; Kristensen, Vessela; Hall, Per; Easton, Douglas F; Pastinen, Tomi; Nord, Silje; Simard, Jacques

2016-12-06

There are significant inter-individual differences in the levels of gene expression. Through modulation of gene expression, cis-acting variants represent an important source of phenotypic variation. Consequently, cis-regulatory SNPs associated with differential allelic expression are functional candidates for further investigation as disease-causing variants. To investigate whether common variants associated with differential allelic expression were involved in breast cancer susceptibility, a list of genes was established on the basis of their involvement in cancer related pathways and/or mechanisms. Thereafter, using data from a genome-wide map of allelic expression associated SNPs, 313 genetic variants were selected and their association with breast cancer risk was then evaluated in 46,451 breast cancer cases and 42,599 controls of European ancestry ascertained from 41 studies participating in the Breast Cancer Association Consortium. The associations were evaluated with overall breast cancer risk and with estrogen receptor negative and positive disease. One novel breast cancer susceptibility locus on 4q21 (rs11099601) was identified (OR = 1.05, P = 5.6x10-6). rs11099601 lies in a 135 kb linkage disequilibrium block containing several genes, including, HELQ, encoding the protein HEL308 a DNA dependant ATPase and DNA Helicase involved in DNA repair, MRPS18C encoding the Mitochondrial Ribosomal Protein S18C and FAM175A (ABRAXAS), encoding a BRCA1 BRCT domain-interacting protein involved in DNA damage response and double-strand break (DSB) repair. Expression QTL analysis in breast cancer tissue showed rs11099601 to be associated with HELQ (P = 8.28x10-14), MRPS18C (P = 1.94x10-27) and FAM175A (P = 3.83x10-3), explaining about 20%, 14% and 1%, respectively of the variance inexpression of these genes in breast carcinomas.

Three TaFAR genes function in the biosynthesis of primary alcohols and the response to abiotic stresses in Triticum aestivum

PubMed Central

Wang, Meiling; Wang, Yong; Wu, Hongqi; Xu, Jing; Li, Tingting; Hegebarth, Daniela; Jetter, Reinhard; Chen, Letian; Wang, Zhonghua

2016-01-01

Cuticular waxes play crucial roles in protecting plants against biotic and abiotic stresses. They are complex mixtures of very-long-chain fatty acids and their derivatives, including C20–C32 fatty alcohols. Here, we report the identification of 32 FAR-like genes and the detailed characterization of TaFAR2, TaFAR3 and TaFAR4, wax biosynthetic genes encoding fatty acyl-coenzyme A reductase (FAR) in wheat leaf cuticle. Heterologous expression of the three TaFARs in wild-type yeast and mutated yeast showed that TaFAR2, TaFAR3 and TaFAR4 were predominantly responsible for the accumulation of C18:0, C28:0 and C24:0 primary alcohols, respectively. Transgenic expression of the three TaFARs in tomato fruit and Arabidopsis cer4 mutant led to increased production of C22:0–C30:0 primary alcohols. GFP-fusion protein injection assay showed that the three encoded TaFAR proteins were localized to the endoplasmic reticulum (ER), the site of wax biosynthesis. The transcriptional expression of the three TaFAR genes was induced by cold, salt, drought and ABA. Low air humidity led to increased expression of TaFAR genes and elevated wax accumulation in wheat leaves. Collectively, these data suggest that TaFAR2, TaFAR3 and TaFAR4 encode active alcohol-forming FARs involved in the synthesis of primary alcohol in wheat leaf and the response to environmental stresses. PMID:27112792

Therapeutic activities of intravenous immunoglobulins in multiple sclerosis involve modulation of chemokine expression.

PubMed

Pigard, Nadine; Elovaara, Irina; Kuusisto, Hanna; Paalavuo, Raija; Dastidar, Prasun; Zimmermann, Klaus; Schwarz, Hans-Peter; Reipert, Birgit

2009-04-30

The objective of this study was to identify genes that are differentially expressed in peripheral T cells of patients with MS exacerbation receiving treatment with IVIG. Using microarray analysis, we identified 360 genes that were at least two-fold up- or down-regulated. The expression of four representative genes (PTGER4, CXCL5, IL11 and CASP2) was confirmed by quantitative PCR. Four of the differentially expressed genes encode chemokines (CXCL3, CXCL5, CCL13 and XCL2) that are involved in directing leukocyte migration. We suggest that the modulation of chemokine expression in peripheral T cells contributes to the beneficial activity of IVIG in patients with MS exacerbation.

Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants.

PubMed

Lou, Xiao-Ming; Yao, Quan-Hong; Zhang, Zhen; Peng, Ri-He; Xiong, Ai-Sheng; Wang, Hua-Kun

2007-04-01

The original hepatitis B virus (HBV) large surface antigen gene was synthesized. In order to optimize the expression of this gene in tomato plants, the tobacco pathogenesis-related protein S signal peptide was fused to the 5' end of the modified gene and the sequence encoding amino acids S, E, K, D, E, and L was placed at the 3' end. The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants. The expression of the antigen from transgenic plants was confirmed by PCR and reverse transcriptase PCR. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed that the maximal level of HBsAg was about 0.02% of the soluble protein in transgenic tomato fruit. The amount of HBsAg in mature fruits was found to be 65- to 171-fold larger than in small or medium fruits and leaf tissues. Examination of transgenic plant samples by transmission electron microscopy proved that HBsAg had been expressed and had accumulated. The HBsAg protein was capable of assembling into capsomers and virus-like particles. To our knowledge, this is the first time the HBV large surface antigen has been expressed in plants. This work suggests the possibility of producing a new alternative vaccine for human HBV.

Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression

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

Olszewski, Pawel K., E-mail: olsze005@umn.edu; Minnesota Obesity Center, Saint Paul, MN 55108; Fredriksson, Robert

2011-05-13

Highlights: {yields} The majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto. {yields} The level of colocalization is similar in the male and female brain. {yields} Fto overexpression in hypothalamic neurons increases oxytocin mRNA levels by 50%. {yields} Oxytocin does not affect Fto expression through negative feedback mechanisms. -- Abstract: Single nucleotide polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. Alterations in Fto expression in transgenic animals affect body weight, energy expenditure and food intake. Fto, a nuclear protein and proposed transcription co-factor, has been speculated to affect energy balance throughmore » a functional relationship with specific genes encoding feeding-related peptides. Herein, we employed double immunohistochemistry and showed that the majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto in the brain of male and female mice. We then overexpressed Fto in a murine hypothalamic cell line and, using qPCR, detected a 50% increase in the level of oxytocin mRNA. Expression levels of several other feeding-related genes, including neuropeptide Y (NPY) and Agouti-related protein (AgRP), were unaffected by the FTO transfection. Addition of 10 and 100 nmol oxytocin to the cell culture medium did not affect Fto expression in hypothalamic cells. We conclude that Fto, a proposed transcription co-factor, influences expression of the gene encoding a satiety mediator, oxytocin.« less

[Cloning, prokaryotic expression and antibacterial assay of Tenecin gene encoding an antibacterial peptide from Tenebrio molitor].

PubMed

Liu, Ying; Jiang, Yu-xin; Li, Chao-pin

2011-12-01

To clone tenecin gene, an antibacterial peptide gene, from Tenebrio molitor for its prokaryotic expression and explore the molecular mechanism for regulating the expression of antibacterial peptide in Tenebrio molitor larvae. The antibacterial peptide was induced from the larvae of Tenebrio molitor by intraperitoneal injection of Escherichia coli DH-5α (1×10(8)/ml). RT-PCR was performed 72 h after the injection to clone Tenecin gene followed by sequencing and bioinformatic analysis. The recombinant expression vector pET-28a(+)-Tenecin was constructed and transformed into E. coli BL21(DE3) cells and the expression of tenecin protein was observed after IPTG induction. Tenecin expression was detected in transformed E.coli using SDS-PAGE after 1 mmol/L IPTG induction. Tenecin gene, which was about 255 bp in length, encoded Tenecin protein with a relative molecular mass of 9 kD. Incubation of E.coli with 80, 60, 40, and 20 µg/ml tenecin for 18 h resulted in a diameter of the inhibition zone of 25.1∓0.03, 20.7∓0.06, 17.2∓0.11 and 9.3∓0.04 mm, respectively. Tenecin protein possesses strong antibacterial activity against E. coli DH-5α, which warrants further study of this protein for its potential as an antibacterial agent in clinical application.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

1998-10-13

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

Expression of Duplicate msa Genes in the Salmonid Pathogen Renibacterium salmoninarum

PubMed Central

Rhodes, Linda D.; Coady, Alison M.; Strom, Mark S.

2002-01-01

Renibacterium salmoninarum is a gram-positive bacterium responsible for bacterial kidney disease of salmon and trout. R. salmoninarum has two identical copies of the gene encoding major soluble antigen (MSA), an immunodominant, extracellular protein. To determine whether one or both copies of msa are expressed, reporter plasmids encoding a fusion of MSA and green fluorescent protein controlled by 0.6 kb of promoter region from msa1 or msa2 were constructed and introduced into R. salmoninarum. Single copies of the reporter plasmids integrated into the chromosome by homologous recombination. Expression of mRNA and protein from the integrated plasmids was detected, and transformed cells were fluorescent, demonstrating that both msa1 and msa2 are expressed under in vitro conditions. This is the first report of successful transformation and homologous recombination in R. salmoninarum. PMID:12406741

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

1998-01-01

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous gene

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2007-03-20

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2000-08-22

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

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

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

Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant.

PubMed Central

Rentsch, D; Hirner, B; Schmelzer, E; Frommer, W B

1996-01-01

A yeast mutant lacking SHR3, a protein specifically required for correct targeting of plasma membrane amino acid permeases, was used to study the targeting of plant transporters and as a tool to isolate new SHR3-independent amino acid transporters. For this purpose, an shr3 mutant was transformed with an Arabidopsis cDNA library. Thirty-four clones were capable of growth under selective conditions, but none showed homology with SHR3. However, genes encoding eight different amino acid transporters belonging to three different transporter families were isolated. Five of these are members of the general amino acid permease (AAP) gene family, one is a member of the NTR family, encoding an oligopeptide transporter, and two belong to a new class of transporter genes. A functional analysis of the latter two genes revealed that they encode specific proline transporters (ProT) that are distantly related to the AAP gene family. ProT1 was found to be expressed in all organs, but highest levels were found in roots, stems, and flowers. Expression in flowers was highest in the floral stalk phloem that enters the carpels and was downregulated after fertilization, indicating a specific role in supplying the ovules with proline. ProT2 transcripts were found ubiquitously throughout the plant, but expression was strongly induced under water or salt stress, implying that ProT2 plays an important role in nitrogen distribution during water stress, unlike members of the AAP gene family whose expression was repressed under the same conditions. These results corroborate the general finding that under water stress, amino acid export is impaired whereas proline export is increased. PMID:8776904

Using co-expression analysis and stress-based screens to uncover Arabidopsis peroxisomal proteins involved in drought response

DOE PAGES

Li, Jiying; Hu, Jianping; Bassham, Diane

2015-09-14

Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Usingmore » microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their coexpression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Lastly, our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to environmental stresses.« less

Three IgH isotypes, IgM, IgA and IgY are expressed in Gentoo penguin and zebra finch.

PubMed

Han, Binyue; Li, Yan; Han, Haitang; Zhao, Yaofeng; Pan, Qingjie; Ren, Liming

2017-01-01

Previous studies on a limited number of birds suggested that the IgD-encoding gene was absent in birds. However, one of our recent studies showed that the gene was definitely expressed in the ostrich and emu. Interestingly, we also identified subclass diversification of IgM and IgY in these two birds. To better understand immunoglobulin genes in birds, in this study, we analyzed the immunoglobulin heavy chain genes in the zebra finch (Taeniopygia guttata) and Gentoo penguin (Pygoscelis papua), belonging respectively to the order Passeriformes, the most successful bird order in terms of species diversity and numbers, and Sphenisciformes, a relatively primitive avian order. Similar to the results obtained in chickens and ducks, only three genes encoding immunoglobulin heavy chain isotypes, IgM, IgA and IgY, were identified in both species. Besides, we detected a transcript encoding a short membrane-bound IgA lacking the last two CH exons in the Gentoo penguin. We did not find any evidence supporting the presence of IgD gene or subclass diversification of IgM/IgY in penguin or zebra finch. The obtained data in our study provide more insights into the immunoglobulin heavy chain genes in birds and may help to better understand the evolution of immunoglobulin genes in tetrapods.

Unbiased View of Synaptic and Neuronal Gene Complement in Ctenophores: Are There Pan-neuronal and Pan-synaptic Genes across Metazoa?

PubMed

Moroz, Leonid L; Kohn, Andrea B

2015-12-01

Hypotheses of origins and evolution of neurons and synapses are controversial, mostly due to limited comparative data. Here, we investigated the genome-wide distribution of the bilaterian "synaptic" and "neuronal" protein-coding genes in non-bilaterian basal metazoans (Ctenophora, Porifera, Placozoa, and Cnidaria). First, there are no recognized genes uniquely expressed in neurons across all metazoan lineages. None of the so-called pan-neuronal genes such as embryonic lethal abnormal vision (ELAV), Musashi, or Neuroglobin are expressed exclusively in neurons of the ctenophore Pleurobrachia. Second, our comparative analysis of about 200 genes encoding canonical presynaptic and postsynaptic proteins in bilaterians suggests that there are no true "pan-synaptic" genes or genes uniquely and specifically attributed to all classes of synapses. The majority of these genes encode receptive and secretory complexes in a broad spectrum of eukaryotes. Trichoplax (Placozoa) an organism without neurons and synapses has more orthologs of bilaterian synapse-related/neuron-related genes than do ctenophores-the group with well-developed neuronal and synaptic organization. Third, the majority of genes encoding ion channels and ionotropic receptors are broadly expressed in unicellular eukaryotes and non-neuronal tissues in metazoans. Therefore, they cannot be viewed as neuronal markers. Nevertheless, the co-expression of multiple types of ion channels and receptors does correlate with the presence of neural and synaptic organization. As an illustrative example, the ctenophore genomes encode a greater diversity of ion channels and ionotropic receptors compared with the genomes of the placozoan Trichoplax and the demosponge Amphimedon. Surprisingly, both placozoans and sponges have a similar number of orthologs of "synaptic" proteins as we identified in the genomes of two ctenophores. Ctenophores have a distinct synaptic organization compared with other animals. Our analysis of transcriptomes from 10 different ctenophores did not detect recognized orthologs of synthetic enzymes encoding several classical, low-molecular-weight (neuro)transmitters; glutamate signaling machinery is one of the few exceptions. Novel peptidergic signaling molecules were predicted for ctenophores, together with the diversity of putative receptors including SCNN1/amiloride-sensitive sodium channel-like channels, many of which could be examples of a lineage-specific expansion within this group. In summary, our analysis supports the hypothesis of independent evolution of neurons and, as corollary, a parallel evolution of synapses. We suggest that the formation of synaptic machinery might occur more than once over 600 million years of animal evolution. © The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Transcription Factors Expressed in Lateral Organ Boundaries: Identification of Downstream Targets

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

Springer, Patricia S

2010-07-12

The processes of lateral organ initiation and patterning are central to the generation of mature plant form. Characterization of the molecular mechanisms underlying these processes is essential to our understanding of plant development. Communication between the shoot apical meristem and initiating organ primordia is important both for functioning of the meristem and for proper organ patterning, and very little is known about this process. In particular, the boundary between meristem and leaf is emerging as a critical region that is important for SAM maintenance and regulation of organogenesis. The goal of this project was to characterize three boundary-expressed genes thatmore » encode predicted transcription factors. Specifically, we have studied LATERAL ORGAN BOUNDARIES (LOB), LATERAL ORGAN FUSION1 (LOF1), and LATERAL ORGAN FUSION2 (LOF2). LOB encodes the founding member of the LOB-DOMAIN (LBD) plant-specific DNA binding transcription factor family and LOF1 and LOF2 encode paralogous MYB-domain transcription factors. We characterized the genetic relationship between these three genes and other boundary and meristem genes. We also used an ectopic inducible expression system to identify direct targets of LOB.« less

SGRL can regulate chlorophyll metabolism and contributes to normal plant growth and development in Pisum sativum L.

PubMed

Bell, Andrew; Moreau, Carol; Chinoy, Catherine; Spanner, Rebecca; Dalmais, Marion; Le Signor, Christine; Bendahmane, Abdel; Klenell, Markus; Domoney, Claire

2015-12-01

Among a set of genes in pea (Pisum sativum L.) that were induced under drought-stress growth conditions, one encoded a protein with significant similarity to a regulator of chlorophyll catabolism, SGR. This gene, SGRL, is distinct from SGR in genomic location, encoded carboxy-terminal motif, and expression through plant and seed development. Divergence of the two encoded proteins is associated with a loss of similarity in intron/exon gene structure. Transient expression of SGRL in leaves of Nicotiana benthamiana promoted the degradation of chlorophyll, in a manner that was distinct from that shown by SGR. Removal of a predicted transmembrane domain from SGRL reduced its activity in transient expression assays, although variants with and without this domain reduced SGR-induced chlorophyll degradation, indicating that the effects of the two proteins are not additive. The combined data suggest that the function of SGRL during growth and development is in chlorophyll re-cycling, and its mode of action is distinct from that of SGR. Studies of pea sgrL mutants revealed that plants had significantly lower stature and yield, a likely consequence of reduced photosynthetic efficiencies in mutant compared with control plants under conditions of high light intensity.

Impact of haloperidol and quetiapine on the expression of genes encoding antioxidant enzymes in human neuroblastoma SH-SY5Y cells.

PubMed

Schmidt, Andreas Johannes; Hemmeter, Ulrich Michael; Krieg, Jürgen-Christian; Vedder, Helmut; Heiser, Philip

2009-05-01

Antipsychotics are known to alter antioxidant activities in vivo. Therefore, the aim of the present study was to examine in the human neuroblastoma SH-SY5Y cell line the impact of a typical (haloperidol) and an atypical (quetiapine) antipsychotic on the expression of genes encoding the key enzymes of the antioxidant metabolism (Cu, Zn superoxide dismutase; Mn superoxide dismutase; glutathione peroxidase; catalase) and enzymes of the glutathione metabolism (gamma-glutamyl cysteine synthetase, glutathione-S-transferase, gamma-glutamyltranspeptidase, glutathione reductase). The cells were incubated for 24h with 0.3, 3, 30 and 300microM haloperidol and quetiapine, respectively; mRNA levels were measured by polymerase chain reaction. In the present study, we observed mostly significant decreases of mRNA contents. With respect to the key pathways, we detected mainly effects on the mRNA levels of the hydrogen peroxide detoxifying enzymes. Among the enzymes of the glutathione metabolism, glutathione-S-transferase- and gamma-glutamyltranspeptidase-mRNA levels showed the most prominent effects. Taken together, our results demonstrate a significantly reduced expression of genes encoding for antioxidant enzymes after treatment with the antipsychotics, haloperidol and quetiapine.

Functional characterization of the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato plants.

PubMed

Yang, Qingjie; Yuan, Dawei; Shi, Lianxuan; Capell, Teresa; Bai, Chao; Wen, Nuan; Lu, Xiaodan; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

2012-10-01

The accumulation of carotenoids in plants depends critically on the spatiotemporal expression profiles of the genes encoding enzymes in the carotenogenic pathway. We cloned and characterized the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter to determine its role in the regulation of carotenogenesis, because the native gene is expressed at high levels in petals, which contain abundant chromoplasts. We transformed tomato (Solanum lycopersicum cv. Micro-Tom) plants with the gusA gene encoding the reporter enzyme β-glucuronidase (GUS) under the control of the GlZEP promoter, and investigated the reporter expression profile at the mRNA and protein levels. We detected high levels of gusA expression and GUS activity in chromoplast-containing flowers and fruits, but minimal levels in immature fruits containing green chloroplasts, in sepals, leaves, stems and roots. GlZEP-gusA expression was strictly associated with fruit development and chromoplast differentiation, suggesting an evolutionarily-conserved link between ZEP and the differentiation of organelles that store carotenoid pigments. The impact of our results on current models for the regulation of carotenogenesis in plants is discussed.

Product-induced gene expression, a product-responsive reporter assay used to screen metagenomic libraries for enzyme-encoding genes.

PubMed

Uchiyama, Taku; Miyazaki, Kentaro

2010-11-01

A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.

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.

Identifying metabolic enzymes with multiple types of association evidence

PubMed Central

Kharchenko, Peter; Chen, Lifeng; Freund, Yoav; Vitkup, Dennis; Church, George M

2006-01-01

Background Existing large-scale metabolic models of sequenced organisms commonly include enzymatic functions which can not be attributed to any gene in that organism. Existing computational strategies for identifying such missing genes rely primarily on sequence homology to known enzyme-encoding genes. Results We present a novel method for identifying genes encoding for a specific metabolic function based on a local structure of metabolic network and multiple types of functional association evidence, including clustering of genes on the chromosome, similarity of phylogenetic profiles, gene expression, protein fusion events and others. Using E. coli and S. cerevisiae metabolic networks, we illustrate predictive ability of each individual type of association evidence and show that significantly better predictions can be obtained based on the combination of all data. In this way our method is able to predict 60% of enzyme-encoding genes of E. coli metabolism within the top 10 (out of 3551) candidates for their enzymatic function, and as a top candidate within 43% of the cases. Conclusion We illustrate that a combination of genome context and other functional association evidence is effective in predicting genes encoding metabolic enzymes. Our approach does not rely on direct sequence homology to known enzyme-encoding genes, and can be used in conjunction with traditional homology-based metabolic reconstruction methods. The method can also be used to target orphan metabolic activities. PMID:16571130

Expression and characterization of hyperthermostable exo-polygalacturonase TtGH28 from Thermotoga thermophilus

USDA-ARS?s Scientific Manuscript database

The gene TtGH28 encoding a putative GH28 polygalacturonase from Pseudothermotoga thermarum DSM 5069 (Theth_0397, NCBI# AEH50492.1) was synthesized, expressed in E. coli, and characterized. Alignment of the amino acid sequence of gene product TtGH28 with other GH28 proteins whose structures and detai...

Analysis of functions of the chitin deacetylase gene family in Tribolium castaneum

USDA-ARS?s Scientific Manuscript database

The expression profiles of nine genes encoding chitin deacetylase (CDA)-like proteins were studied during development and in various tissues of the red flour beetle, Tribolium castaneum, by RT-PCR. TcCDA1, TcCDA2 and TcCDA5 were expressed throughout all stages of development, while TcCDA6 – 9 were ...

Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor gene family encode potential rice allergens.

PubMed

Alvarez, A M; Fukuhara, E; Nakase, M; Adachi, T; Aoki, N; Nakamura, R; Matsuda, T

1995-07-01

Four rice seed proteins encoded by cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.

Gene Mining for Proline Based Signaling Proteins in Cell Wall of Arabidopsis thaliana

PubMed Central

Ihsan, Muhammad Z.; Ahmad, Samina J. N.; Shah, Zahid Hussain; Rehman, Hafiz M.; Aslam, Zubair; Ahuja, Ishita; Bones, Atle M.; Ahmad, Jam N.

2017-01-01

The cell wall (CW) as a first line of defense against biotic and abiotic stresses is of primary importance in plant biology. The proteins associated with cell walls play a significant role in determining a plant's sustainability to adverse environmental conditions. In this work, the genes encoding cell wall proteins (CWPs) in Arabidopsis were identified and functionally classified using geneMANIA and GENEVESTIGATOR with published microarrays data. This yielded 1605 genes, out of which 58 genes encoded proline-rich proteins (PRPs) and glycine-rich proteins (GRPs). Here, we have focused on the cellular compartmentalization, biological processes, and molecular functioning of proline-rich CWPs along with their expression at different plant developmental stages. The mined genes were categorized into five classes on the basis of the type of PRPs encoded in the cell wall of Arabidopsis thaliana. We review the domain structure and function of each class of protein, many with respect to the developmental stages of the plant. We have then used networks, hierarchical clustering and correlations to analyze co-expression, co-localization, genetic, and physical interactions and shared protein domains of these PRPs. This has given us further insight into these functionally important CWPs and identified a number of potentially new cell-wall related proteins in A. thaliana. PMID:28289422

Environmental stress alters genes expression and induces ovule abortion: reactive oxygen species appear as ovules commit to abort.

PubMed

Sun, Kelian; Cui, Yuehua; Hauser, Bernard A

2005-11-01

Environmental stress dramatically reduces plant reproduction. Previous results showed that placing roots in 200 mM NaCl for 12 h caused 90% of the developing Arabidopsis ovules to abort (Sun et al. in Plant Physiol 135:2358-2367, 2004). To discover the molecular responses that occur during ovule abortion, gene expression was monitored using Affymetrix 24k genome arrays. Transcript levels were measured in pistils that were stressed for 6, 12, 18, and 24 h, then compared with the levels in healthy pistils. Over the course of this experiment, a total of 535 salt-responsive genes were identified. Cluster analysis showed that differentially expressed genes exhibited reproducible changes in expression. The expression of 65 transcription factors, some of which are known to be involved in stress responses, were modulated during ovule abortion. In flowers, salt stress led to a 30-fold increase in Na+ ions and modest, but significant, decreases in the accumulation of other ions. The expression of cation exchangers and ion transporters were induced, presumably to reestablish ion homeostasis following salt stress. Genes that encode enzymes that detoxify reactive oxygen species (ROS), including ascorbate peroxidase and peroxidase, were downregulated after ovules committed to abort. These changes in gene expression coincided with the synthesis of ROS in female gametophytes. One day after salt stress, ROS spread from the gametophytes to the maternal chalaza and integuments. In addition, genes encoding proteins that regulate ethylene responses, including ethylene biosynthesis, ethylene signal transduction and ethylene-responsive transcription factors, were upregulated after stress. Hypotheses are proposed on the basis of this expression analysis, which will be evaluated further in future experiments.

Activation of ethylene-responsive p-hydroxyphenylpyruvate dioxygenase leads to increased tocopherol levels during ripening in mango

PubMed Central

Singh, Rajesh K.; Ali, Sharique A.; Nath, Pravendra; Sane, Vidhu A.

2011-01-01

Mango is characterized by high tocopherol and carotenoid content during ripening. From a cDNA screen of differentially expressing genes during mango ripening, a full-length p-hydroxyphenylpyruvate dioxygenase (MiHPPD) gene homologue was isolated that encodes a key enzyme in the biosynthesis of tocopherols. The gene encoded a 432-amino-acid protein. Transcript analysis during different stages of ripening revealed that the gene is ripening related and rapidly induced by ethylene. The increase in MiHPPD transcript accumulation was followed by an increase in tocopherol levels during ripening. The ripening-related increase in MiHPPD expression was also seen in response to abscisic acid and to alesser extent to indole-3-acetic acid. The expression of MiHPPD was not restricted to fruits but was also seen in other tissues such as leaves particularly during senescence. The strong ethylene induction of MiHPPD was also seen in young leaves indicating that ethylene induction of MiHPPD is tissue independent. Promoter analysis of MiHPPD gene in tomato discs and leaves of stable transgenic lines of Arabidopsis showed that the cis elements for ripening-related, ethylene-responsive, and senescence-related expression resided within the 1590 nt region upstream of the ATG codon. Functionality of the gene was demonstrated by the ability of the expressed protein in bacteria to convert p-hydroxyphenylpyruvate to homogentisate. These results provide the first evidence for HPPD expression during ripening of a climacteric fruit. PMID:21430290

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

PubMed

Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

2017-08-01

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

GiFRD encodes a protein involved in anaerobic growth in the arbuscular mycorrhizal fungus Glomus intraradices.

PubMed

Sędzielewska, Kinga A; Vetter, Katja; Bode, Rüdiger; Baronian, Keith; Watzke, Roland; Kunze, Gotthard

2012-04-01

Fumarate reductase is a protein involved in the maintenance of redox balance during oxygen deficiency. This enzyme irreversibly catalyzes the reduction of fumarate to succinate and requires flavin cofactors as electron donors. Two examples are the soluble mitochondrial and the cytosolic fumarate reductases of Saccharomyces cerevisiae encoded by the OSM1 and FRDS1 genes, respectively. This work reports the identification and characterization of the gene encoding cytosolic fumarate reductase enzyme in the arbuscular mycorrhizal fungus, Glomus intraradices and the establishment of its physiological role. Using a yeast expression system, we demonstrate that G. intraradices GiFRD encodes a protein that has fumarate reductase activity which can functionally substitute for the S. cerevisiae fumarate reductases. Additionally, we showed that GiFRD transformants are not affected by presence of salt in medium, indicating that the presence of this gene has no effect on yeast behavior under osmotic stress. The fact that GiFRD expression and enzymatic activity was present only in asymbiotic stage confirmed existence of at least one anaerobic metabolic pathway in this phase of fungus life cycle. This suggests that the AMF behave as facultative anaerobes in the asymbiotic stage. Copyright © 2012 Elsevier Inc. All rights reserved.

Plasimids containing the gene for DNA polymerase I from Streptococcus pneumoniae

DOEpatents

Lacks, Sanford A.; Martinez, Susana; Lopez, Paloma; Espinosa, Manuel

1991-01-01

A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of Streptococcus pneumoniae. Plasmid pSM22, the vector containing the pneumocccal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme.

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

The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells.

PubMed

Jiwaji, Meesbah; Daly, Rónán; Pansare, Kshama; McLean, Pauline; Yang, Jingli; Kolch, Walter; Pitt, Andrew R

2010-12-31

The importance of appropriate normalization controls in quantitative real-time polymerase chain reaction (qPCR) experiments has become more apparent as the number of biological studies using this methodology has increased. In developing a system to study gene expression from transiently transfected plasmids, it became clear that normalization using chromosomally encoded genes is not ideal, at it does not take into account the transfection efficiency and the significantly lower expression levels of the plasmids. We have developed and validated a normalization method for qPCR using a co-transfected plasmid. The best chromosomal gene for normalization in the presence of the transcriptional activators used in this study, cadmium, dexamethasone, forskolin and phorbol-12-myristate 13-acetate was first identified. qPCR data was analyzed using geNorm, Normfinder and BestKeeper. Each software application was found to rank the normalization controls differently with no clear correlation. Including a co-transfected plasmid encoding the Renilla luciferase gene (Rluc) in this analysis showed that its calculated stability was not as good as the optimised chromosomal genes, most likely as a result of the lower expression levels and transfection variability. Finally, we validated these analyses by testing two chromosomal genes (B2M and ActB) and a co-transfected gene (Rluc) under biological conditions. When analyzing co-transfected plasmids, Rluc normalization gave the smallest errors compared to the chromosomal reference genes. Our data demonstrates that transfected Rluc is the most appropriate normalization reference gene for transient transfection qPCR analysis; it significantly reduces the standard deviation within biological experiments as it takes into account the transfection efficiencies and has easily controllable expression levels. This improves reproducibility, data validity and most importantly, enables accurate interpretation of qPCR data.

Bovine Herpes Virus 1 (BHV-1) and Herpes Simplex Virus Type 1 (HSV-1) Promote Survival of Latently Infected Sensory Neurons, in Part by Inhibiting Apoptosis

PubMed Central

Jones, Clinton

2013-01-01

α-Herpesvirinae subfamily members, including herpes simplex virus type 1 (HSV-1) and bovine herpes virus 1 (BHV-1), initiate infection in mucosal surfaces. BHV-1 and HSV-1 enter sensory neurons by cell-cell spread where a burst of viral gene expression occurs. When compared to non-neuronal cells, viral gene expression is quickly extinguished in sensory neurons resulting in neuronal survival and latency. The HSV-1 latency associated transcript (LAT), which is abundantly expressed in latently infected neurons, inhibits apoptosis, viral transcription, and productive infection, and directly or indirectly enhances reactivation from latency in small animal models. Three anti-apoptosis genes can be substituted for LAT, which will restore wild type levels of reactivation from latency to a LAT null mutant virus. Two small non-coding RNAs encoded by LAT possess anti-apoptosis functions in transfected cells. The BHV-1 latency related RNA (LR-RNA), like LAT, is abundantly expressed during latency. The LR-RNA encodes a protein (ORF2) and two microRNAs that are expressed in certain latently infected neurons. Wild-type expression of LR gene products is required for stress-induced reactivation from latency in cattle. ORF2 has anti-apoptosis functions and interacts with certain cellular transcription factors that stimulate viral transcription and productive infection. ORF2 is predicted to promote survival of infected neurons by inhibiting apoptosis and sequestering cellular transcription factors which stimulate productive infection. In addition, the LR encoded microRNAs inhibit viral transcription and apoptosis. In summary, the ability of BHV-1 and HSV-1 to interfere with apoptosis and productive infection in sensory neurons is crucial for the life-long latency-reactivation cycle in their respective hosts. PMID:25278776

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 novel, broad-range, CTXΦ-derived stable integrative expression vector for functional studies.

PubMed

Das, Bhabatosh; Kumari, Reena; Pant, Archana; Sen Gupta, Sourav; Saxena, Shruti; Mehta, Ojasvi; Nair, Gopinath Balakrish

2014-12-01

CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Overlapping Podospora anserina Transcriptional Responses to Bacterial and Fungal Non Self Indicate a Multilayered Innate Immune Response

PubMed Central

Lamacchia, Marina; Dyrka, Witold; Breton, Annick; Saupe, Sven J.; Paoletti, Mathieu

2016-01-01

Recognition and response to non self is essential to development and survival of all organisms. It can occur between individuals of the same species or between different organisms. Fungi are established models for conspecific non self recognition in the form of vegetative incompatibility (VI), a genetically controlled process initiating a programmed cell death (PCD) leading to the rejection of a fusion cell between genetically different isolates of the same species. In Podospora anserina VI is controlled by members of the hnwd gene family encoding for proteins analogous to NOD Like Receptors (NLR) immune receptors in eukaryotes. It was hypothesized that the hnwd controlled VI reaction was derived from the fungal innate immune response. Here we analyze the P. anserina transcriptional responses to two bacterial species, Serratia fonticola to which P. anserina survives and S. marcescens to which P. anserina succumbs, and compare these to the transcriptional response induced under VI conditions. Transcriptional responses to both bacteria largely overlap, however the number of genes regulated and magnitude of regulation is more important when P. anserina survives. Transcriptional responses to bacteria also overlap with the VI reaction for both up or down regulated gene sets. Genes up regulated tend to be clustered in the genome, and display limited phylogenetic distribution. In all three responses we observed genes related to autophagy to be up-regulated. Autophagy contributes to the fungal survival in all three conditions. Genes encoding for secondary metabolites and histidine kinase signaling are also up regulated in all three conditions. Transcriptional responses also display differences. Genes involved in response to oxidative stress, or encoding small secreted proteins are essentially expressed in response to bacteria, while genes encoding NLR proteins are expressed during VI. Most functions encoded in response to bacteria favor survival of the fungus while most functions up regulated during VI would lead to cell death. These differences are discussed in the frame of a multilayered response to non self in fungi. PMID:27148175

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

Stols, L.; Donnelly, M.I.; Kulkarni, G.

The malic enzyme gene of Ascaris suum was cloned into the vector pTRC99a in two forms encoding alternative amino-termini. The resulting plasmids, pMEA1 and pMEA2, were introduced into Escherichia coli NZN111, a strain that is unable to grow fermentatively because of inactivation of the genes encoding pyruvate dissimilation. Induction of pMEA1, which encodes the native animoterminus, gave better overexpression of malic enzyme, approx 12-fold compared to uninduced cells. Under the appropriate culture conditions, expression of malic enzyme allowed the fermentative dissimilation of glucose by NZN111. The major fermentation product formed in induced cultures was succinic acid.

Role of fruA and csgA genes in gene expression during development of Myxococcus xanthus. Analysis by two-dimensional gel electrophoresis.

PubMed

Horiuchi, Takayuki; Taoka, Masato; Isobe, Toshiaki; Komano, Teruya; Inouye, Sumiko

2002-07-26

Two genes, fruA and csgA, encoding a putative transcription factor and C-factor, respectively, are essential for fruiting body formation of Myxococcus xanthus. To investigate the role of fruA and csgA genes in developmental gene expression, developing cells as well as vegetative cells of M. xanthus wild-type, fruA::Tc, and csgA731 strains were pulse-labeled with [(35)S]methionine, and the whole cell proteins were analyzed using two-dimensional immobilized pH gradient/SDS-PAGE. Differences in protein synthesis patterns among more than 700 protein spots were detected during development of the three strains. Fourteen proteins showing distinctly different expression patterns in mutant cells were analyzed in more detail. Five of the 14 proteins were identified as elongation factor Tu (EF-Tu), Dru, DofA, FruA, and protein S by immunoblot analysis and mass spectroscopy. A gene encoding DofA was cloned and sequenced. Although both fruA and csgA genes regulate early development of M. xanthus, they were found to differently regulate expression of several developmental genes. The production of six proteins, including DofA and protein S, was dependent on fruA, whereas the production of two proteins was dependent on csgA, and one protein was dependent on both fruA and csgA. To explain the present findings, a new model was presented in which different levels of FruA phosphorylation may distinctively regulate the expression of two groups of developmental genes.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

PubMed Central

2010-01-01

Background Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. Conclusions Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon. PMID:20799976

FluG affects secretion in colonies of Aspergillus niger.

PubMed

Wang, Fengfeng; Krijgsheld, Pauline; Hulsman, Marc; de Bekker, Charissa; Müller, Wally H; Reinders, Marcel; de Vries, Ronald P; Wösten, Han A B

2015-01-01

Colonies of Aspergillus niger are characterized by zonal heterogeneity in growth, sporulation, gene expression and secretion. For instance, the glucoamylase gene glaA is more highly expressed at the periphery of colonies when compared to the center. As a consequence, its encoded protein GlaA is mainly secreted at the outer part of the colony. Here, multiple copies of amyR were introduced in A. niger. Most transformants over-expressing this regulatory gene of amylolytic genes still displayed heterogeneous glaA expression and GlaA secretion. However, heterogeneity was abolished in transformant UU-A001.13 by expressing glaA and secreting GlaA throughout the mycelium. Sequencing the genome of UU-A001.13 revealed that transformation had been accompanied by deletion of part of the fluG gene and disrupting its 3' end by integration of a transformation vector. Inactivation of fluG in the wild-type background of A. niger also resulted in breakdown of starch under the whole colony. Asexual development of the ∆fluG strain was not affected, unlike what was previously shown in Aspergillus nidulans. Genes encoding proteins with a signal sequence for secretion, including part of the amylolytic genes, were more often downregulated in the central zone of maltose-grown ∆fluG colonies and upregulated in the intermediate part and periphery when compared to the wild-type. Together, these data indicate that FluG of A. niger is a repressor of secretion.

Functional genomic responses to cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR(delta508) in the lung.

PubMed

Xu, Yan; Liu, Cong; Clark, Jean C; Whitsett, Jeffrey A

2006-04-21

Cystic fibrosis (CF), a common lethal pulmonary disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that disturbs fluid homeostasis and host defense in target organs. The effects of CFTR and delta508-CFTR were assessed in transgenic mice that 1) lack CFTR expression (Cftr-/-); 2) express the human delta508 CFTR (CFTR(delta508)); 3) overexpress the normal human CFTR (CFTR(tg)) in respiratory epithelial cells. Genes were selected from Affymetrix Murine Gene-Chips analysis and subjected to functional classification, k-means clustering, promoter cis-elements/modules searching, literature mining, and pathway exploring. Genomic responses to Cftr-/- were not corrected by expression of CFTR(delta508). Genes regulating host defense, inflammation, fluid and electrolyte transport were similarly altered in Cftr-/- and CFTR(delta508) mice. CFTR(delta508) induced a primary disturbance in expression of genes regulating redox and antioxidant systems. Genomic responses to CFTR(tg) were modest and were not associated with lung pathology. CFTR(tg) and CFTR(delta508) induced genes encoding heat shock proteins and other chaperones but did not activate the endoplasmic reticulum-associated degradation pathway. RNAs encoding proteins that directly interact with CFTR were identified in each of the CFTR mouse models, supporting the hypothesis that CFTR functions within a multiprotein complex whose members interact at the level of protein-protein interactions and gene expression. Promoters of genes influenced by CFTR shared common regulatory elements, suggesting that their co-expression may be mediated by shared regulatory mechanisms. Genes and pathways involved in the response to CFTR may be of interest as modifiers of CF.

Modulation of Gene Expression in Actinobacillus pleuropneumoniae Exposed to Bronchoalveolar Fluid

PubMed Central

Lone, Abdul G.; Deslandes, Vincent; Nash, John H. E.; Jacques, Mario; MacInnes, Janet I.

2009-01-01

Background Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia, is an important pathogen of swine throughout the world. It must rapidly overcome the innate pulmonary immune defenses of the pig to cause disease. To better understand this process, the objective of this study was to identify genes that are differentially expressed in a medium that mimics the lung environment early in the infection process. Methods and Principal Findings Since bronchoalveolar lavage fluid (BALF) contains innate immune and other components found in the lungs, we examined gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after a 30 min exposure to BALF, using DNA microarrays and real-time PCR. The functional classes of genes found to be up-regulated most often in BALF were those encoding proteins involved in energy metabolism, especially anaerobic metabolism, and in cell envelope, DNA, and protein biosynthesis. Transcription of a number of known virulence genes including apxIVA and the gene for SapF, a protein which is involved in resistance to antimicrobial peptides, was also up-regulated in BALF. Seventy-nine percent of the genes that were up-regulated in BALF encoded a known protein product, and of these, 44% had been reported to be either expressed in vivo and/or involved in virulence. Conclusions The results of this study suggest that in early stages of infection, A. pleuropneumoniae may modulate expression of genes involved in anaerobic energy generation and in the synthesis of proteins involved in cell wall biogenesis, as well as established virulence factors. Given that many of these genes are thought to be expressed in vivo or involved in virulence, incubation in BALF appears, at least partially, to simulate in vivo conditions and may provide a useful medium for the discovery of novel vaccine or therapeutic targets. PMID:19578537

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae.

PubMed

Schoeman, H; Vivier, M A; Du Toit, M; Dicks, L M; Pretorius, I S

1999-06-15

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end-products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast-based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1.0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA-1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA-1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA-1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1P-MFa1S-pedA-ADH1T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1P) and terminator (ADH1T). Secretion of the PED1-encoded pediocin PA-1 was directed by the yeast mating pheromone alpha-factor's secretion signal (MFa1S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4.6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria.

Gene co-expression analysis identifies gene clusters associated with isotropic and polarized growth in Aspergillus fumigatus conidia.

PubMed

Baltussen, Tim J H; Coolen, Jordy P M; Zoll, Jan; Verweij, Paul E; Melchers, Willem J G

2018-04-26

Aspergillus fumigatus is a saprophytic fungus that extensively produces conidia. These microscopic asexually reproductive structures are small enough to reach the lungs. Germination of conidia followed by hyphal growth inside human lungs is a key step in the establishment of infection in immunocompromised patients. RNA-Seq was used to analyze the transcriptome of dormant and germinating A. fumigatus conidia. Construction of a gene co-expression network revealed four gene clusters (modules) correlated with a growth phase (dormant, isotropic growth, polarized growth). Transcripts levels of genes encoding for secondary metabolites were high in dormant conidia. During isotropic growth, transcript levels of genes involved in cell wall modifications increased. Two modules encoding for growth and cell cycle/DNA processing were associated with polarized growth. In addition, the co-expression network was used to identify highly connected intermodular hub genes. These genes may have a pivotal role in the respective module and could therefore be compelling therapeutic targets. Generally, cell wall remodeling is an important process during isotropic and polarized growth, characterized by an increase of transcripts coding for hyphal growth and cell cycle/DNA processing when polarized growth is initiated. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Evolutionary and Expression Analyses of the Apple Basic Leucine Zipper Transcription Factor Family

PubMed Central

Zhao, Jiao; Guo, Rongrong; Guo, Chunlei; Hou, Hongmin; Wang, Xiping; Gao, Hua

2016-01-01

Transcription factors (TFs) play essential roles in the regulatory networks controlling many developmental processes in plants. Members of the basic leucine (Leu) zipper (bZIP) TF family, which is unique to eukaryotes, are involved in regulating diverse processes, including flower and vascular development, seed maturation, stress signaling, and defense responses to pathogens. The bZIP proteins have a characteristic bZIP domain composed of a DNA-binding basic region and a Leu zipper dimerization region. In this study, we identified 112 apple (Malus domestica Borkh) bZIP TF-encoding genes, termed MdbZIP genes. Synteny analysis indicated that segmental and tandem duplication events, as well as whole genome duplication, have contributed to the expansion of the apple bZIP family. The family could be divided into 11 groups based on structural features of the encoded proteins, as well as on the phylogenetic relationship of the apple bZIP proteins to those of the model plant Arabidopsis thaliana (AtbZIP genes). Synteny analysis revealed that several paired MdbZIP genes and AtbZIP gene homologs were located in syntenic genomic regions. Furthermore, expression analyses of group A MdbZIP genes showed distinct expression levels in 10 different organs. Moreover, changes in these expression profiles in response to abiotic stress conditions and various hormone treatments identified MdbZIP genes that were responsive to high salinity and drought, as well as to different phytohormones. PMID:27066030

Evolutionary and Expression Analyses of the Apple Basic Leucine Zipper Transcription Factor Family.

PubMed

Zhao, Jiao; Guo, Rongrong; Guo, Chunlei; Hou, Hongmin; Wang, Xiping; Gao, Hua

2016-01-01

Transcription factors (TFs) play essential roles in the regulatory networks controlling many developmental processes in plants. Members of the basic leucine (Leu) zipper (bZIP) TF family, which is unique to eukaryotes, are involved in regulating diverse processes, including flower and vascular development, seed maturation, stress signaling, and defense responses to pathogens. The bZIP proteins have a characteristic bZIP domain composed of a DNA-binding basic region and a Leu zipper dimerization region. In this study, we identified 112 apple (Malus domestica Borkh) bZIP TF-encoding genes, termed MdbZIP genes. Synteny analysis indicated that segmental and tandem duplication events, as well as whole genome duplication, have contributed to the expansion of the apple bZIP family. The family could be divided into 11 groups based on structural features of the encoded proteins, as well as on the phylogenetic relationship of the apple bZIP proteins to those of the model plant Arabidopsis thaliana (AtbZIP genes). Synteny analysis revealed that several paired MdbZIP genes and AtbZIP gene homologs were located in syntenic genomic regions. Furthermore, expression analyses of group A MdbZIP genes showed distinct expression levels in 10 different organs. Moreover, changes in these expression profiles in response to abiotic stress conditions and various hormone treatments identified MdbZIP genes that were responsive to high salinity and drought, as well as to different phytohormones.

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

PubMed Central

Raabe, Carsten A.; Sanchez, Cecilia P.; Randau, Gerrit; Robeck, Thomas; Skryabin, Boris V.; Chinni, Suresh V.; Kube, Michael; Reinhardt, Richard; Ng, Guey Hooi; Manickam, Ravichandran; Kuryshev, Vladimir Y.; Lanzer, Michael; Brosius, Juergen; Tang, Thean Hock; Rozhdestvensky, Timofey S.

2010-01-01

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors. PMID:19864253

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum.

PubMed

Raabe, Carsten A; Sanchez, Cecilia P; Randau, Gerrit; Robeck, Thomas; Skryabin, Boris V; Chinni, Suresh V; Kube, Michael; Reinhardt, Richard; Ng, Guey Hooi; Manickam, Ravichandran; Kuryshev, Vladimir Y; Lanzer, Michael; Brosius, Juergen; Tang, Thean Hock; Rozhdestvensky, Timofey S

2010-01-01

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense-antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors.

Senescence-inducible LEC2 enhances triacylglycerol accumulation in leaves without negatively affecting plant growth

PubMed Central

Kim, Hyun Uk; Lee, Kyeong-Ryeol; Jung, Su-Jin; Shin, Hyun A; Go, Young Sam; Suh, Mi-Chung; Kim, Jong Bum

2017-01-01

Summary The synthesis of fatty acids and glycerolipids in wild-type Arabidopsis leaves do not typically lead to strong triacylglycerol (TAG) accumulation. LEAFY COTYLEDON2 (LEC2) is a master regulator of seed maturation and oil accumulation in seeds. Constitutive ectopic LEC2 expression causes somatic embryogenesis and defects in seedling growth. Here, we report that senescence-inducible LEC2 expression caused a 3-fold increase in TAG levels in transgenic leaves compared with that in the leaves of wild-type plants. Plant growth was not severely affected by the accumulation the TAG in response to LEC2 expression. The levels of plastid-synthesized lipids, mono- and di-galactosyldiacylglycerol and phosphatidylglycerol, were reduced more in senescence-induced LEC2 than endoplasmic reticulum-synthesized lipids, including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Senescence-induced LEC2 upregulated the expression of many genes involved in fatty acid and TAG biosynthesis at precise times in senescent leaves, including WRINKLED1 (WRI1), which encodes a fatty acid transcription factor. The expression of glycerol-3-phosphate dehydrogenase 1 and phospholipid:diacylglycerol 2 were increased in the transgenic leaves. Five seed-type oleosin-encoding genes, expressed during oil-body formation, and the seed-specific FAE1 gene, which encodes the enzyme responsible for the synthesis of C20:1 and C22:1 fatty acids, were also expressed at higher levels in senescing transgenic leaves than in wild-type leaves. Senescence-inducible LEC2 triggers the key metabolic steps that increase TAG accumulation in vegetative tissues. PMID:25790072

[Cloning and expressing of cyclophilin B gene from Schistosoma japonnicum and the analysis of immunoprotective effect].

PubMed

Peng, Jinbiao; Han, Hongxiao; Hong, Yang; Wang, Yan; Guo, Fanji; Shi, Yaojun; Fu, Zhiqiang; Liu, Jinming; Cheng, Guofeng; Lin, Jiaojiao

2010-03-01

The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.

Cyclic Tensile Strain Upregulates Pro-Inflammatory Cytokine Expression Via FAK-MAPK Signaling in Chondrocytes.

PubMed

Yanoshita, Makoto; Hirose, Naoto; Okamoto, Yuki; Sumi, Chikako; Takano, Mami; Nishiyama, Sayuri; Asakawa-Tanne, Yuki; Horie, Kayo; Onishi, Azusa; Yamauchi, Yuka; Mitsuyoshi, Tomomi; Kunimatsu, Ryo; Tanimoto, Kotaro

2018-05-08

Excessive mechanical stimulation is considered an important factor in the destruction of chondrocytes. Focal adhesion kinase (FAK) is non-receptor tyrosine kinase related to a number of different signaling proteins. Little is known about the function of FAK in chondrocytes under mechanical stimulation. In the present study, we investigated the function of FAK in mechanical signal transduction and the mechanism through which cyclic tensile strain (CTS) induces expression of inflammation-related factors. Mouse ATDC5 chondrogenic cells were subjected to CTS of 0.5 Hz to 10% cell elongation with an FAK inhibitor. The expression of genes encoding COX-2, IL-1β, and TNF-α was examined using real-time RT-PCR after CTS application with FAK inhibitor. Phosphorylation of p-38, ERK, and JNK was analyzed by Western blotting. Differences in COX-2 expression following pretreatment with FAK, p-38, ERK, and JNK inhibitors were compared by Western blotting. We found that CTS increased the expression of genes encoding COX-2, IL-1β, and TNF-α and activated the phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with an FAK inhibitor for 2 h reduced the expression of genes encoding COX-2, IL-1β, and TNF-α induced by CTS-associated inflammation and decreased phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with FAK, p-38, ERK, and JNK inhibitors markedly suppressed COX-2 and IL-1β protein expression. In conclusion, FAK appears to regulate inflammation in chondrocytes under CTS via MAPK pathways.

Senescence-inducible LEC2 enhances triacylglycerol accumulation in leaves without negatively affecting plant growth.

PubMed

Kim, Hyun Uk; Lee, Kyeong-Ryeol; Jung, Su-Jin; Shin, Hyun A; Go, Young Sam; Suh, Mi-Chung; Kim, Jong Bum

2015-12-01

The synthesis of fatty acids and glycerolipids in wild-type Arabidopsis leaves does not typically lead to strong triacylglycerol (TAG) accumulation. LEAFY COTYLEDON2 (LEC2) is a master regulator of seed maturation and oil accumulation in seeds. Constitutive ectopic LEC2 expression causes somatic embryogenesis and defects in seedling growth. Here, we report that senescence-inducible LEC2 expression caused a threefold increase in TAG levels in transgenic leaves compared with that in the leaves of wild-type plants. Plant growth was not severely affected by the accumulation the TAG in response to LEC2 expression. The levels of plastid-synthesized lipids, mono- and di-galactosyldiacylglycerol and phosphatidylglycerol were reduced more in senescence-induced LEC2 than in endoplasmic reticulum-synthesized lipids, including phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. Senescence-induced LEC2 up-regulated the expression of many genes involved in fatty acid and TAG biosynthesis at precise times in senescent leaves, including WRINKLED1 (WRI1), which encodes a fatty acid transcription factor. The expressions of glycerol-3-phosphate dehydrogenase 1 and phospholipid:diacylglycerol 2 were increased in the transgenic leaves. Five seed-type oleosin-encoding genes, expressed during oil-body formation, and the seed-specific FAE1 gene, which encodes the enzyme responsible for the synthesis of C20:1 and C22:1 fatty acids, were also expressed at higher levels in senescing transgenic leaves than in wild-type leaves. Senescence-inducible LEC2 triggers the key metabolic steps that increase TAG accumulation in vegetative tissues. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

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

Koczor, Christopher A., E-mail: ckoczor@emory.edu; Fields, Earl; Jedrzejczak, Mark J.

This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides formore » calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of CACNA1C (Cav1.2).« less

Expression of a highly basic peroxidase gene in NaCl-adapted tomato cell suspensions.

PubMed

Medina, M I; Botella, M A; Quesada, M A; Valpuesta, V

1997-05-05

A tomato peroxidase gene, TPX2, that is only weakly expressed in the roots of young tomato seedlings is highly expressed in tomato suspension cells adapted to high external NaCl concentration. The protein encoded by this gene, with an isolectric point value of approximately 9.6, is found in the culture medium of the growing cells. Our data suggest that the expression of TPX2 in the salt-adapted cells is not the result of the elicitation imposed by the in vitro culture or the presence of high NaCl concentration in the medium.

Intragenome Diversity of Gene Families Encoding Toxin-like Proteins in Venomous Animals.

PubMed

Rodríguez de la Vega, Ricardo C; Giraud, Tatiana

2016-11-01

The evolution of venoms is the story of how toxins arise and of the processes that generate and maintain their diversity. For animal venoms these processes include recruitment for expression in the venom gland, neofunctionalization, paralogous expansions, and functional divergence. The systematic study of these processes requires the reliable identification of the venom components involved in antagonistic interactions. High-throughput sequencing has the potential of uncovering the entire set of toxins in a given organism, yet the existence of non-venom toxin paralogs and the misleading effects of partial census of the molecular diversity of toxins make necessary to collect complementary evidence to distinguish true toxins from their non-venom paralogs. Here, we analyzed the whole genomes of two scorpions, one spider and one snake, aiming at the identification of the full repertoires of genes encoding toxin-like proteins. We classified the entire set of protein-coding genes into paralogous groups and monotypic genes, identified genes encoding toxin-like proteins based on known toxin families, and quantified their expression in both venom-glands and pooled tissues. Our results confirm that genes encoding toxin-like proteins are part of multigene families, and that these families arise by recruitment events from non-toxin genes followed by limited expansions of the toxin-like protein coding genes. We also show that failing to account for sequence similarity with non-toxin proteins has a considerable misleading effect that can be greatly reduced by comparative transcriptomics. Our study overall contributes to the understanding of the evolutionary dynamics of proteins involved in antagonistic interactions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Zebrafish Dmrta2 regulates neurogenesis in the telencephalon.

PubMed

Yoshizawa, Akio; Nakahara, Yoshinari; Izawa, Toshiaki; Ishitani, Tohru; Tsutsumi, Makiko; Kuroiwa, Atsushi; Itoh, Motoyuki; Kikuchi, Yutaka

2011-11-01

Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab-3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB-3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior-dorsal telencephalon) is markedly reduced in this mutant at the 14-somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes-related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over-expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior-dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior-dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior-dorsal telencephalic cell fate during somitogenesis. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Characterization of big bang, a novel gene encoding for PDZ domain-containing proteins that are dynamically expressed throughout Drosophila development.

PubMed

Kim, Sabrina Y; Renihan, Maia K; Boulianne, Gabrielle L

2006-06-01

PDZ (PSD-95, Discs-large, ZO-1) domain proteins often function as scaffolding proteins and have been shown to play important roles in diverse cellular processes such as the establishment and maintenance of cell polarity, and signal transduction. Here, we report the identification and cloning of a novel Drosophila melanogaster gene that is predicted to produce several different PDZ domain-containing proteins through alternative promoter usage and alternative splicing. This gene, that we have named big bang (bbg), was first identified as C96-GAL4, a GAL4 enhancer trap line that was generated in our lab. To further characterize bbg, its expression pattern was examined in ovaries, embryos, and late third instar larvae using UAS reporter gene constructs, in situ hybridization, or immunocytochemistry. In addition, the expression of alternatively spliced transcripts was examined in more detail using in situ hybridization. We find that during embryogenesis bbg is predominantly expressed in the developing gut, but it is also expressed in external sensory organs found in the epidermis. In the late third instar larva, bbg is expressed along the presumptive wing margin in the wing disc, broadly in the eye disc, and in other imaginal discs as well as in the brain. The expression patterns observed are dynamic and specific during development, suggesting that like other genes that encode for several different PDZ domain protein isoforms, bbg likely plays important roles in multiple developmental processes.

Identification and characterization of a second CD4-like gene in teleost fish.

PubMed

Dijkstra, Johannes Martinus; Somamoto, Tomonori; Moore, Lindsey; Hordvik, Ivar; Ototake, Mitsuru; Fischer, Uwe

2006-02-01

In fish, T cell subdivision is not well studied, although CD8 and CD4 homologues have been reported. This study describes a second teleost CD4-like gene, CD4-like 2 (CD4L-2). Two rainbow trout copies of this gene were found, -2a and -2b, encoding molecules sharing 81% aa identity. The 2a/2b duplication may be related to tetraploid ancestry of salmonid fishes. In the Fugu genome CD4L-2 lies head to tail with an earlier reported, very different CD4-like gene [Suetake, H., Araki, K., Suzuki, Y., 2004. Cloning, expression, and characterization of fugu CD4, the first ectothermic animal CD4. Immunogenetics 56, 368-374], which was designated CD4L-1 in the present article. The flanking genes of the Fugu CD4L-1 and CD4L-2 are reminiscent of the genes surrounding CD4 and LAG-3 in mammals. However, neither synteny nor phylogenetic analysis could decide between CD4 and LAG-3 identity for the fish CD4L genes. CD4L-1 and CD4L-2 share a tyrosine protein kinase p56(lck) binding motif in the cytoplasmic tail with CD4 but not with LAG-3. Trout CD4L-2 expression is highest in the thymus, similar to mammalian and chicken CD4, whereas Fugu CD4L-1 expression was highest in the spleen. However, CD4L-2 encodes only two IG-like domains, whereas CD4L-1, CD4 and LAG-3 encode four. The CD4-like genes 1 and 2 in fish apparently went through an evolution different from that of LAG-3 and CD4 in higher vertebrates.

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 characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of genes encoding membrane proteins of Borrelia burgdorferi.

PubMed Central

Ramamoorthy, R; Povinelli, L; Philipp M, T

1996-01-01

An expression library made with Borrelia burgdorferi DNA in the vector lambda ZapII was screened with serum from a monkey infected with the Lyme disease agent. This serum killed B. burgdorferi in vitro by an antibody-dependent, complement-mediated mechanism and contained antibodies to at least seven spirochetal antigens, none of which were the major outer surface proteins OspA or OspB. Among several positive clones, a clone containing the B. burgdorferi bmpA gene encoding the immunodominant antigen P39 was obtained. Chromosome walking and DNA sequence analysis permitted the identification of two additional upstream genes homologous to the bmpA gene and its related companion, bmpB. The first of these was the recently characterized bmpC gene, and adjacent to it was the fourth and new member of this class, which has been designated bmpD. The gene product encoded by bmpD is 34l residues long, contains a signal sequence with a potential signal peptidase II cleavage site, and has 26% identity with TmpC of Treponema pallidum. Southern blotting confirmed the tandem arrangement of all four bmp genes in the chromosome of B. burgdorferi JD1. However, Northern (RNA) blotting revealed that bmpD is expressed as a monocistronic transcript, which indicates that it is not part of an operon at the bmp locus. The bmpD gene was found to be conserved in representative members of the three species of the B. burgdorferi sensu lato complex, suggesting that it serves an important biological function in the spirochete. PMID:8606088

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls

PubMed Central

Fassah, Dilla Mareistia; Jeong, Jin Young

2018-01-01

Objective This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Methods Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10) and steers (n = 10) of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Results Castration increased the mRNA (3.6 fold; p<0.01) and protein levels (1.4 fold; p< 0.05) of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05). Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05) and lactate dehydrogenase B (2.2 fold; p<0.01) genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05) and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05) genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial) (3.5 fold; p<0.01) and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06) genes for propionate incorporation. Conclusion Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular fat deposition. PMID:29502393