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.

Characterization of key triacylglycerol biosynthesis processes in rhodococci

DOE PAGES

Amara, Sawsan; Seghezzi, Nicolas; Otani, Hiroshi; ...

2016-04-29

In this study, oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcriptsmore » were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δ atf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production.« less

Characterization of key triacylglycerol biosynthesis processes in rhodococci

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

Amara, Sawsan; Seghezzi, Nicolas; Otani, Hiroshi

In this study, oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcriptsmore » were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δ atf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production.« less

The Saccharomyces cerevisiae ETH1 Gene, an Inducible Homolog of Exonuclease III That Provides Resistance to DNA-Damaging Agents and Limits Spontaneous Mutagenesis

PubMed Central

Bennett, Richard A. O.

1999-01-01

The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1+ [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were ∼3-fold more sensitive to MMS and ∼10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were ∼15-fold more sensitive to MMS and ∼2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains. Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein. PMID:10022867

Genes encoding a callose synthase and phytochrome A are adjacent to a MAP3Ka-like gene in Beta vulgaris USH20

USDA-ARS?s Scientific Manuscript database

MAP3Ka encodes a key conserved protein kinase responsible for orchestrating a rapid cascade of cellular events ultimately leading to localized cell death. Hypersensitive response, as it is termed, enables genetically-resistant plants to limit microbial invasion under the right environmental conditio...

Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

PubMed

Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

2016-02-17

Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Evolutionary Characteristics of Missing Proteins: Insights into the Evolution of Human Chromosomes Related to Missing-Protein-Encoding Genes.

PubMed

Xu, Aishi; Li, Guang; Yang, Dong; Wu, Songfeng; Ouyang, Hongsheng; Xu, Ping; He, Fuchu

2015-12-04

Although the "missing protein" is a temporary concept in C-HPP, the biological information for their "missing" could be an important clue in evolutionary studies. Here we classified missing-protein-encoding genes into two groups, the genes encoding PE2 proteins (with transcript evidence) and the genes encoding PE3/4 proteins (with no transcript evidence). These missing-protein-encoding genes distribute unevenly among different chromosomes, chromosomal regions, or gene clusters. In the view of evolutionary features, PE3/4 genes tend to be young, spreading at the nonhomology chromosomal regions and evolving at higher rates. Interestingly, there is a higher proportion of singletons in PE3/4 genes than the proportion of singletons in all genes (background) and OTCSGs (organ, tissue, cell type-specific genes). More importantly, most of the paralogous PE3/4 genes belong to the newly duplicated members of the paralogous gene groups, which mainly contribute to special biological functions, such as "smell perception". These functions are heavily restricted into specific type of cells, tissues, or specific developmental stages, acting as the new functional requirements that facilitated the emergence of the missing-protein-encoding genes during evolution. In addition, the criteria for the extremely special physical-chemical proteins were first set up based on the properties of PE2 proteins, and the evolutionary characteristics of those proteins were explored. Overall, the evolutionary analyses of missing-protein-encoding genes are expected to be highly instructive for proteomics and functional studies in the future.

Exploring the Limits for Reduction of Plastid Genomes: A Case Study of the Mycoheterotrophic Orchids Epipogium aphyllum and Epipogium roseum

PubMed Central

Schelkunov, Mikhail I.; Shtratnikova, Viktoria Yu; Nuraliev, Maxim S.; Selosse, Marc-Andre; Penin, Aleksey A.; Logacheva, Maria D.

2015-01-01

The question on the patterns and limits of reduction of plastid genomes in nonphotosynthetic plants and the reasons of their conservation is one of the intriguing topics in plant genome evolution. Here, we report sequencing and analysis of plastid genome in nonphotosynthetic orchids Epipogium aphyllum and Epipogium roseum, which, with sizes of 31 and 19 kbp, respectively, represent the smallest plastid genomes characterized by now. Besides drastic reduction, which is expected, we found several unusual features of these “minimal” plastomes: Multiple rearrangements, highly biased nucleotide composition, and unprecedentedly high substitution rate. Only 27 and 29 genes remained intact in the plastomes of E. aphyllum and E. roseum—those encoding ribosomal components, transfer RNAs, and three additional housekeeping genes (infA, clpP, and accD). We found no signs of relaxed selection acting on these genes. We hypothesize that the main reason for retention of plastid genomes in Epipogium is the necessity to translate messenger RNAs (mRNAs) of accD and/or clpP proteins which are essential for cell metabolism. However, these genes are absent in plastomes of several plant species; their absence is compensated by the presence of a functional copy arisen by gene transfer from plastid to the nuclear genome. This suggests that there is no single set of plastid-encoded essential genes, but rather different sets for different species and that the retention of a gene in the plastome depends on the interaction between the nucleus and plastids. PMID:25635040

Genome-wide association study of acute post-surgical pain in humans

PubMed Central

Kim, Hyungsuk; Ramsay, Edward; Lee, Hyewon; Wahl, Sharon; Dionne, Raymond A

2009-01-01

Aims Testing a relatively small genomic region with a few hundred SNPs provides limited information. Genome-wide association studies (GWAS) provide an opportunity to overcome the limitation of candidate gene association studies. Here, we report the results of a GWAS for the responses to an NSAID analgesic. Materials & methods European Americans (60 females and 52 males) undergoing oral surgery were genotyped with Affymetrix 500K SNP assay. Additional SNP genotyping was performed from the gene in linkage disequilibrium with the candidate SNP revealed by the GWAS. Results GWAS revealed a candidate SNP (rs2562456) associated with analgesic onset, which is in linkage disequilibrium with a gene encoding a zinc finger protein. Additional SNP genotyping of ZNF429 confirmed the association with analgesic onset in humans (p = 1.8 × 10−10, degrees of freedom = 103, F = 28.3). We also found candidate loci for the maximum post-operative pain rating (rs17122021, p = 6.9 × 10−7) and post-operative pain onset time (rs6693882, p = 2.1 × 10−6), however, correcting for multiple comparisons did not sustain these genetic associations. Conclusion GWAS for acute clinical pain followed by additional SNP genotyping of a neighboring gene suggests that genetic variations in or near the loci encoding DNA binding proteins play a role in the individual variations in responses to analgesic drugs. PMID:19207018

Plastid-Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae.

PubMed

Weng, Mao-Lun; Ruhlman, Tracey A; Jansen, Robert K

2016-06-27

Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid-nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

FLAVODIIRON2 and FLAVODIIRON4 Proteins Mediate an Oxygen-Dependent Alternative Electron Flow in Synechocystis sp. PCC 6803 under CO2-Limited Conditions1[OPEN

PubMed Central

Shimakawa, Ginga; Shaku, Keiichiro; Nishi, Akiko; Hayashi, Ryosuke; Yamamoto, Hiroshi; Sakamoto, Katsuhiko; Makino, Amane; Miyake, Chikahiro

2015-01-01

This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO2-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO2 limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO2-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO2 are not available. PMID:25540330

Preclinical Assessment of wt GNE Gene Plasmid for Management of Hereditary Inclusion Body Myopathy 2 (HIBM2)

PubMed Central

Jay, Chris; Nemunaitis, Gregory; Nemunaitis, John; Senzer, Neil; Hinderlich, Stephan; Darvish, Daniel; Ogden, Julie; Eager, John; Tong, Alex; Maples, Phillip B

2008-01-01

Hereditary Inclusion Body Myopathy (HIBM2) is a chronic progressive skeletal muscle wasting disorder which generally leads to complete disability before the age of 50 years. There is currently no effective therapeutic treatment for HIBM2. Development of this disease is related to expression in family members of an autosomal recessive mutation of the GNE gene, which encodes the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE/MNK). This is the rate limiting bifunctional enzyme that catalyzes the first 2 steps of sialic acid biosynthesis. Decreased sialic acid production, consequently leads to decreased sialyation of a variety of glycoproteins including the critical muscle protein alpha-dystroglycan (α-DG). This in turn severely cripples muscle function and leads to the onset of the syndrome. We hypothesize that replacing the mutated GNE gene with the wildtype gene may restore functional capacity of GNE/MNK and therefore production of sialic acid, allowing for improvement in muscle function and/or delay in rate of muscle deterioration. We have constructed three GNE gene/CMV promoter plasmids (encoding the wildtype, HIBM2, and Sialuria forms of GNE) and demonstrated enhanced GNE gene activity following delivery to GNE-deficient CHO-Lec3 cells. GNE/MNK enzyme function was significantly increased and subsequent induction of sialic acid production was demonstrated after transfection into Lec3 cells with the wild type or R266Q mutant GNE vector. These data form the foundation for future preclinical and clinical studies for GNE gene transfer to treat HIBM2 patients. PMID:19787087

Genetic stability of Ross River virus during epidemic spread in nonimmune humans.

PubMed

Burness, A T; Pardoe, I; Faragher, S G; Vrati, S; Dalgarno, L

1988-12-01

We have examined the rate of evolution of Ross River virus, a mosquito-borne RNA virus, during epidemic spread through tens of thousands of nonimmune humans over a period of 10 months. Two regions of the Ross River virus genome were sequenced: the E2 gene (1.2 kb in length), which encodes the major neutralization determinant of the virus, and 0.4 kb of the 3'-untranslated region. In the E2 gene, a single nucleotide change was selected which led to a predicted amino acid change at residue 219. No changes were selected in the 3'-untranslated region. By comparison with rates of evolution reported for non-arthropod-borne RNA viruses, the rate for Ross River virus is surprisingly low. We identify three features of the Ross River virus replication and transmission cycle which may limit the rate of evolution of arthropod-borne viruses in the field.

Zinc Deficiency Impacts CO2 Assimilation and Disrupts Copper Homeostasis in Chlamydomonas reinhardtii*

PubMed Central

Malasarn, Davin; Kropat, Janette; Hsieh, Scott I.; Finazzi, Giovanni; Casero, David; Loo, Joseph A.; Pellegrini, Matteo; Wollman, Francis-André; Merchant, Sabeeha S.

2013-01-01

Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition-responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon-concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc deficiency, probably due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3, and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to copper limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc-deficient cells are functionally copper-deficient, although they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester copper in a biounavailable form, perhaps to prevent mismetallation of critical zinc sites. PMID:23439652

Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients.

PubMed

Motallebi, Mitra; Jabalameli, Fereshteh; Asadollahi, Kheirollah; Taherikalani, Morovat; Emaneini, Mohammad

2016-08-01

The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differential regulation of mnp2, a new manganese peroxidase-encoding gene from the ligninolytic fungus Trametes versicolor PRL 572

Treesearch

Tomas Johansson; Per Olof Nyman; Daniel Cullen

2002-01-01

A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO4 to cultures increased...

Lignin peroxidase gene family of Phanerochaete chrysosporium : complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome

Treesearch

Philip Stewart; Philip Kersten; Amber J. Vanden Wymelenberg; Jill A. Gaskell; Daniel Cullen

1992-01-01

Lignin peroxidases (LiP) of Phanerochaete chrysosporium are encoded by a family of six closely related genes. Five LiP genes have been localized to the same dimorphic chromosome. In this investigation, relative transcript levels of the LiP genes were determined. Transcripts of the LiPA, LiPB, and 0282 genes were at similar levels in both carbon-and nitrogen-limited...

Limited Dissemination of Extended-Spectrum β-Lactamase- and Plasmid-Encoded AmpC-Producing Escherichia coli from Food and Farm Animals, Sweden.

PubMed

Börjesson, Stefan; Ny, Sofia; Egervärn, Maria; Bergström, Jakob; Rosengren, Åsa; Englund, Stina; Löfmark, Sonja; Byfors, Sara

2016-04-01

Extended-spectrum β-lactamase (ESBL)- and plasmid-encoded ampC (pAmpC)-producing Enterobacteriaceae might spread from farm animals to humans through food. However, most studies have been limited in number of isolates tested and areas studied. We examined genetic relatedness of 716 isolates from 4,854 samples collected from humans, farm animals, and foods in Sweden to determine whether foods and farm animals might act as reservoirs and dissemination routes for ESBL/pAmpC-producing Escherichia coli. Results showed that clonal spread to humans appears unlikely. However, we found limited dissemination of genes encoding ESBL/pAmpC and plasmids carrying these genes from foods and farm animals to healthy humans and patients. Poultry and chicken meat might be a reservoir and dissemination route to humans. Although we found no evidence of clonal spread of ESBL/pAmpC-producing E. coli from farm animals or foods to humans, ESBL/pAmpC-producing E. coli with identical genes and plasmids were present in farm animals, foods, and humans.

Functional analysis of the Brassica napus L. phytoene synthase (PSY) gene family.

PubMed

López-Emparán, Ada; Quezada-Martinez, Daniela; Zúñiga-Bustos, Matías; Cifuentes, Víctor; Iñiguez-Luy, Federico; Federico, María Laura

2014-01-01

Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three "Arabidopsis-like" subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.

Design of a recombinant Escherichia coli for producing L-phenylalanine from glycerol.

PubMed

Thongchuang, Mayura; Pongsawasdi, Piamsook; Chisti, Yusuf; Packdibamrung, Kanoktip

2012-10-01

A recombinant Escherichia coli was engineered to produce the commercially important amino acid L-phenylalanine (L-Phe) using glycerol as the carbon source. Compared to the conventionally used glucose and sucrose, glycerol is a less expensive carbon source. As phenylalanine dehydrogenase (PheDH) activity is involved in the last step of L-Phe synthesis in E. coli, a phenylalanine dehydrogenase gene (phedh) from the thermotolerant Bacillus lentus was cloned into pRSFDuet-1 (pPheDH) and expressed in E. coli BL21(DE3). The resulting clone had a limited ability to produce L-Phe from glycerol, possibly because of a poor glycerol uptake by the cell, or an inability to excrete L-Phe, or both. Therefore, yddG gene encoding an aromatic amino acid exporter and glpF gene encoding a glycerol transport facilitator were coexpressed with the phedh in a reengineered E. coli. In a glycerol medium, the maximum L-Phe production rates of the clones pPY (phedh and yddG genes) and pPYF (phedh, yddG and glpF genes) were 1.4- and 1.8-fold higher than the maximum production rate of the pPheDH clone. The better producing pPYF clone was further evaluated in a 5 l stirred-tank fermenter (37 °C, an aeration rate of 1 vvm, an agitation speed of 400 rpm). In the fermenter, the maximum concentration of L-Phe (366 mg/l) was achieved in a much shorter period compared to in the shake flasks. In the latter, the highest titer of L-Phe was only 76 % of the maximum value attained in the fermenter.

Exploring the limits for reduction of plastid genomes: a case study of the mycoheterotrophic orchids Epipogium aphyllum and Epipogium roseum.

PubMed

Schelkunov, Mikhail I; Shtratnikova, Viktoria Yu; Nuraliev, Maxim S; Selosse, Marc-Andre; Penin, Aleksey A; Logacheva, Maria D

2015-01-28

The question on the patterns and limits of reduction of plastid genomes in nonphotosynthetic plants and the reasons of their conservation is one of the intriguing topics in plant genome evolution. Here, we report sequencing and analysis of plastid genome in nonphotosynthetic orchids Epipogium aphyllum and Epipogium roseum, which, with sizes of 31 and 19 kbp, respectively, represent the smallest plastid genomes characterized by now. Besides drastic reduction, which is expected, we found several unusual features of these "minimal" plastomes: Multiple rearrangements, highly biased nucleotide composition, and unprecedentedly high substitution rate. Only 27 and 29 genes remained intact in the plastomes of E. aphyllum and E. roseum-those encoding ribosomal components, transfer RNAs, and three additional housekeeping genes (infA, clpP, and accD). We found no signs of relaxed selection acting on these genes. We hypothesize that the main reason for retention of plastid genomes in Epipogium is the necessity to translate messenger RNAs (mRNAs) of accD and/or clpP proteins which are essential for cell metabolism. However, these genes are absent in plastomes of several plant species; their absence is compensated by the presence of a functional copy arisen by gene transfer from plastid to the nuclear genome. This suggests that there is no single set of plastid-encoded essential genes, but rather different sets for different species and that the retention of a gene in the plastome depends on the interaction between the nucleus and plastids. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Coordinated Rates of Evolution between Interacting Plastid and Nuclear Genes in Geraniaceae

PubMed Central

Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal; Blazier, J. Chris; Jansen, Robert K.

2015-01-01

Although gene coevolution has been widely observed within individuals and between different organisms, rarely has this phenomenon been investigated within a phylogenetic framework. The Geraniaceae is an attractive system in which to study plastid-nuclear genome coevolution due to the highly elevated evolutionary rates in plastid genomes. In plants, the plastid-encoded RNA polymerase (PEP) is a protein complex composed of subunits encoded by both plastid (rpoA, rpoB, rpoC1, and rpoC2) and nuclear genes (sig1-6). We used transcriptome and genomic data for 27 species of Geraniales in a systematic evaluation of coevolution between genes encoding subunits of the PEP holoenzyme. We detected strong correlations of dN (nonsynonymous substitutions) but not dS (synonymous substitutions) within rpoB/sig1 and rpoC2/sig2, but not for other plastid/nuclear gene pairs, and identified the correlation of dN/dS ratio between rpoB/C1/C2 and sig1/5/6, rpoC1/C2 and sig2, and rpoB/C2 and sig3 genes. Correlated rates between interacting plastid and nuclear sequences across the Geraniales could result from plastid-nuclear genome coevolution. Analyses of coevolved amino acid positions suggest that structurally mediated coevolution is not the major driver of plastid-nuclear coevolution. The detection of strong correlation of evolutionary rates between SIG and RNAP genes suggests a plausible explanation for plastome-genome incompatibility in Geraniaceae. PMID:25724640

Acclimation of Saccharomyces cerevisiae to low temperature: a chemostat-based transcriptome analysis.

PubMed

Tai, Siew Leng; Daran-Lapujade, Pascale; Walsh, Michael C; Pronk, Jack T; Daran, Jean-Marc

2007-12-01

Effects of suboptimal temperatures on transcriptional regulation in yeast have been extensively studied in batch cultures. To eliminate indirect effects of specific growth rates that are inherent to batch-cultivation studies, genome-wide transcriptional responses to low temperatures were analyzed in steady-state chemostats, grown at a fixed specific growth rate (0.03 h(-1)). Although in vivo metabolic fluxes were essentially the same in cultures grown at 12 and at 30 degrees C, concentrations of the growth-limiting nutrients (glucose or ammonia) were higher at 12 degrees C. This difference was reflected by transcript levels of genes that encode transporters for the growth-limiting nutrients. Several transcriptional responses to low temperature occurred under both nutrient-limitation regimes. Increased transcription of ribosome-biogenesis genes emphasized the importance of adapting protein-synthesis capacity to low temperature. In contrast to observations in cold-shock and batch-culture studies, transcript levels of environmental stress response genes were reduced at 12 degrees C. Transcription of trehalose-biosynthesis genes and intracellular trehalose levels indicated that, in contrast to its role in cold-shock adaptation, trehalose is not involved in steady-state low-temperature adaptation. Comparison of the chemostat-based transcriptome data with literature data revealed large differences between transcriptional reprogramming during long-term low-temperature acclimation and the transcriptional responses to a rapid transition to low temperature.

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.

The ribosomal protein genes and Minute loci of Drosophila melanogaster

PubMed Central

Marygold, Steven J; Roote, John; Reuter, Gunter; Lambertsson, Andrew; Ashburner, Michael; Millburn, Gillian H; Harrison, Paul M; Yu, Zhan; Kenmochi, Naoya; Kaufman, Thomas C; Leevers, Sally J; Cook, Kevin R

2007-01-01

Background Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. Results We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. Conclusion This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome. PMID:17927810

Enhanced Bioconversion of Cellobiose by Industrial Saccharomyces cerevisiae Used for Cellulose Utilization

PubMed Central

Hu, Meng-Long; Zha, Jian; He, Lin-Wei; Lv, Ya-Jin; Shen, Ming-Hua; Zhong, Cheng; Li, Bing-Zhi; Yuan, Ying-Jin

2016-01-01

Cellobiose accumulation and the compromised temperature for yeast fermentation are the main limiting factors of enzymatic hydrolysis process during simultaneous saccharification and fermentation (SSF). In this study, genes encoding cellobiose transporter and β-glucosidase were introduced into an industrial Saccharomyces cerevisiae strain, and evolution engineering was carried out to improve the cellobiose utilization of the engineered yeast strain. The evolved strain exhibited significantly higher cellobiose consumption rate (2.8-fold) and ethanol productivity (4.9-fold) compared with its parent strain. Besides, the evolved strain showed a high cellobiose consumption rate of 3.67 g/L/h at 34°C and 3.04 g/L/h at 38°C. Moreover, little cellobiose was accumulated during SSF of Avicel using the evolved strain at 38°C, and the ethanol yield from Avicel increased by 23% from 0.34 to 0.42 g ethanol/g cellulose. Overexpression of the genes encoding cellobiose transporter and β-glucosidase accelerated cellobiose utilization, and the improvement depended on the strain background. The results proved that fast cellobiose utilization enhanced ethanol production by reducing cellobiose accumulation during SSF at high temperature. PMID:26973619

Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed

USGS Publications Warehouse

Rocca, Jennifer D.; Hall, Edward K.; Lennon, Jay T.; Evans, Sarah E.; Waldrop, Mark P.; Cotner, James B.; Nemergut, Diana R.; Graham, Emily B.; Wallenstein, Matthew D.

2015-01-01

For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes.

Coordinated rates of evolution between interacting plastid and nuclear genes in Geraniaceae.

PubMed

Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal; Blazier, J Chris; Jansen, Robert K

2015-03-01

Although gene coevolution has been widely observed within individuals and between different organisms, rarely has this phenomenon been investigated within a phylogenetic framework. The Geraniaceae is an attractive system in which to study plastid-nuclear genome coevolution due to the highly elevated evolutionary rates in plastid genomes. In plants, the plastid-encoded RNA polymerase (PEP) is a protein complex composed of subunits encoded by both plastid (rpoA, rpoB, rpoC1, and rpoC2) and nuclear genes (sig1-6). We used transcriptome and genomic data for 27 species of Geraniales in a systematic evaluation of coevolution between genes encoding subunits of the PEP holoenzyme. We detected strong correlations of dN (nonsynonymous substitutions) but not dS (synonymous substitutions) within rpoB/sig1 and rpoC2/sig2, but not for other plastid/nuclear gene pairs, and identified the correlation of dN/dS ratio between rpoB/C1/C2 and sig1/5/6, rpoC1/C2 and sig2, and rpoB/C2 and sig3 genes. Correlated rates between interacting plastid and nuclear sequences across the Geraniales could result from plastid-nuclear genome coevolution. Analyses of coevolved amino acid positions suggest that structurally mediated coevolution is not the major driver of plastid-nuclear coevolution. The detection of strong correlation of evolutionary rates between SIG and RNAP genes suggests a plausible explanation for plastome-genome incompatibility in Geraniaceae. © 2015 American Society of Plant Biologists. All rights reserved.

A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index

PubMed Central

Xia, Xuhua

2015-01-01

Two alternative hypotheses attribute different benefits to codon-anticodon adaptation. The first assumes that protein production is rate limited by both initiation and elongation and that codon-anticodon adaptation would result in higher elongation efficiency and more efficient and accurate protein production, especially for highly expressed genes. The second claims that protein production is rate limited only by initiation efficiency but that improved codon adaptation and, consequently, increased elongation efficiency have the benefit of increasing ribosomal availability for global translation. To test these hypotheses, a recent study engineered a synthetic library of 154 genes, all encoding the same protein but differing in degrees of codon adaptation, to quantify the effect of differential codon adaptation on protein production in Escherichia coli. The surprising conclusion that “codon bias did not correlate with gene expression” and that “translation initiation, not elongation, is rate-limiting for gene expression” contradicts the conclusion reached by many other empirical studies. In this paper, I resolve the contradiction by reanalyzing the data from the 154 sequences. I demonstrate that translation elongation accounts for about 17% of total variation in protein production and that the previous conclusion is due to the use of a codon adaptation index (CAI) that does not account for the mutation bias in characterizing codon adaptation. The effect of translation elongation becomes undetectable only when translation initiation is unrealistically slow. A new index of translation elongation ITE is formulated to facilitate studies on the efficiency and evolution of the translation machinery. PMID:25480780

Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment

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

Norton, Jeanette M.; Klotz, Martin G; Stein, Lisa Y

2008-01-01

The complete genome of the ammonia-oxidizing bacterium, Nitrosospira multiformis (ATCC 25196T), consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2827 putative proteins. Of these, 2026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and N. eutropha were the best match for 42% of the predicted genes in N. multiformis. The genome contains three nearly identical copies of amo and hao gene clusters as large repeats. Distinguishing features compared to N. europaea include: the presencemore » of gene clusters encoding urease and hydrogenase, a RuBisCO-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced AOB genomes. Gene clusters encoding proteins associated with outer membrane and cell envelope functions including transporters, porins, exopolysaccharide synthesis, capsule formation and protein sorting/export were abundant. Numerous sensory transduction and response regulator gene systems directed towards sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate and cyanophycin storage and utilization were identified providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.« less

Positive Selection in Rapidly Evolving Plastid–Nuclear Enzyme Complexes

PubMed Central

Rockenbach, Kate; Havird, Justin C.; Monroe, J. Grey; Triant, Deborah A.; Taylor, Douglas R.; Sloan, Daniel B.

2016-01-01

Rates of sequence evolution in plastid genomes are generally low, but numerous angiosperm lineages exhibit accelerated evolutionary rates in similar subsets of plastid genes. These genes include clpP1 and accD, which encode components of the caseinolytic protease (CLP) and acetyl-coA carboxylase (ACCase) complexes, respectively. Whether these extreme and repeated accelerations in rates of plastid genome evolution result from adaptive change in proteins (i.e., positive selection) or simply a loss of functional constraint (i.e., relaxed purifying selection) is a source of ongoing controversy. To address this, we have taken advantage of the multiple independent accelerations that have occurred within the genus Silene (Caryophyllaceae) by examining phylogenetic and population genetic variation in the nuclear genes that encode subunits of the CLP and ACCase complexes. We found that, in species with accelerated plastid genome evolution, the nuclear-encoded subunits in the CLP and ACCase complexes are also evolving rapidly, especially those involved in direct physical interactions with plastid-encoded proteins. A massive excess of nonsynonymous substitutions between species relative to levels of intraspecific polymorphism indicated a history of strong positive selection (particularly in CLP genes). Interestingly, however, some species are likely undergoing loss of the native (heteromeric) plastid ACCase and putative functional replacement by a duplicated cytosolic (homomeric) ACCase. Overall, the patterns of molecular evolution in these plastid–nuclear complexes are unusual for anciently conserved enzymes. They instead resemble cases of antagonistic coevolution between pathogens and host immune genes. We discuss a possible role of plastid–nuclear conflict as a novel cause of accelerated evolution. PMID:27707788

Microbial Metabolic Potential for Carbon Degradation and Nutrient (Nitrogen and Phosphorus) Acquisition in an Ombrotrophic Peatland

PubMed Central

Tfaily, Malak M.; Green, Stefan J.; Steinweg, J. Megan; Chanton, Patrick; Imvittaya, Aopeau; Chanton, Jeffrey P.; Cooper, William; Schadt, Christopher

2014-01-01

This study integrated metagenomic and nuclear magnetic resonance (NMR) spectroscopic approaches to investigate microbial metabolic potential for organic matter decomposition and nitrogen (N) and phosphorus (P) acquisition in soils of an ombrotrophic peatland in the Marcell Experimental Forest (MEF), Minnesota, USA. This analysis revealed vertical stratification in key enzymatic pathways and taxa containing these pathways. Metagenomic analyses revealed that genes encoding laccases and dioxygenases, involved in aromatic compound degradation, declined in relative abundance with depth, while the relative abundance of genes encoding metabolism of amino sugars and all four saccharide groups increased with depth in parallel with a 50% reduction in carbohydrate content. Most Cu-oxidases were closely related to genes from Proteobacteria and Acidobacteria, and type 4 laccase-like Cu-oxidase genes were >8 times more abundant than type 3 genes, suggesting an important and overlooked role for type 4 Cu-oxidase in phenolic compound degradation. Genes associated with sulfate reduction and methanogenesis were the most abundant anaerobic respiration genes in these systems, with low levels of detection observed for genes of denitrification and Fe(III) reduction. Fermentation genes increased in relative abundance with depth and were largely affiliated with Syntrophobacter. Methylocystaceae-like small-subunit (SSU) rRNA genes, pmoA, and mmoX genes were more abundant among methanotrophs. Genes encoding N2 fixation, P uptake, and P regulons were significantly enriched in the surface peat and in comparison to other ecosystems, indicating N and P limitation. Persistence of inorganic orthophosphate throughout the peat profile in this P-limiting environment indicates that P may be bound to recalcitrant organic compounds, thus limiting P bioavailability in the subsurface. Comparative metagenomic analysis revealed a high metabolic potential for P transport and starvation, N2 fixation, and oligosaccharide degradation at MEF relative to other wetland and soil environments, consistent with the nutrient-poor and carbohydrate-rich conditions found in this Sphagnum-dominated boreal peatland. PMID:24682299

Microbial metabolic potential for carbon degradation and nutrient (nitrogen and phosphorus) acquisition in an ombrotrophic peatland.

PubMed

Lin, Xueju; Tfaily, Malak M; Green, Stefan J; Steinweg, J Megan; Chanton, Patrick; Imvittaya, Aopeau; Chanton, Jeffrey P; Cooper, William; Schadt, Christopher; Kostka, Joel E

2014-06-01

This study integrated metagenomic and nuclear magnetic resonance (NMR) spectroscopic approaches to investigate microbial metabolic potential for organic matter decomposition and nitrogen (N) and phosphorus (P) acquisition in soils of an ombrotrophic peatland in the Marcell Experimental Forest (MEF), Minnesota, USA. This analysis revealed vertical stratification in key enzymatic pathways and taxa containing these pathways. Metagenomic analyses revealed that genes encoding laccases and dioxygenases, involved in aromatic compound degradation, declined in relative abundance with depth, while the relative abundance of genes encoding metabolism of amino sugars and all four saccharide groups increased with depth in parallel with a 50% reduction in carbohydrate content. Most Cu-oxidases were closely related to genes from Proteobacteria and Acidobacteria, and type 4 laccase-like Cu-oxidase genes were >8 times more abundant than type 3 genes, suggesting an important and overlooked role for type 4 Cu-oxidase in phenolic compound degradation. Genes associated with sulfate reduction and methanogenesis were the most abundant anaerobic respiration genes in these systems, with low levels of detection observed for genes of denitrification and Fe(III) reduction. Fermentation genes increased in relative abundance with depth and were largely affiliated with Syntrophobacter. Methylocystaceae-like small-subunit (SSU) rRNA genes, pmoA, and mmoX genes were more abundant among methanotrophs. Genes encoding N2 fixation, P uptake, and P regulons were significantly enriched in the surface peat and in comparison to other ecosystems, indicating N and P limitation. Persistence of inorganic orthophosphate throughout the peat profile in this P-limiting environment indicates that P may be bound to recalcitrant organic compounds, thus limiting P bioavailability in the subsurface. Comparative metagenomic analysis revealed a high metabolic potential for P transport and starvation, N2 fixation, and oligosaccharide degradation at MEF relative to other wetland and soil environments, consistent with the nutrient-poor and carbohydrate-rich conditions found in this Sphagnum-dominated boreal peatland.

High Rates of Antimicrobial Drug Resistance Gene Acquisition after International Travel, the Netherlands

PubMed Central

von Wintersdorff, Christian J.H.; Penders, John; Stobberingh, Ellen E.; Lashof, Astrid M.L. Oude; Hoebe, Christian J.P.A.; Savelkoul, Paul H.M.

2014-01-01

We investigated the effect of international travel on the gut resistome of 122 healthy travelers from the Netherlands by using a targeted metagenomic approach. Our results confirm high acquisition rates of the extended-spectrum β-lactamase encoding gene blaCTX-M, documenting a rise in prevalence from 9.0% before travel to 33.6% after travel (p<0.001). The prevalence of quinolone resistance encoding genes qnrB and qnrS increased from 6.6% and 8.2% before travel to 36.9% and 55.7% after travel, respectively (both p<0.001). Travel to Southeast Asia and the Indian subcontinent was associated with the highest acquisition rates of qnrS and both blaCTX-M and qnrS, respectively. Investigation of the associations between the acquisitions of the blaCTX-M and qnr genes showed that acquisition of a blaCTX-M gene was not associated with that of a qnrB (p = 0.305) or qnrS (p = 0.080) gene. These findings support the increasing evidence that travelers contribute to the spread of antimicrobial drug resistance. PMID:24655888

High rates of antimicrobial drug resistance gene acquisition after international travel, The Netherlands.

PubMed

von Wintersdorff, Christian J H; Penders, John; Stobberingh, Ellen E; Oude Lashof, Astrid M L; Hoebe, Christian J P A; Savelkoul, Paul H M; Wolffs, Petra F G

2014-04-01

We investigated the effect of international travel on the gut resistome of 122 healthy travelers from the Netherlands by using a targeted metagenomic approach. Our results confirm high acquisition rates of the extended-spectrum β-lactamase encoding gene blaCTX-M, documenting a rise in prevalence from 9.0% before travel to 33.6% after travel (p<0.001). The prevalence of quinolone resistance encoding genes qnrB and qnrS increased from 6.6% and 8.2% before travel to 36.9% and 55.7% after travel, respectively (both p<0.001). Travel to Southeast Asia and the Indian subcontinent was associated with the highest acquisition rates of qnrS and both blaCTX-M and qnrS, respectively. Investigation of the associations between the acquisitions of the blaCTX-M and qnr genes showed that acquisition of a blaCTX-M gene was not associated with that of a qnrB (p = 0.305) or qnrS (p = 0.080) gene. These findings support the increasing evidence that travelers contribute to the spread of antimicrobial drug resistance.

Remodeling of Hepatic Metabolism and Hyperaminoacidemia in Mice Deficient in Proglucagon-Derived Peptides

PubMed Central

Watanabe, Chika; Seino, Yusuke; Miyahira, Hiroki; Yamamoto, Michiyo; Fukami, Ayako; Ozaki, Nobuaki; Takagishi, Yoshiko; Sato, Jun; Fukuwatari, Tsutomu; Shibata, Katsumi; Oiso, Yutaka; Murata, Yoshiharu; Hayashi, Yoshitaka

2012-01-01

Glucagon is believed to be one of the most important peptides for upregulating blood glucose levels. However, homozygous glucagon–green fluorescent protein (gfp) knock-in mice (Gcggfp/gfp: GCGKO) are normoglycemic despite the absence of proglucagon-derived peptides, including glucagon. To characterize metabolism in the GCGKO mice, we analyzed gene expression and metabolome in the liver. The expression of genes encoding rate-limiting enzymes for gluconeogenesis was only marginally altered. On the other hand, genes encoding enzymes involved in conversion of amino acids to metabolites available for the tricarboxylic acid cycle and/or gluconeogenesis showed lower expression in the GCGKO liver. The expression of genes involved in the metabolism of fatty acids and nicotinamide was also altered. Concentrations of the metabolites in the GCGKO liver were altered in manners concordant with alteration in the gene expression patterns, and the plasma concentrations of amino acids were elevated in the GCGKO mice. The insulin concentration in serum and phosphorylation of Akt protein kinase in liver were reduced in GCGKO mice. These results indicated that proglucagon-derived peptides should play important roles in regulating various metabolic pathways, especially that of amino acids. Serum insulin concentration is lowered to compensate the impacts of absent proglucagon-derived peptide on glucose metabolism. On the other hand, impacts on other metabolic pathways are only partially compensated by reduced insulin action. PMID:22187375

Insertional Mutations in the Hydrogenase vhc and frc Operons Encoding Selenium-Free Hydrogenases in Methanococcus voltae

PubMed Central

Berghofer, Y.; Klein, A.

1995-01-01

Methanococcus voltae, which contains four different gene groups that encode [NiFe]-hydrogenases, was transformed with integration vectors to achieve polar inactivation of two of the four hydrogenase operons that encode the selenium-free enzymes Vhc and Frc. Transformants which were selected by their acquired puromycin resistance showed site-specific insertions in either the vhc or frc operon by single crossover events. Southern hybridization revealed tandem integrations of whole vectors in the vhc operon, whereas only one vector copy was found in the frc operon. Northern (RNA) hybridizations showed a pac transcript of defined size, indicating strong termination in front of the hydrogenase genes downstream. In spite of the apparent abolition of expression of selenium-free hydrogenases through these polar insertions, they were not lethal to cells upon growth in selenium-deprived minimal medium, which we had previously shown to strongly induce transcription of the respective operons in M. voltae. Instead, like wild-type control cultures, transformants responded to selenium deprivation only with a reduction in growth rate. We conclude that loss of the potential to express a selenium-free hydrogenase can nevertheless be balanced by very small amounts of selenium hydrogenases under laboratory conditions in which the hydrogen supply is not likely to be a limiting growth factor. PMID:16535019

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae▿

PubMed Central

Tokuhiro, Kenro; Muramatsu, Masayoshi; Ohto, Chikara; Kawaguchi, Toshiya; Obata, Shusei; Muramoto, Nobuhiko; Hirai, Masana; Takahashi, Haruo; Kondo, Akihiko; Sakuradani, Eiji; Shimizu, Sakayu

2009-01-01

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering. PMID:19592534

Seed-Specific Expression of OsDWF4, a Rate-Limiting Gene Involved in Brassinosteroids Biosynthesis, Improves Both Grain Yield and Quality in Rice.

PubMed

Li, Qian-Feng; Yu, Jia-Wen; Lu, Jun; Fei, Hong-Yuan; Luo, Ming; Cao, Bu-Wei; Huang, Li-Chun; Zhang, Chang-Quan; Liu, Qiao-Quan

2018-04-18

Brassinosteroids (BRs) are essential plant-specific steroidal hormones that regulate diverse growth and developmental processes in plants. We evaluated the effects of OsDWF4, a gene that encodes a rate-limiting enzyme in BR biosynthesis, on both rice yield and quality when driven by the Gt1 or Ubi promoter, which correspond to seed-specific or constitutive expression, respectively. Generally, transgenic plants expressing OsDWF4 showed increased grain yield with more tillers and longer and heavier seeds. Moreover, the starch physicochemical properties of the transgenic rice were also improved. Interestingly, OsDWF4 was found to exert different effects on either rice yield or quality when driven by the different promoters. The overall performance of the pGt1::OsDWF4 lines was better than that of the pUbi::OsDWF4 lines. Our data not only demonstrate the effects of OsDWF4 overexpression on both rice yield and quality but also suggest that a seed-specific promoter is a good choice in BR-mediated rice breeding programs.

Epstein–Barr virus latent genes

PubMed Central

Kang, Myung-Soo; Kieff, Elliott

2015-01-01

Latent Epstein–Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized. PMID:25613728

The Effect of Iron Limitation on the Transcriptome and Proteome of Pseudomonas fluorescens Pf-5

PubMed Central

Lim, Chee Kent; Hassan, Karl A.; Tetu, Sasha G.; Loper, Joyce E.; Paulsen, Ian T.

2012-01-01

One of the most important micronutrients for bacterial growth is iron, whose bioavailability in soil is limited. Consequently, rhizospheric bacteria such as Pseudomonas fluorescens employ a range of mechanisms to acquire or compete for iron. We investigated the transcriptomic and proteomic effects of iron limitation on P. fluorescens Pf-5 by employing microarray and iTRAQ techniques, respectively. Analysis of this data revealed that genes encoding functions related to iron homeostasis, including pyoverdine and enantio-pyochelin biosynthesis, a number of TonB-dependent receptor systems, as well as some inner-membrane transporters, were significantly up-regulated in response to iron limitation. Transcription of a ribosomal protein L36-encoding gene was also highly up-regulated during iron limitation. Certain genes or proteins involved in biosynthesis of secondary metabolites such as 2,4-diacetylphloroglucinol (DAPG), orfamide A and pyrrolnitrin, as well as a chitinase, were over-expressed under iron-limited conditions. In contrast, we observed that expression of genes involved in hydrogen cyanide production and flagellar biosynthesis were down-regulated in an iron-depleted culture medium. Phenotypic tests revealed that Pf-5 had reduced swarming motility on semi-solid agar in response to iron limitation. Comparison of the transcriptomic data with the proteomic data suggested that iron acquisition is regulated at both the transcriptional and post-transcriptional levels. PMID:22723948

Functional Analysis of the Brassica napus L. Phytoene Synthase (PSY) Gene Family

PubMed Central

López-Emparán, Ada; Quezada-Martinez, Daniela; Zúñiga-Bustos, Matías; Cifuentes, Víctor; Iñiguez-Luy, Federico; Federico, María Laura

2014-01-01

Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three “Arabidopsis-like” subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed. PMID:25506829

Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

PubMed Central

Báez-Viveros, José Luis; Flores, Noemí; Juárez, Katy; Castillo-España, Patricia; Bolivar, Francisco; Gosset, Guillermo

2007-01-01

Background The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed. Results MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway. Conclusion The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains. PMID:17880710

Enhanced hexose fermentation by Saccharomyces cerevisiae through integration of stoichiometric modeling and genetic screening.

PubMed

Quarterman, Josh; Kim, Soo Rin; Kim, Pan-Jun; Jin, Yong-Su

2015-01-20

In order to determine beneficial gene deletions for ethanol production by the yeast Saccharomyces cerevisiae, we performed an in silico gene deletion experiment based on a genome-scale metabolic model. Genes coding for two oxidative phosphorylation reactions (cytochrome c oxidase and ubiquinol cytochrome c reductase) were identified by the model-based simulation as potential deletion targets for enhancing ethanol production and maintaining acceptable overall growth rate in oxygen-limited conditions. Since the two target enzymes are composed of multiple subunits, we conducted a genetic screening study to evaluate the in silico results and compare the effect of deleting various portions of the respiratory enzyme complexes. Over two-thirds of the knockout mutants identified by the in silico study did exhibit experimental behavior in qualitative agreement with model predictions, but the exceptions illustrate the limitation of using a purely stoichiometric model-based approach. Furthermore, there was a substantial quantitative variation in phenotype among the various respiration-deficient mutants that were screened in this study, and three genes encoding respiratory enzyme subunits were identified as the best knockout targets for improving hexose fermentation in microaerobic conditions. Specifically, deletion of either COX9 or QCR9 resulted in higher ethanol production rates than the parental strain by 37% and 27%, respectively, with slight growth disadvantages. Also, deletion of QCR6 led to improved ethanol production rate by 24% with no growth disadvantage. The beneficial effects of these gene deletions were consistently demonstrated in different strain backgrounds and with four common hexoses. The combination of stoichiometric modeling and genetic screening using a systematic knockout collection was useful for narrowing a large set of gene targets and identifying targets of interest. Copyright © 2014 Elsevier B.V. All rights reserved.

Fatty Acid Synthesis and Pyruvate Metabolism Pathways Remain Active in Dihydroartemisinin-Induced Dormant Ring Stages of Plasmodium falciparum

PubMed Central

Chen, Nanhua; LaCrue, Alexis N.; Teuscher, Franka; Waters, Norman C.; Gatton, Michelle L.; Kyle, Dennis E.

2014-01-01

Artemisinin (ART)-based combination therapy (ACT) is used as the first-line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action, there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART-induced ring-stage dormancy and recovery have been implicated as possible causes of recrudescence; however, little is known about the characteristics of dormant parasites, including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA)-induced dormancy and recovery. Transcription analysis showed an immediate downregulation for 10 genes following exposure to DHA but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly of genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, was also maintained. Additions of inhibitors for biotin acetyl-coenzyme A (CoA) carboxylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively, following DHA treatment. Our results demonstrate that most metabolic pathways are downregulated in DHA-induced dormant parasites. In contrast, fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment. PMID:24913167

Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes.

PubMed Central

van Endert, P M; Lopez, M T; Patel, S D; Monaco, J J; McDevitt, H O

1992-01-01

Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes. Images PMID:1360671

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.

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells

PubMed Central

Min, Irene M.; Waterfall, Joshua J.; Core, Leighton J.; Munroe, Robert J.; Schimenti, John; Lis, John T.

2011-01-01

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, “bivalent” ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5′ proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation. PMID:21460038

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.

Individual differences in rate of encoding predict estimates of visual short-term memory capacity (K).

PubMed

Jannati, Ali; McDonald, John J; Di Lollo, Vincent

2015-06-01

The capacity of visual short-term memory (VSTM) is commonly estimated by K scores obtained with a change-detection task. Contrary to common belief, K may be influenced not only by capacity but also by the rate at which stimuli are encoded into VSTM. Experiment 1 showed that, contrary to earlier conclusions, estimates of VSTM capacity obtained with a change-detection task are constrained by temporal limitations. In Experiment 2, we used change-detection and backward-masking tasks to obtain separate within-subject estimates of K and of rate of encoding, respectively. A median split based on rate of encoding revealed significantly higher K estimates for fast encoders. Moreover, a significant correlation was found between K and the estimated rate of encoding. The present findings raise the prospect that the reported relationships between K and such cognitive concepts as fluid intelligence may be mediated not only by VSTM capacity but also by rate of encoding. (c) 2015 APA, all rights reserved).

Term placenta shows methylation independent down regulation of Cyp19 gene in animals with retained fetal membranes.

PubMed

Ghai, Sandeep; Monga, Rachna; Mohanty, T K; Chauhan, M S; Singh, Dheer

2012-02-01

Retention of fetal membranes (RFM) is the major post-partum disorder in dairy cattle. Cyp19 gene encodes the aromatase enzyme responsible for catalyzing the rate limiting step in estrogen biosynthesis, an important hormone for placental maturation and expulsion. The present study was aimed for comparative analysis of Cyp19 gene expression and its epigenetic regulation in placental cotyledons of animals with and without RFM. Significantly lower expression of Cyp19 gene was found in placental samples of RFM affected animals in comparison to normal animals. Methylation analysis of 5 CpG dinucleotides of placenta specific Cyp19 gene promoter I.1 and proximal promoter, PII showed hypo-methylation of both PI.1 and PII in term placenta of normal and diseased animals. In conclusion, a mechanism other than promoter methylation is responsible for decreased aromatase expression in placental cotyledons of animals suffering from RFM. Copyright © 2010 Elsevier Ltd. All rights reserved.

Association study of the tryptophan hydroxylase gene and bipolar affective disorder using family-based internal controls.

PubMed

Rietschel, M; Schorr, A; Albus, M; Franzek, E; Kreiner, R; Held, T; Knapp, M; Müller, D J; Schulze, T G; Propping, P; Maier, W; Nöthen, M M

2000-06-12

The tryptophan hydroxylase (TPH) gene encodes for the rate-limiting enzyme of the serotonin metabolism and, therefore, has to be considered a major candidate for association studies in affective disorders. Recently, an association between this gene and bipolar affective disorder has been reported in a French population. We sought to replicate this finding in a German sample. Allele frequencies of a biallelic polymorphism (A218C) of the TPH gene were determined in 95 bipolar I patients and their parents. Preferential transmission of alleles from heterozygous parents to bipolar offspring was tested with the "transmission disequilibrium test" (TDT), which eliminates the contribution of population stratification to an association finding. Our sample yielded a power >90% to detect the originally reported effect. Neither allele 218A nor allele 218C were preferentially transmitted from heterozygous parents to bipolar offspring. Our results, therefore, do not support the hypothesis that the TPH gene is involved in the etiology of bipolar disorder.

A splicing mutation in the gene encoding phytoene synthase causes orange coloration in Habanero pepper fruits.

PubMed

Kim, Ok Rye; Cho, Myeong-Cheoul; Kim, Byung-Dong; Huh, Jin Hoe

2010-12-01

Peppers (Capsicum spp.) display a variety of fruit colors that are reflected by the composition and amount of diverse carotenoid pigments accumulated in the pericarp. Three independent loci, c1, c2, and y, are known to determine the mature color of pepper fruits by their allelic combinations. We examined the inheritance of fruit color in recombinant inbred lines (RILs) derived from an interspecific cross between C. annuum cv. TF68 (red) and C. chinense cv. Habanero (orange). The c2 gene encodes phytoene synthase (PSY), a rate-limiting enzyme in the carotenoid biosynthesis pathway. TF68 has a dominant c2+ allele whereas Habanero is homozygous for the recessive c2 allele, which determined RIL fruit color. Here we report that the recessive c2 allele has a point mutation in the PSY gene that occurs at a splice acceptor site of the fifth intron leading to both a frame shift and premature translational termination, suggesting that impaired activity of PSY is responsible for orange fruit color. During ripening, PSY is expressed at a significantly high level in orange colored fruits compared to red ones. Interestingly, the PSY gene of red Habanero has a conserved splice acceptor dinucleotide AG. Further analysis suggests that red Habanero is a wild type revertant of the PSY mutant orange Habanero.

Polyploidy Enhances F1 Pollen Sterility Loci Interactions That Increase Meiosis Abnormalities and Pollen Sterility in Autotetraploid Rice1[OPEN

PubMed Central

Wu, Jinwen; Chen, Lin; Chen, Zhixiong; Wang, Lan; Lu, Yonggen

2015-01-01

Intersubspecific autotetraploid rice (Oryza sativa ssp. indica × japonica) hybrids have greater biological and yield potentials than diploid rice. However, the low fertility of intersubspecific autotetraploid hybrids, which is largely caused by high pollen abortion rates, limits their commercial utility. To decipher the cytological and molecular mechanisms underlying allelic interactions in autotetraploid rice, we developed an autotetraploid rice hybrid that was heterozygous (SiSj) at F1 pollen sterility loci (Sa, Sb, and Sc) using near-isogenic lines. Cytological studies showed that the autotetraploid had higher percentages (>30%) of abnormal chromosome behavior and aberrant meiocytes (>50%) during meiosis than did the diploid rice hybrid control. Analysis of gene expression profiles revealed 1,888 genes that were differentially expressed between the autotetraploid and diploid hybrid lines at the meiotic stage, among which 889 and 999 were up- and down-regulated, respectively. Of the 999 down-regulated genes, 940 were associated with the combined effect of polyploidy and pollen sterility loci interactions (IPE). Gene Ontology enrichment analysis identified a prominent functional gene class consisting of seven genes related to photosystem I (Gene Ontology 0009522). Moreover, 55 meiosis-related or meiosis stage-specific genes were associated with IPE in autotetraploid rice, including Os02g0497500, which encodes a DNA repair-recombination protein, and Os02g0490000, which encodes a component of the ubiquitin-proteasome pathway. These results suggest that polyploidy enhances epistatic interactions between alleles of pollen sterility loci, thereby altering the expression profiles of important meiosis-related or meiosis stage-specific genes and resulting in high pollen sterility. PMID:26511913

Polyploidy Enhances F1 Pollen Sterility Loci Interactions That Increase Meiosis Abnormalities and Pollen Sterility in Autotetraploid Rice.

PubMed

Wu, Jinwen; Shahid, Muhammad Qasim; Chen, Lin; Chen, Zhixiong; Wang, Lan; Liu, Xiangdong; Lu, Yonggen

2015-12-01

Intersubspecific autotetraploid rice (Oryza sativa ssp. indica × japonica) hybrids have greater biological and yield potentials than diploid rice. However, the low fertility of intersubspecific autotetraploid hybrids, which is largely caused by high pollen abortion rates, limits their commercial utility. To decipher the cytological and molecular mechanisms underlying allelic interactions in autotetraploid rice, we developed an autotetraploid rice hybrid that was heterozygous (S(i)S(j)) at F1 pollen sterility loci (Sa, Sb, and Sc) using near-isogenic lines. Cytological studies showed that the autotetraploid had higher percentages (>30%) of abnormal chromosome behavior and aberrant meiocytes (>50%) during meiosis than did the diploid rice hybrid control. Analysis of gene expression profiles revealed 1,888 genes that were differentially expressed between the autotetraploid and diploid hybrid lines at the meiotic stage, among which 889 and 999 were up- and down-regulated, respectively. Of the 999 down-regulated genes, 940 were associated with the combined effect of polyploidy and pollen sterility loci interactions (IPE). Gene Ontology enrichment analysis identified a prominent functional gene class consisting of seven genes related to photosystem I (Gene Ontology 0009522). Moreover, 55 meiosis-related or meiosis stage-specific genes were associated with IPE in autotetraploid rice, including Os02g0497500, which encodes a DNA repair-recombination protein, and Os02g0490000, which encodes a component of the ubiquitin-proteasome pathway. These results suggest that polyploidy enhances epistatic interactions between alleles of pollen sterility loci, thereby altering the expression profiles of important meiosis-related or meiosis stage-specific genes and resulting in high pollen sterility. © 2015 American Society of Plant Biologists. All Rights Reserved.

Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates, Using a Novel Multilocus Sequence Analysis Scheme and (GTG)5-PCR Fingerprinting▿

PubMed Central

Rademaker, Jan L. W.; Herbet, Hélène; Starrenburg, Marjo J. C.; Naser, Sabri M.; Gevers, Dirk; Kelly, William J.; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E. T.

2007-01-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)5-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene. PMID:17890345

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting.

PubMed

Rademaker, Jan L W; Herbet, Hélène; Starrenburg, Marjo J C; Naser, Sabri M; Gevers, Dirk; Kelly, William J; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E T

2007-11-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.

Utilization of urea and expression profiles of related genes in the dinoflagellate Prorocentrum donghaiense

PubMed Central

Jing, Xiaoli; Lin, Senjie; Zhang, Huan; Koerting, Claudia; Yu, Zhigang

2017-01-01

Urea has been shown to contribute more than half of total nitrogen (N) required by phytoplankton in some estuaries and coastal waters and to provide a substantial portion of the N demand for many harmful algal blooms (HABs) of dinoflagellates. In this study, we investigated the physiological and transcriptional responses in Prorocentrum donghaiense to changes in nitrate and urea availability. We found that this species could efficiently utilize urea as sole N source and achieve comparable growth rate and photosynthesis capability as it did under nitrate. These physiological parameters were markedly lower in cultures grown under nitrate- or urea-limited conditions. P. donghaiense N content was similarly low under nitrate- or urea-limited culture condition, but was markedly higher under urea-replete condition than under nitrate-replete condition. Carbon (C) content was consistently elevated under N-limited condition. Consequently, the C:N ratio was as high as 21:1 under nitrate- or urea-limitation, but 7:1 under urea-replete condition and 9:1 to 10:1 under nitrate-replete condition. Using quantitative reverse transcription PCR, we investigated the expression pattern for four genes involved in N transport and assimilation. The results indicated that genes encoding nitrate transport, urea hydrolysis, and nickel transporter gene were sensitive to changes in general N nutrient availability whereas the urea transporter gene responded much more strongly to changes in urea concentration. Taken together, our study shows the high bioavailability of urea, its impact on C:N stoichiometry, and the sensitivity of urea transporter gene expression to urea availability. PMID:29117255

Utilization of urea and expression profiles of related genes in the dinoflagellate Prorocentrum donghaiense.

PubMed

Jing, Xiaoli; Lin, Senjie; Zhang, Huan; Koerting, Claudia; Yu, Zhigang

2017-01-01

Urea has been shown to contribute more than half of total nitrogen (N) required by phytoplankton in some estuaries and coastal waters and to provide a substantial portion of the N demand for many harmful algal blooms (HABs) of dinoflagellates. In this study, we investigated the physiological and transcriptional responses in Prorocentrum donghaiense to changes in nitrate and urea availability. We found that this species could efficiently utilize urea as sole N source and achieve comparable growth rate and photosynthesis capability as it did under nitrate. These physiological parameters were markedly lower in cultures grown under nitrate- or urea-limited conditions. P. donghaiense N content was similarly low under nitrate- or urea-limited culture condition, but was markedly higher under urea-replete condition than under nitrate-replete condition. Carbon (C) content was consistently elevated under N-limited condition. Consequently, the C:N ratio was as high as 21:1 under nitrate- or urea-limitation, but 7:1 under urea-replete condition and 9:1 to 10:1 under nitrate-replete condition. Using quantitative reverse transcription PCR, we investigated the expression pattern for four genes involved in N transport and assimilation. The results indicated that genes encoding nitrate transport, urea hydrolysis, and nickel transporter gene were sensitive to changes in general N nutrient availability whereas the urea transporter gene responded much more strongly to changes in urea concentration. Taken together, our study shows the high bioavailability of urea, its impact on C:N stoichiometry, and the sensitivity of urea transporter gene expression to urea availability.

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

SNPs in the 5'-regulatory region of the tyrosinase gene do not affect plumage color in ducks (Anas platyrhynchos).

PubMed

Zhang, N N; Hu, J W; Liu, H H; Xu, H Y; He, H; Li, L

2015-12-29

Tyrosinase, encoded by the TYR gene, is the rate-limiting enzyme in the production of melanin pigment. In this study, plumage color separation was observed in Cherry Valley duck line D and F1 and F2 hybrid generations of Liancheng white ducks. Gene sequencing and bioinformatic analysis were applied to the 5'-regulatory region of TYR, to explore the connection between TYR sequence variation and duck plumage color. Four SNPs were found in the 5'-regulatory region. The SNPs were in tight linkage and formed three haplotypes. However, the genotype distribution in groups with different plumage color was not significantly different, and there were no changes in the transcription factor binding sites between the different genotypes. In conclusion, these SNP variations may not cause the differences in feather color observed in this test group.

The draft genome of sweet orange (Citrus sinensis).

PubMed

Xu, Qiang; Chen, Ling-Ling; Ruan, Xiaoan; Chen, Dijun; Zhu, Andan; Chen, Chunli; Bertrand, Denis; Jiao, Wen-Biao; Hao, Bao-Hai; Lyon, Matthew P; Chen, Jiongjiong; Gao, Song; Xing, Feng; Lan, Hong; Chang, Ji-Wei; Ge, Xianhong; Lei, Yang; Hu, Qun; Miao, Yin; Wang, Lun; Xiao, Shixin; Biswas, Manosh Kumar; Zeng, Wenfang; Guo, Fei; Cao, Hongbo; Yang, Xiaoming; Xu, Xi-Wen; Cheng, Yun-Jiang; Xu, Juan; Liu, Ji-Hong; Luo, Oscar Junhong; Tang, Zhonghui; Guo, Wen-Wu; Kuang, Hanhui; Zhang, Hong-Yu; Roose, Mikeal L; Nagarajan, Niranjan; Deng, Xiu-Xin; Ruan, Yijun

2013-01-01

Oranges are an important nutritional source for human health and have immense economic value. Here we present a comprehensive analysis of the draft genome of sweet orange (Citrus sinensis). The assembled sequence covers 87.3% of the estimated orange genome, which is relatively compact, as 20% is composed of repetitive elements. We predicted 29,445 protein-coding genes, half of which are in the heterozygous state. With additional sequencing of two more citrus species and comparative analyses of seven citrus genomes, we present evidence to suggest that sweet orange originated from a backcross hybrid between pummelo and mandarin. Focused analysis on genes involved in vitamin C metabolism showed that GalUR, encoding the rate-limiting enzyme of the galacturonate pathway, is significantly upregulated in orange fruit, and the recent expansion of this gene family may provide a genomic basis. This draft genome represents a valuable resource for understanding and improving many important citrus traits in the future.

Acetate scavenging activity in Escherichia coli: interplay of acetyl-CoA synthetase and the PEP-glyoxylate cycle in chemostat cultures.

PubMed

Renilla, Sergio; Bernal, Vicente; Fuhrer, Tobias; Castaño-Cerezo, Sara; Pastor, José M; Iborra, José L; Sauer, Uwe; Cánovas, Manuel

2012-03-01

Impairment of acetate production in Escherichia coli is crucial for the performance of many biotechnological processes. Aerobic production of acetate (or acetate overflow) results from changes in the expression of central metabolism genes. Acetyl-CoA synthetase scavenges extracellular acetate in glucose-limited cultures. Once converted to acetyl-CoA, it can be catabolized by the tricarboxylic acid cycle or the glyoxylate pathway. In this work, we assessed the significance of these pathways on acetate overflow during glucose excess and limitation. Gene expression, enzyme activities, and metabolic fluxes were studied in E. coli knock-out mutants related to the glyoxylate pathway operon and its regulators. The relevance of post-translational regulation by AceK-mediated phosphorylation of isocitrate dehydrogenase for pathway functionality was underlined. In chemostat cultures performed at increasing dilution rates, acetate overflow occurs when growing over a threshold glucose uptake rate. This threshold was not affected in a glyoxylate-pathway-deficient strain (lacking isocitrate lyase, the first enzyme of the pathway), indicating that it is not relevant for acetate overflow. In carbon-limited chemostat cultures, gluconeogenesis (maeB, sfcA, and pck), the glyoxylate operon and, especially, acetyl-CoA synthetase are upregulated. A mutant in acs (encoding acetyl-CoA synthetase) produced acetate at all dilution rates. This work demonstrates that, in E. coli, acetate production occurs at all dilution rates and that overflow is the result of unbalanced synthesis and scavenging activities. The over-expression of acetyl-CoA synthetase by cAMP-CRP-dependent induction limits this phenomenon in cultures consuming glucose at low rate, ensuring the recycling of the acetyl-CoA and acetyl-phosphate pools, although establishing an energy-dissipating substrate cycle.

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

Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin-induced dormant ring stages of Plasmodium falciparum.

PubMed

Chen, Nanhua; LaCrue, Alexis N; Teuscher, Franka; Waters, Norman C; Gatton, Michelle L; Kyle, Dennis E; Cheng, Qin

2014-08-01

Artemisinin (ART)-based combination therapy (ACT) is used as the first-line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action, there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART-induced ring-stage dormancy and recovery have been implicated as possible causes of recrudescence; however, little is known about the characteristics of dormant parasites, including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA)-induced dormancy and recovery. Transcription analysis showed an immediate downregulation for 10 genes following exposure to DHA but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly of genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, was also maintained. Additions of inhibitors for biotin acetyl-coenzyme A (CoA) carboxylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively, following DHA treatment. Our results demonstrate that most metabolic pathways are downregulated in DHA-induced dormant parasites. In contrast, fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Investigating the Molecular Mechanism of TSO1 Function in Arabidopsis cell division and meristem development

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

Zhongchi Liu

2004-10-01

Unlike animals, plants are constantly exposed to environmental mutagens including ultraviolet light and reactive oxygen species. Further, plant cells are totipotent with highly plastic developmental programs. An understanding of molecular mechanisms underlying the ability of plants to monitor and repair its DNA and to eliminate damaged cells are of great importance. Previously we have identified two genes, TSO1 and TSO2, from a flowering plant Arabidopsis thaliana. Mutations in these two genes cause callus-like flowers, fasciated shoot apical meristems, and abnormal cell division, indicating that TSO1 and TSO2 may encode important cell cycle regulators. Previous funding from DOE led to themore » molecular cloning of TSO1, which was shown to encode a novel nuclear protein with two CXC domains suspected to bind DNA. This DOE grant has allowed us to characterize and isolate TSO2 that encodes the small subunit of the ribonucleotide reductase (RNR). RNR comprises two large subunits (R1) an d two small subunits (R2), catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA replication and repair. Previous studies in yeast and mammals indicated that defective RNR often led to cell cycle arrest, growth retardation and p53-dependent apoptosis while abnormally elevated RNR activities led to higher mutation rates. Subsequently, we identified two additional R2 genes, R2A and R2B in the Arabidopsis genome. Using reverse genetics, mutations in R2A and R2B were isolated, and double and triple mutants among the three R2 genes (TSO2, R2A and R2B) were constructed and analyzed. We showed that Arabidopsis tso2 mutants, with reduced dNTP levels, were more sensitive to UV-C. While r2a or r2b single mutants did not exhibit any phenotypes, tso2 r2b double mutants were embryonic lethal and tso2 r2a double mutants were seedling lethal indicating redundant functions among the three R2 genes. Furthermore, tso2 r2a double mutants exhibited increased DNA dam age, massive programmed cell death, and the release of transcriptional gene silencing. Our data suggests that plants can initiate programmed cell death to eliminate damaged cells despite the absence of p53 in plant genome.« less

Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley

USDA-ARS?s Scientific Manuscript database

The SQUAMOSA-promoter binding like (SPL) gene family encodes transcription factors shown in a number of species to influence plant growth and development, but information about these genes in barley is limited. This study identified 13 barley SPL genes, within five distinct groups, that are ortholog...

Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

PubMed

Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

2016-03-01

The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

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

PubMed Central

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

2017-01-01

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

MobileASL: intelligibility of sign language video over mobile phones.

PubMed

Cavender, Anna; Vanam, Rahul; Barney, Dane K; Ladner, Richard E; Riskin, Eve A

2008-01-01

For Deaf people, access to the mobile telephone network in the United States is currently limited to text messaging, forcing communication in English as opposed to American Sign Language (ASL), the preferred language. Because ASL is a visual language, mobile video phones have the potential to give Deaf people access to real-time mobile communication in their preferred language. However, even today's best video compression techniques can not yield intelligible ASL at limited cell phone network bandwidths. Motivated by this constraint, we conducted one focus group and two user studies with members of the Deaf Community to determine the intelligibility effects of video compression techniques that exploit the visual nature of sign language. Inspired by eye tracking results that show high resolution foveal vision is maintained around the face, we studied region-of-interest encodings (where the face is encoded at higher quality) as well as reduced frame rates (where fewer, better quality, frames are displayed every second). At all bit rates studied here, participants preferred moderate quality increases in the face region, sacrificing quality in other regions. They also preferred slightly lower frame rates because they yield better quality frames for a fixed bit rate. The limited processing power of cell phones is a serious concern because a real-time video encoder and decoder will be needed. Choosing less complex settings for the encoder can reduce encoding time, but will affect video quality. We studied the intelligibility effects of this tradeoff and found that we can significantly speed up encoding time without severely affecting intelligibility. These results show promise for real-time access to the current low-bandwidth cell phone network through sign-language-specific encoding techniques.

Modification of glucose import capacity in Escherichia coli: physiologic consequences and utility for improving DNA vaccine production

PubMed Central

2013-01-01

Background The bacterium Escherichia coli can be grown employing various carbohydrates as sole carbon and energy source. Among them, glucose affords the highest growth rate. This sugar is nowadays widely employed as raw material in industrial fermentations. When E. coli grows in a medium containing non-limiting concentrations of glucose, a metabolic imbalance occurs whose main consequence is acetate secretion. The production of this toxic organic acid reduces strain productivity and viability. Solutions to this problem include reducing glucose concentration by substrate feeding strategies or the generation of mutant strains with impaired glucose import capacity. In this work, a collection of E. coli strains with inactive genes encoding proteins involved in glucose transport where generated to determine the effects of reduced glucose import capacity on growth rate, biomass yield, acetate and production of an experimental plasmid DNA vaccine (pHN). Results A group of 15 isogenic derivatives of E. coli W3110 were generated with single and multiple deletions of genes encoding glucose, mannose, beta-glucoside, maltose and N-acetylglucosamine components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), as well as the galactose symporter and the Mgl galactose/glucose ABC transporter. These strains were characterized by growing them in mineral salts medium supplemented with 2.5 g/L glucose. Maximum specific rates of glucose consumption (qs) spanning from 1.33 to 0.32 g/g h were displayed by the group of mutants and W3110, which resulted in specific growth rates ranging from 0.65-0.18 h-1. Acetate accumulation was reduced or abolished in cultures with all mutant strains. W3110 and five selected mutant derivatives were transformed with pHN. A 3.2-fold increase in pHN yield on biomass was observed in cultures of a mutant strain with deletion of genes encoding the glucose and mannose PTS components, as well as Mgl. Conclusions The group of E. coli mutants generated in this study displayed a reduction or elimination of overflow metabolism and a linear correlation between qs and the maximum specific growth rate as well as the acetate production rate. By comparing DNA vaccine production parameters among some of these mutants, it was possible to identify a near-optimal glucose import rate value for this particular application. The strains employed in this study should be a useful resource for studying the effects of different predefined qs values on production capacity for various biotechnological products. PMID:23638701

Activation of the Yeast UBI4 Polyubiquitin Gene by Zap1 Transcription Factor via an Intragenic Promoter Is Critical for Zinc-deficient Growth*

PubMed Central

MacDiarmid, Colin W.; Taggart, Janet; Jeong, Jeeyon; Kerdsomboon, Kittikhun; Eide, David J.

2016-01-01

Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4Δ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4Δ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2E301K) suppressed the ubi4Δ growth defect. The rpt2E301K mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4Δ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2E301K mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ∼5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4Δ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells. PMID:27432887

Variants in the human intestinal fatty acid binding protein 2 gene in obese subjects.

PubMed

Sipiläinen, R; Uusitupa, M; Heikkinen, S; Rissanen, A; Laakso, M

1997-08-01

Fatty acid binding protein 2 gene (FABP2) has been proposed to be an important candidate gene for insulin resistance; therefore, it also could be a promising candidate gene for obesity. We screened the whole coding region of the FABP2 gene in 40 obese nondiabetic Finnish subjects. Furthermore, we investigated the effects of the codon 54 polymorphism of this gene (Ala-->Thr) on insulin levels and basal metabolic rate in 170 obese subjects. The frequencies of the variants found in exon 4 (GTA-->GTG) and 3'-noncoding region (GCGCA-->GCACA), as well as the allele frequencies for the variable lengths of the ATT repeat sequence in intron 2 did not differ between the obese subjects and nonobese controls. The frequency of threonine-encoding allele in codon 54 of the FABP2 gene did not differ between obese and control subjects (28 vs. 29%, respectively). In the obese group there were no differences in gender distribution, age, weight, body mass index, lean body mass, percentage of body fat, waist circumference, and waist-to-hip ratio among the individuals homozygous for Ala54, heterozygous for Thr54, and homozygous for Thr54-encoding alleles. Similarly, fasting serum insulin, glucose, lipids and lipoprotein concentrations, basal metabolic rate (adjusted for lean body mass and age), respiratory quotient, and rates of glucose and lipid oxidation did not differ among the groups. We conclude that obesity is not associated with specific variants in the FABP2 gene. Furthermore, the codon 54 Ala to Thr polymorphism of this gene does not influence insulin levels or basal metabolic rate in obese Finns.

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

PubMed

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

2017-01-01

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

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.

Tailor-made gene silencing of Staphylococcus aureus clinical isolates by CRISPR interference

PubMed Central

Sato’o, Yusuke; Hisatsune, Junzo; Yu, Liansheng; Sakuma, Tetsushi; Yamamoto, Takashi

2018-01-01

Preparing the genetically modified organisms have required much time and labor, making it the rate-limiting step but CRISPR/Cas9 technology appearance has changed this difficulty. Although reports on CRISPR/Cas9 technology such as genome editing and CRISPR interference (CRISPRi) in eukaryotes increased, those in prokaryotes especially in Staphylococci were limited. Thus, its potential in the bacteriology remains unexplored. This is attributed to ecological difference between eukaryotes and prokaryotes. Here, we constructed a novel CRISPRi plasmid vector, pBACi for Staphylococcus aureus. The transformation efficiency of S. aureus was ~104 CFU/μg DNA using a vector extracted from dcm negative, which encoded one of DNA modification genes, E. coli. Further, pBACi was introduced into various clinical isolates including that not accepting the conventional temperature-sensitive vector. dcas9 in the vector was expressed throughout the growth phases of S. aureus and this vector decreased various gene mRNA expressions based on the crRNA targeting sequences and altered the knockdown strains’ phenotypes. The targeted genes included various virulence and antibiotic resistant genes. Bioinformatics suggest this vector can be introduced into wide range of low-GC Gram-positive bacteria. Because this new CRISPR/Cas9-based vector can easily prepare knockdown strains, we believe the novel vector will facilitate the characterization of the function of genes from S. aureus and other Gram-positive bacteria. PMID:29377933

Molecular Cloning and Characterization of Two Genes Encoding Dihydroflavonol-4-Reductase from Populus trichocarpa

PubMed Central

Jia, Zhichun; Yang, Li; Sun, Yimin; Xiao, Xunyan; Song, Feng; Luo, Keming

2012-01-01

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a rate-limited enzyme in the biosynthesis of anthocyanins and condensed tannins (proanthocyanidins) that catalyzes the reduction of dihydroflavonols to leucoanthocyanins. In this study, two full-length transcripts encoding for PtrDFR1 and PtrDFR2 were isolated from Populus trichocarpa. Sequence alignment of the two PtrDFRs with other known DFRs reveals the homology of these genes. The expression profile of PtrDFRs was investigated in various tissues of P. trichocarpa. To determine their functions, two PtrDFRs were overexpressed in tobacco (Nicotiana tabacum) via Agrobacterium-mediated transformation. The associated color change in the flowers was observed in all 35S:PtrDFR1 lines, but not in 35S:PtrDFR2 lines. Compared to the wild-type control, a significantly higher accumulation of anthocyanins was detected in transgenic plants harboring the PtrDFR1. Furthermore, overexpressing PtrDFR1 in Chinese white poplar (P. tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation, indicating the potential regulation of condensed tannins by PtrDFR2 in the biosynthetic pathway in poplars. PMID:22363429

Assessing the resistance and bioremediation ability of selected bacterial and protozoan species to heavy metals in metal-rich industrial wastewater

PubMed Central

2013-01-01

Background Heavy-metals exert considerable stress on the environment worldwide. This study assessed the resistance to and bioremediation of heavy-metals by selected protozoan and bacterial species in highly polluted industrial-wastewater. Specific variables (i.e. chemical oxygen demand, pH, dissolved oxygen) and the growth/die-off-rates of test organisms were measured using standard methods. Heavy-metal removals were determined in biomass and supernatant by the Inductively Couple Plasma Optical Emission Spectrometer. A parallel experiment was performed with dead microbial cells to assess the biosorption ability of test isolates. Results The results revealed that the industrial-wastewater samples were highly polluted with heavy-metal concentrations exceeding by far the maximum limits (in mg/l) of 0.05-Co, 0.2-Ni, 0.1-Mn, 0.1-V, 0.01-Pb, 0.01-Cu, 0.1-Zn and 0.005-Cd, prescribed by the UN-FAO. Industrial-wastewater had no major effects on Pseudomonas putida, Bacillus licheniformis and Peranema sp. (growth rates up to 1.81, 1.45 and 1.43 d-1, respectively) compared to other test isolates. This was also revealed with significant COD increases (p < 0.05) in culture media inoculated with living bacterial isolates (over 100%) compared to protozoan isolates (up to 24% increase). Living Pseudomonas putida demonstrated the highest removal rates of heavy metals (Co-71%, Ni-51%, Mn-45%, V-83%, Pb-96%, Ti-100% and Cu-49%) followed by Bacillus licheniformis (Al-23% and Zn-53%) and Peranema sp. (Cd-42%). None of the dead cells were able to remove more than 25% of the heavy metals. Bacterial isolates contained the genes copC, chrB, cnrA3 and nccA encoding the resistance to Cu, Cr, Co-Ni and Cd-Ni-Co, respectively. Protozoan isolates contained only the genes encoding Cu and Cr resistance (copC and chrB genes). Peranema sp. was the only protozoan isolate which had an additional resistant gene cnrA3 encoding Co-Ni resistance. Conclusion Significant differences (p < 0.05) observed between dead and living microbial cells for metal-removal and the presence of certain metal-resistant genes indicated that the selected microbial isolates used both passive (biosorptive) and active (bioaccumulation) mechanisms to remove heavy metals from industrial wastewater. This study advocates the use of Peranema sp. as a potential candidate for the bioremediation of heavy-metals in wastewater treatment, in addition to Pseudomonas putida and Bacillus licheniformis. PMID:23387904

Assessing the resistance and bioremediation ability of selected bacterial and protozoan species to heavy metals in metal-rich industrial wastewater.

PubMed

Kamika, Ilunga; Momba, Maggy N B

2013-02-06

Heavy-metals exert considerable stress on the environment worldwide. This study assessed the resistance to and bioremediation of heavy-metals by selected protozoan and bacterial species in highly polluted industrial-wastewater. Specific variables (i.e. chemical oxygen demand, pH, dissolved oxygen) and the growth/die-off-rates of test organisms were measured using standard methods. Heavy-metal removals were determined in biomass and supernatant by the Inductively Couple Plasma Optical Emission Spectrometer. A parallel experiment was performed with dead microbial cells to assess the biosorption ability of test isolates. The results revealed that the industrial-wastewater samples were highly polluted with heavy-metal concentrations exceeding by far the maximum limits (in mg/l) of 0.05-Co, 0.2-Ni, 0.1-Mn, 0.1-V, 0.01-Pb, 0.01-Cu, 0.1-Zn and 0.005-Cd, prescribed by the UN-FAO. Industrial-wastewater had no major effects on Pseudomonas putida, Bacillus licheniformis and Peranema sp. (growth rates up to 1.81, 1.45 and 1.43 d-1, respectively) compared to other test isolates. This was also revealed with significant COD increases (p < 0.05) in culture media inoculated with living bacterial isolates (over 100%) compared to protozoan isolates (up to 24% increase). Living Pseudomonas putida demonstrated the highest removal rates of heavy metals (Co-71%, Ni-51%, Mn-45%, V-83%, Pb-96%, Ti-100% and Cu-49%) followed by Bacillus licheniformis (Al-23% and Zn-53%) and Peranema sp. (Cd-42%). None of the dead cells were able to remove more than 25% of the heavy metals. Bacterial isolates contained the genes copC, chrB, cnrA3 and nccA encoding the resistance to Cu, Cr, Co-Ni and Cd-Ni-Co, respectively. Protozoan isolates contained only the genes encoding Cu and Cr resistance (copC and chrB genes). Peranema sp. was the only protozoan isolate which had an additional resistant gene cnrA3 encoding Co-Ni resistance. Significant differences (p < 0.05) observed between dead and living microbial cells for metal-removal and the presence of certain metal-resistant genes indicated that the selected microbial isolates used both passive (biosorptive) and active (bioaccumulation) mechanisms to remove heavy metals from industrial wastewater. This study advocates the use of Peranema sp. as a potential candidate for the bioremediation of heavy-metals in wastewater treatment, in addition to Pseudomonas putida and Bacillus licheniformis.

Producing human ceramide-NS by metabolic engineering using yeast Saccharomyces cerevisiae.

PubMed

Murakami, Suguru; Shimamoto, Toshi; Nagano, Hideaki; Tsuruno, Masahiro; Okuhara, Hiroaki; Hatanaka, Haruyo; Tojo, Hiromasa; Kodama, Yukiko; Funato, Kouichi

2015-11-17

Ceramide is one of the most important intercellular components responsible for the barrier and moisture retention functions of the skin. Because of the risks involved with using products of animal origin and the low productivity of plants, the availability of ceramides is currently limited. In this study, we successfully developed a system that produces sphingosine-containing human ceramide-NS in the yeast Saccharomyces cerevisiae by eliminating the genes for yeast sphingolipid hydroxylases (encoded by SUR2 and SCS7) and introducing the gene for a human sphingolipid desaturase (encoded by DES1). The inactivation of the ceramidase gene YDC1, overexpression of the inositol phosphosphingolipid phospholipase C gene ISC1, and endoplasmic reticulum localization of the DES1 gene product resulted in enhanced production of ceramide-NS. The engineered yeast strains can serve as hosts not only for providing a sustainable source of ceramide-NS but also for developing further systems to produce sphingosine-containing sphingolipids.

Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: Many losses of photosynthesis and complex patterns of rate variation

PubMed Central

dePamphilis, Claude W.; Young, Nelson D.; Wolfe, Andrea D.

1997-01-01

The plastid genomes of some nonphotosynthetic parasitic plants have experienced an extreme reduction in gene content and an increase in evolutionary rate of remaining genes. Nothing is known of the dynamics of these events or whether either is a direct outcome of the loss of photosynthesis. The parasitic Scrophulariaceae and Orobanchaceae, representing a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to nonphotosynthetic holoparasites, are used to investigate these issues. We present a phylogenetic hypothesis for parasitic Scrophulariaceae and Orobanchaceae based on sequences of the plastid gene rps2, encoding the S2 subunit of the plastid ribosome. Parasitic Scrophulariaceae and Orobanchaceae form a monophyletic group in which parasitism can be inferred to have evolved once. Holoparasitism has evolved independently at least five times, with certain holoparasitic lineages representing single species, genera, and collections of nonphotosynthetic genera. Evolutionary loss of the photosynthetic gene rbcL is limited to a subset of holoparasitic lineages, with several holoparasites retaining a full length rbcL sequence. In contrast, the translational gene rps2 is retained in all plants investigated but has experienced rate accelerations in several hemi- as well as holoparasitic lineages, suggesting that there may be substantial molecular evolutionary changes to the plastid genome of parasites before the loss of photosynthesis. Independent patterns of synonymous and nonsynonymous rate acceleration in rps2 point to distinct mechanisms underlying rate variation in different lineages. Parasitic Scrophulariaceae (including the traditional Orobanchaceae) provide a rich platform for the investigation of molecular evolutionary process, gene function, and the evolution of parasitism. PMID:9207097

Prevalence and Characterization of Carbapenem-Resistant Enterobacteriaceae Isolated from Mulago National Referral Hospital, Uganda

PubMed Central

Okoche, Deogratius; Asiimwe, Benon B.; Katabazi, Fred Ashaba; Kato, Laban; Najjuka, Christine F.

2015-01-01

Introduction Carbapenemases have increasingly been reported in enterobacteriaceae worldwide. Most carbapenemases are plasmid encoded hence resistance can easily spread. Carbapenem-resistant enterobacteriaceae are reported to cause mortality in up to 50% of patients who acquire bloodstream infections. We set out to determine the burden of carbapenem resistance as well as establish genes encoding for carbapenemases in enterobacteriaceae clinical isolates obtained from Mulago National Referral Hospital, Uganda. Methods This was a cross-sectional study with a total of 196 clinical isolates previously collected from pus swabs, urine, blood, sputum, tracheal aspirates, cervical swabs, endomentrial aspirates, rectal swabs, Vaginal swabs, ear swabs, products of conception, wound biopsy and amniotic fluid. All isolates were subjected to phenotypic carbapenemase screening using Boronic acid-based inhibition, Modified Hodge and EDTA double combined disk test. In addition, all the isolates were subjected to PCR assay to confirm presence of carbapenemase encoding genes. Results The study found carbapenemase prevalence of 22.4% (44/196) in the isolates using phenotypic tests, with the genotypic prevalence slightly higher at 28.6% (56/196). Over all, the most prevalent gene was blaVIM (21,10.7%), followed by blaOXA-48 (19, 9.7%), blaIMP (12, 6.1%), blaKPC (10, 5.1%) and blaNDM-1 (5, 2.6%). Among 56 isolates positive for 67 carbapenemase encoding genes, Klebsiella pneumonia was the species with the highest number (52.2%). Most 32/67(47.7%) of these resistance genes were in bacteria isolated from pus swabs. Conclusion There is a high prevalence of carbapenemases and carbapenem-resistance encoding genes among third generation cephalosporins resistant Enterobacteriaceae in Uganda, indicating a danger of limited treatment options in this setting in the near future. PMID:26284519

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.

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.

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

PubMed Central

Smit, Bart A.; van Hylckama Vlieg, Johan E. T.; Engels, Wim J. M.; Meijer, Laura; Wouters, Jan T. M.; Smit, Gerrit

2005-01-01

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions. PMID:15640202

Information analysis of posterior canal afferents in the turtle, Trachemys scripta elegans.

PubMed

Rowe, Michael H; Neiman, Alexander B

2012-01-24

We have used sinusoidal and band-limited Gaussian noise stimuli along with information measures to characterize the linear and non-linear responses of morpho-physiologically identified posterior canal (PC) afferents and to examine the relationship between mutual information rate and other physiological parameters. Our major findings are: 1) spike generation in most PC afferents is effectively a stochastic renewal process, and spontaneous discharges are fully characterized by their first order statistics; 2) a regular discharge, as measured by normalized coefficient of variation (cv*), reduces intrinsic noise in afferent discharges at frequencies below the mean firing rate; 3) coherence and mutual information rates, calculated from responses to band-limited Gaussian noise, are jointly determined by gain and intrinsic noise (discharge regularity), the two major determinants of signal to noise ratio in the afferent response; 4) measures of optimal non-linear encoding were only moderately greater than optimal linear encoding, indicating that linear stimulus encoding is limited primarily by internal noise rather than by non-linearities; and 5) a leaky integrate and fire model reproduces these results and supports the suggestion that the combination of high discharge regularity and high discharge rates serves to extend the linear encoding range of afferents to higher frequencies. These results provide a framework for future assessments of afferent encoding of signals generated during natural head movements and for comparison with coding strategies used by other sensory systems. This article is part of a Special Issue entitled: Neural Coding. Copyright © 2011 Elsevier B.V. All rights reserved.

A genomic approach to the understanding of Xylella fastidiosa pathogenicity.

PubMed

Lambais, M R; Goldman, M H; Camargo, L E; Goldman, G H

2000-10-01

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes several economically important plant diseases, including citrus variegated chlorosis (CVC). X. fastidiosa is the first plant pathogen to have its genome completely sequenced. In addition, it is probably the least previously studied of any organism for which the complete genome sequence is available. Several pathogenicity-related genes have been identified in the X. fastidiosa genome by similarity with other bacterial genes involved in pathogenesis in plants, as well as in animals. The X. fastidiosa genome encodes different classes of proteins directly or indirectly involved in cell-cell interactions, degradation of plant cell walls, iron homeostasis, anti-oxidant responses, synthesis of toxins, and regulation of pathogenicity. Neither genes encoding members of the type III protein secretion system nor avirulence-like genes have been identified in X. fastidiosa.

Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from Pseudomonas rhodesiae CIP 107491 and Production of the Recombinant Protein in Escherichia coli.

PubMed

Dubessay, Pascal; Larroche, Christian; Fontanille, Pierre

2017-12-28

The alpha-pinene oxide lyase (Prα-POL) from Pseudomonas rhodesiae CIP107491 belongs to catabolic alpha-pinene degradation pathway. In this study, the gene encoding Prα-POL has been identified using mapping approach combined to inverse PCR (iPCR) strategy. The Prα-POL gene included a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7 kDa protein, with a theoretical isoelectric point (pI) of 5.23. The amino acids sequence analysis showed homologies with those of proteins with unknown function from GammaProteobacteria group. Identification of a conserved domain in amino acid in positions 18 to 190 permitted to classify Prα-POL among the nuclear transport factor 2 (NTF2) protein superfamily. Heterologous expression of Prα-POL, both under its native form and with a histidin tag, was successfully performed in Escherichia coli, and enzymatic kinetics were analyzed. Bioconversion assay using recombinant E. coli strain allowed to reach a rate of isonovalal production per gramme of biomass about 40-fold higher than the rate obtained with P. rhodesiae.

A Mutation in the Bacillus subtilis rsbU Gene That Limits RNA Synthesis during Sporulation.

PubMed

Rothstein, David M; Lazinski, David; Osburne, Marcia S; Sonenshein, Abraham L

2017-07-15

Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32 P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate. IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU , a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes. Copyright © 2017 American Society for Microbiology.

Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China.

PubMed

Zhao, Huanqiang; Hu, Fupin; Jin, Shu; Xu, Xiaogang; Zou, Yuhan; Ding, Baixing; He, Chunyan; Gong, Fang; Liu, Qingzhong

2016-01-01

Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

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

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.

Systematic Analysis and Comparison of Nucleotide-Binding Site Disease Resistance Genes in a Diploid Cotton Gossypium raimondii

PubMed Central

Wei, Hengling; Li, Wei; Sun, Xiwei; Zhu, Shuijin; Zhu, Jun

2013-01-01

Plant disease resistance genes are a key component of defending plants from a range of pathogens. The majority of these resistance genes belong to the super-family that harbors a Nucleotide-binding site (NBS). A number of studies have focused on NBS-encoding genes in disease resistant breeding programs for diverse plants. However, little information has been reported with an emphasis on systematic analysis and comparison of NBS-encoding genes in cotton. To fill this gap of knowledge, in this study, we identified and investigated the NBS-encoding resistance genes in cotton using the whole genome sequence information of Gossypium raimondii. Totally, 355 NBS-encoding resistance genes were identified. Analyses of the conserved motifs and structural diversity showed that the most two distinct features for these genes are the high proportion of non-regular NBS genes and the high diversity of N-termini domains. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication of disease resistance genes could play an important role in cotton by leading to an increase in the functional diversity of the cotton NBS-encoding genes. Analyses of phylogenetic comparisons indicated that, in cotton, the NBS-encoding genes with TIR domain not only have their own evolution pattern different from those of genes without TIR domain, but also have their own species-specific pattern that differs from those of TIR genes in other plants. Analyses of the correlation between disease resistance QTL and NBS-encoding resistance genes showed that there could be more than half of the disease resistance QTL associated to the NBS-encoding genes in cotton, which agrees with previous studies establishing that more than half of plant resistance genes are NBS-encoding genes. PMID:23936305

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana.

PubMed

Yu, Jingyin; Tehrim, Sadia; Zhang, Fengqi; Tong, Chaobo; Huang, Junyan; Cheng, Xiaohui; Dong, Caihua; Zhou, Yanqiu; Qin, Rui; Hua, Wei; Liu, Shengyi

2014-01-03

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana. Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species. This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Effects of Elevated CO2 on Levels of Primary Metabolites and Transcripts of Genes Encoding Respiratory Enzymes and Their Diurnal Patterns in Arabidopsis thaliana: Possible Relationships with Respiratory Rates

PubMed Central

Watanabe, Chihiro K.; Sato, Shigeru; Yanagisawa, Shuichi; Uesono, Yukifumi; Terashima, Ichiro; Noguchi, Ko

2014-01-01

Elevated CO2 affects plant growth and photosynthesis, which results in changes in plant respiration. However, the mechanisms underlying the responses of plant respiration to elevated CO2 are poorly understood. In this study, we measured diurnal changes in the transcript levels of genes encoding respiratory enzymes, the maximal activities of the enzymes and primary metabolite levels in shoots of Arabidopsis thaliana grown under moderate or elevated CO2 conditions (390 or 780 parts per million by volume CO2, respectively). We examined the relationships between these changes and respiratory rates. Under elevated CO2, the transcript levels of several genes encoding respiratory enzymes increased at the end of the light period, but these increases did not result in changes in the maximal activities of the corresponding enzymes. The levels of some primary metabolites such as starch and sugar phosphates increased under elevated CO2, particularly at the end of the light period. The O2 uptake rate at the end of the dark period was higher under elevated CO2 than under moderate CO2, but higher under moderate CO2 than under elevated CO2 at the end of the light period. These results indicate that the changes in O2 uptake rates are not directly related to changes in maximal enzyme activities and primary metabolite levels. Instead, elevated CO2 may affect anabolic processes that consume respiratory ATP, thereby affecting O2 uptake rates. PMID:24319073

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

PubMed Central

Khosla, C; McDaniel, R; Ebert-Khosla, S; Torres, R; Sherman, D H; Bibb, M J; Hopwood, D A

1993-01-01

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases. Images PMID:8468280

Effects of dietary neutral detergent fiber and starch ratio on rumen epithelial cell morphological structure and gene expression in dairy cows.

PubMed

Ma, L; Zhao, M; Zhao, L S; Xu, J C; Loor, J J; Bu, D P

2017-05-01

This study was designed to investigate the effect of dietary neutral detergent fiber to starch ratio on rumen epithelial morphological structure and gene expression. Eight primiparous dairy cows including 4 ruminally fistulated cows were assigned to 4 total mixed rations with neutral detergent fiber to starch ratios of 0.86, 1.18, 1.63, and 2.34 in a replicated 4 × 4 Latin square design. The duration of each period was 21 d including 14 d for adaptation and 7 d for sampling. Rumen epithelial papillae were collected from the ruminally fistulated cows for morphological structure examination and mRNA expression analysis using quantitative real-time PCR of several genes related to volatile fatty acid absorption and metabolism, and cellular growth. Increasing dietary neutral detergent fiber to starch ratio resulted in a linear increase in the thickness of the stratum spinosum and basale. In contrast, expression of HMGCS2 (encoding the rate-limiting enzyme in the synthesis of ketone bodies) decreased linearly, whereas the expression of MCT2 (encoding a transporter of volatile fatty acid) increased linearly with increasing dietary neutral detergent fiber to starch ratio. As dietary neutral detergent fiber to starch ratio increased, expression of IGFBP5 (a gene related to the growth of rumen epithelial papillae) decreased, whereas IGFBP6 expression increased. Both of these IGFBP genes are regulated by short-chain fatty acids. Overall, the data indicate that dietary neutral detergent fiber to starch ratio can alter the thickness of the rumen epithelial papillae partly through changes in expression of genes associated with regulating volatile fatty acid absorption, metabolism, and cell growth. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The transcriptomic fingerprint of glucoamylase over-expression in Aspergillus niger

PubMed Central

2012-01-01

Background Filamentous fungi such as Aspergillus niger are well known for their exceptionally high capacity for secretion of proteins, organic acids, and secondary metabolites and they are therefore used in biotechnology as versatile microbial production platforms. However, system-wide insights into their metabolic and secretory capacities are sparse and rational strain improvement approaches are therefore limited. In order to gain a genome-wide view on the transcriptional regulation of the protein secretory pathway of A. niger, we investigated the transcriptome of A. niger when it was forced to overexpression the glaA gene (encoding glucoamylase, GlaA) and secrete GlaA to high level. Results An A. niger wild-type strain and a GlaA over-expressing strain, containing multiple copies of the glaA gene, were cultivated under maltose-limited chemostat conditions (specific growth rate 0.1 h-1). Elevated glaA mRNA and extracellular GlaA levels in the over-expressing strain were accompanied by elevated transcript levels from 772 genes and lowered transcript levels from 815 genes when compared to the wild-type strain. Using GO term enrichment analysis, four higher-order categories were identified in the up-regulated gene set: i) endoplasmic reticulum (ER) membrane translocation, ii) protein glycosylation, iii) vesicle transport, and iv) ion homeostasis. Among these, about 130 genes had predicted functions for the passage of proteins through the ER and those genes included target genes of the HacA transcription factor that mediates the unfolded protein response (UPR), e.g. bipA, clxA, prpA, tigA and pdiA. In order to identify those genes that are important for high-level secretion of proteins by A. niger, we compared the transcriptome of the GlaA overexpression strain of A. niger with six other relevant transcriptomes of A. niger. Overall, 40 genes were found to have either elevated (from 36 genes) or lowered (from 4 genes) transcript levels under all conditions that were examined, thus defining the core set of genes important for ensuring high protein traffic through the secretory pathway. Conclusion We have defined the A. niger genes that respond to elevated secretion of GlaA and, furthermore, we have defined a core set of genes that appear to be involved more generally in the intensified traffic of proteins through the secretory pathway of A. niger. The consistent up-regulation of a gene encoding the acetyl-coenzyme A transporter suggests a possible role for transient acetylation to ensure correct folding of secreted proteins. PMID:23237452

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.

Effects of H2 and Formate on Growth Yield and Regulation of Methanogenesis in Methanococcus maripaludis

PubMed Central

Costa, Kyle C.; Yoon, Sung Ho; Pan, Min; Burn, June A.; Baliga, Nitin S.

2013-01-01

Hydrogenotrophic methanogenic Archaea are defined by an H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. While certain responses of these organisms to hydrogen availability have been characterized, responses to formate starvation have not been reported. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess. Analysis of the growth yields of several mutants suggests that this phenomenon occurs independently of the storage of intracellular carbon or a transcriptional response to methanogenesis. Using microarray analysis, we found that the expression of genes encoding coenzyme F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess. PMID:23335420

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

Smith, Daniel P.; Nicora, Carrie D.; Carini, Paul

The alphaproteobacterium “CandidatusPelagibacter ubique” strain HTCC1062 and most other members of the SAR11 clade lack genes for assimilatory sulfate reduction, making them dependent on organosulfur compounds that occur naturally in seawater. To investigate how these cells adapt to sulfur limitation, batch cultures were grown in defined medium containing either limiting or nonlimiting amounts of dimethylsulfoniopropionate (DMSP) as the sole sulfur source. Protein and mRNA expression were measured before, during, and after the transition from exponential growth to stationary phase. Two distinct responses were observed, one as DMSP became exhausted and another as the cells acclimated to a sulfur-limited environment. Themore » first response was characterized by increased transcription and translation of all “Ca. Pelagibacter ubique” genes downstream from the previously confirmedS-adenosyl methionine (SAM) riboswitchesbhmT,mmuM, andmetY. The proteins encoded by these genes were up to 33 times more abundant as DMSP became limiting. Their predicted function is to shunt all available sulfur to methionine. The secondary response, observed during sulfur-limited stationary phase, was a 6- to 10-fold increase in the transcription of the hemecshuttle-encoding geneccmCand two small genes of unknown function (SAR11_1163andSAR11_1164). This bacterium’s strategy for coping with sulfur stress appears to be intracellular redistribution to support methionine biosynthesis rather than increasing organosulfur import. Many of the genes and SAM riboswitches involved in this response are located in a hypervariable genome region (HVR). One of these HVR genes,ordL, is located downstream from a conserved motif that evidence suggests is a novel riboswitch. IMPORTANCE“Ca. Pelagibacter ubique” is a key driver of marine biogeochemistry cycles and a model for understanding how minimal genomes evolved in free-living anucleate organisms. This study explores the unusual sulfur acquisition strategy that has evolved in these cells, which lack assimilatory sulfate reduction and instead rely on reduced sulfur compounds found in oxic marine environments to meet their cellular quotas. Our findings demonstrate that the sulfur acquisition systems are constitutively expressed but the enzymatic steps leading to the essential sulfur-containing amino acid methionine are regulated by a unique array of riboswitches and genes, many of which are encoded in a rapidly evolving genome region. These findings support mounting evidence that streamlined cells have evolved regulatory mechanisms that minimize transcriptional switching and, unexpectedly, localize essential sulfur acquisition genes in a genome region normally associated with adaption to environmental variation.« less

ATP Synthase Repression in Tobacco Restricts Photosynthetic Electron Transport, CO2 Assimilation, and Plant Growth by Overacidification of the Thylakoid Lumen[OA

PubMed Central

Rott, Markus; Martins, Nádia F.; Thiele, Wolfram; Lein, Wolfgang; Bock, Ralph; Kramer, David M.; Schöttler, Mark A.

2011-01-01

Tobacco (Nicotiana tabacum) plants strictly adjust the contents of both ATP synthase and cytochrome b6f complex to the metabolic demand for ATP and NADPH. While the cytochrome b6f complex catalyzes the rate-limiting step of photosynthetic electron flux and thereby controls assimilation, the functional significance of the ATP synthase adjustment is unknown. Here, we reduced ATP synthase accumulation by an antisense approach directed against the essential nuclear-encoded γ-subunit (AtpC) and by the introduction of point mutations into the translation initiation codon of the plastid-encoded atpB gene (encoding the essential β-subunit) via chloroplast transformation. Both strategies yielded transformants with ATP synthase contents ranging from 100 to <10% of wild-type levels. While the accumulation of the components of the linear electron transport chain was largely unaltered, linear electron flux was strongly inhibited due to decreased rates of plastoquinol reoxidation at the cytochrome b6f complex (photosynthetic control). Also, nonphotochemical quenching was triggered at very low light intensities, strongly reducing the quantum efficiency of CO2 fixation. We show evidence that this is due to an increased steady state proton motive force, resulting in strong lumen overacidification, which in turn represses photosynthesis due to photosynthetic control and dissipation of excitation energy in the antenna bed. PMID:21278125

Language-Dependent Pitch Encoding Advantage in the Brainstem Is Not Limited to Acceleration Rates that Occur in Natural Speech

ERIC Educational Resources Information Center

Krishnan, Ananthanarayan; Gandour, Jackson T.; Smalt, Christopher J.; Bidelman, Gavin M.

2010-01-01

Experience-dependent enhancement of neural encoding of pitch in the auditory brainstem has been observed for only specific portions of native pitch contours exhibiting high rates of pitch acceleration, irrespective of speech or nonspeech contexts. This experiment allows us to determine whether this language-dependent advantage transfers to…

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

PubMed

Yassin, Atteyet F; Langenberg, Stefan; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Mukherjee, Supratim; Reddy, T B K; Daum, Chris; Shapiro, Nicole; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos C

2017-01-01

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Rates of Spontaneous Mutation in Bacteriophage T4 Are Independent of Host Fidelity Determinants

PubMed Central

Santos, M. E.; Drake, J. W.

1994-01-01

Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity. PMID:7851754

Differential conservation of transcriptional domains of mammalian Prophet of Pit-1 proteins revealed by structural studies of the bovine gene and comparative functional analysis of the protein.

PubMed

Showalter, Aaron D; Smith, Timothy P L; Bennett, Gary L; Sloop, Kyle W; Whitsett, Julie A; Rhodes, Simon J

2002-05-29

The Prophet of Pit-1 (PROP1) gene encodes a paired class homeodomain transcription factor that is exclusively expressed in the developing mammalian pituitary gland. PROP1 function is essential for anterior pituitary organogenesis, and heritable mutations in the gene are associated with combined pituitary hormone deficiency in human patients and animals. By cloning the bovine PROP1 gene and by comparative analysis, we demonstrate that the homeodomains and carboxyl termini of mammalian PROP1 proteins are highly conserved while the amino termini are diverged. Whereas the carboxyl termini of the human and bovine PROP1 proteins contain potent transcriptional activation domains, the amino termini and homeodomains have repressive activities. The bovine PROP1 gene has four exons and three introns and maps to a region of chromosome seven carrying a quantitative trait locus affecting ovulation rate. Two alleles of the bovine gene were found that encode distinct protein products with different DNA binding and transcriptional activities. These experiments demonstrate that mammalian PROP1 genes encode proteins with complex regulatory capacities and that modest changes in protein sequence can significantly alter the activity of this pituitary developmental transcription factor.

Molecular and Metabolic Adaptations of Lactococcus lactis at Near-Zero Growth Rates

PubMed Central

Ercan, Onur; Wels, Michiel; Smid, Eddy J.

2014-01-01

This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY in L. lactis KF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end products) study provides molecular understanding of the adaptation of L. lactis to conditions supporting low growth rates and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates toward gene regulation patterns involved in zero-growth adaptation. PMID:25344239

Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach.

PubMed

Koutinas, Michalis; Kiparissides, Alexandros; Silva-Rocha, Rafael; Lam, Ming-Chi; Martins Dos Santos, Vitor A P; de Lorenzo, Victor; Pistikopoulos, Efstratios N; Mantalaris, Athanasios

2011-07-01

The majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses. Copyright © 2011 Elsevier Inc. All rights reserved.

Trehalose synthesis in Aspergillus niger: characterization of six homologous genes, all with conserved orthologs in related species

PubMed Central

2014-01-01

Background The disaccharide trehalose is a major component of fungal spores and is released upon germination. Moreover, the sugar is well known for is protective functions, e.g. against thermal stress and dehydration. The properties and synthesis of trehalose have been well investigated in the bakers’ yeast Saccharomyces cerevisiae. In filamentous fungi, such knowledge is limited, although several gene products have been identified. Results Using Aspergillus niger as a model fungus, the aim of this study was to provide an overview of all genes involved in trehalose synthesis. This fungus has three potential trehalose-6-phosphate synthase encoding genes, tpsA-C, and three putative trehalose phosphate phosphatase encoding genes, tppA-C, of which two have not previously been identified. Expression of all six genes was confirmed using real-time PCR, and conserved orthologs could be identified in related Aspergilli. Using a two-hybrid approach, there is a strong indication that four of the proteins physically interact, as has previously been shown in S. cerevisiae. When creating null mutants of all the six genes, three of them, ΔtpsA, ΔtppA and ΔtppB, had lower internal trehalose contents. The only mutant with a pronounced morphological difference was ΔtppA, in which sporulation was severely reduced with abnormal conidiophores. This was also the only mutant with accumulated levels of trehalose-6-phosphate, indicating that the encoded protein is the main phosphatase under normal conditions. Besides ΔtppA, the most studied deletion mutant in this work was ΔtppB. This gene encodes a protein conserved in filamentous Ascomycota. The ΔtppB mutant displayed a low, but not depleted, internal trehalose content, and conidia were more susceptible to thermal stress. Conclusion A. niger contains at least 6 genes putatively involved in trehalose synthesis. Gene expressions related to germination have been quantified and deletion mutants characterized: Mutants lacking tpsA, tppA or tppB have reduced internal trehalose contents. Furthermore, tppA, under normal conditions, encodes the functional trehalose-6-phosphate-phosphatase. PMID:24725382

A study of Staphylococcus aureusnasal carriage, antibacterial resistance and virulence factor encoding genes in a tertiary care hospital, Kayseri, Turkey.

PubMed

Oguzkaya-Artan, M; Artan, C; Baykan, Z; Sakalar, C; Turan, A; Aksu, H

2015-01-01

This study was to determine the virulence encoding genes, and the antibiotic resistance patterns of the Staphylococcus aureus isolates, which were isolated from the nasal samples of chest clinic patients. The nasal samples of the in-patients (431) and out-patients (1857) in Kayseri Training and Research Hospital's Chest Clinic, Kayseri, Turkey, were cultured on CHROMagar (Biolife, Italiana) S. aureus, and subcultured on sheep blood agar for the isolation of S. aureus. Disc diffusion method was used for antimicrobial susceptibility testing. The occurrence of the staphylococcal virulence encoding genes (enterotoksins [sea, seb, sec, see, seg, seh, sei, sej], fibronectin-binding proteins A, B [fnbA, fnbB], toxic shock syndrome toxin-1 [tst]) were detected by polymerase chain reaction. Forty-five of the 55 (81.8%) S. aureus isolates from inpatients, and 319 (90.6%) isolates from tested 352 out-patient's isolates were suspected to all the antibiotics tested. methicillin-resistant S. aureus (MRSA) was detected in 1.2% of S. aureus isolates. Rifampin, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, gentamicin resistance rates were 1.2%, 1.7%, 2.0%, 8.8%, and 1.2%, respectively. The isolates were susceptible to teicoplanin and vancomycin. The genes most frequently found were tst (92.7%), seg (85.8%), sea (83.6%), fnbA (70.9%). There was no statistical significance detected between MRSA and mecA-negative S. aureus isolates in encoding genes distribution (P > 0.05). Our results show that virulence factor encoding genes were prevalent in patients with S. aureus carriage, whereas antibiotic resistance was low. These virulence determinants may increase the risk for subsequent invasive infections in carriers.

A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons.

PubMed

Aschrafi, Armaz; Kar, Amar N; Gale, Jenna R; Elkahloun, Abdel G; Vargas, Jose Noberto S; Sales, Naomi; Wilson, Gabriel; Tompkins, Miranda; Gioio, Anthony E; Kaplan, Barry B

2016-09-01

Mitochondria are enriched in subcellular regions of high energy consumption, such as axons and pre-synaptic nerve endings. Accumulating evidence suggests that mitochondrial maintenance in these distal structural/functional domains of the neuron depends on the "in-situ" translation of nuclear-encoded mitochondrial mRNAs. In support of this notion, we recently provided evidence for the axonal targeting of several nuclear-encoded mRNAs, such as cytochrome c oxidase, subunit 4 (COXIV) and ATP synthase, H+ transporting and mitochondrial Fo complex, subunit C1 (ATP5G1). Furthermore, we showed that axonal trafficking and local translation of these mRNAs plays a critical role in the generation of axonal ATP. Using a global gene expression analysis, this study identified a highly diverse population of nuclear-encoded mRNAs that were enriched in the axon and presynaptic nerve terminals. Among this population of mRNAs, fifty seven were found to be at least two-fold more abundant in distal axons, as compared with the parental cell bodies. Gene ontology analysis of the nuclear-encoded mitochondrial mRNAs suggested functions for these gene products in molecular and biological processes, including but not limited to oxidoreductase and electron carrier activity and proton transport. Based on these results, we postulate that local translation of nuclear-encoded mitochondrial mRNAs present in the axons may play an essential role in local energy production and maintenance of mitochondrial function. Published by Elsevier B.V.

The sh2-R allele of the maize shrunken-2 locus was caused by a complex chromosomal rearrangement.

PubMed

Kramer, Vance; Shaw, Janine R; Senior, M Lynn; Hannah, L Curtis

2015-03-01

The mutant that originally defined the shrunken - 2 locus of maize is shown here to be the product of a complex chromosomal rearrangement. The maize shrunken-2 gene (sh2) encodes the large subunit of the heterotetrameric enzyme, adenosine diphosphate glucose pyrophosphorylases and a rate-limiting enzyme in starch biosynthesis. The sh2 gene was defined approximately 72 years ago by the isolation of a loss-of-function allele conditioning a shrunken, but viable seed. In subsequent years, the realization that this allele, termed zsh2-R or sh2-Reference, causes an extremely high level of sucrose to accumulate in the developing seed led to a revolution in the sweet corn industry. Now, the vast majority of sweet corns grown throughout the world contain this mutant allele. Through initial Southern analysis followed by genomic sequencing, the work reported here shows that this allele arose through a complex set of events involving at least three breaks of chromosome 3 as well as an intra-chromosomal inversion. These findings provide an explanation for some previously reported, unexpected observations concerning rates of recombination within and between genes in this region.

Probing the Boundaries of Orthology: The Unanticipated Rapid Evolution of Drosophila centrosomin

PubMed Central

Eisman, Robert C.; Kaufman, Thomas C.

2013-01-01

The rapid evolution of essential developmental genes and their protein products is both intriguing and problematic. The rapid evolution of gene products with simple protein folds and a lack of well-characterized functional domains typically result in a low discovery rate of orthologous genes. Additionally, in the absence of orthologs it is difficult to study the processes and mechanisms underlying rapid evolution. In this study, we have investigated the rapid evolution of centrosomin (cnn), an essential gene encoding centrosomal protein isoforms required during syncytial development in Drosophila melanogaster. Until recently the rapid divergence of cnn made identification of orthologs difficult and questionable because Cnn violates many of the assumptions underlying models for protein evolution. To overcome these limitations, we have identified a group of insect orthologs and present conserved features likely to be required for the functions attributed to cnn in D. melanogaster. We also show that the rapid divergence of Cnn isoforms is apparently due to frequent coding sequence indels and an accelerated rate of intronic additions and eliminations. These changes appear to be buffered by multi-exon and multi-reading frame maximum potential ORFs, simple protein folds, and the splicing machinery. These buffering features also occur in other genes in Drosophila and may help prevent potentially deleterious mutations due to indels in genes with large coding exons and exon-dense regions separated by small introns. This work promises to be useful for future investigations of cnn and potentially other rapidly evolving genes and proteins. PMID:23749319

Gene Expression Changes in Phosphorus Deficient Potato (Solanum tuberosum L.) Leaves and the Potential for Diagnostic Gene Expression Markers

PubMed Central

Hammond, John P.; Broadley, Martin R.; Bowen, Helen C.; Spracklen, William P.; Hayden, Rory M.; White, Philip J.

2011-01-01

Background There are compelling economic and environmental reasons to reduce our reliance on inorganic phosphate (Pi) fertilisers. Better management of Pi fertiliser applications is one option to improve the efficiency of Pi fertiliser use, whilst maintaining crop yields. Application rates of Pi fertilisers are traditionally determined from analyses of soil or plant tissues. Alternatively, diagnostic genes with altered expression under Pi limiting conditions that suggest a physiological requirement for Pi fertilisation, could be used to manage Pifertiliser applications, and might be more precise than indirect measurements of soil or tissue samples. Results We grew potato (Solanum tuberosum L.) plants hydroponically, under glasshouse conditions, to control their nutrient status accurately. Samples of total leaf RNA taken periodically after Pi was removed from the nutrient solution were labelled and hybridised to potato oligonucleotide arrays. A total of 1,659 genes were significantly differentially expressed following Pi withdrawal. These included genes that encode proteins involved in lipid, protein, and carbohydrate metabolism, characteristic of Pi deficient leaves and included potential novel roles for genes encoding patatin like proteins in potatoes. The array data were analysed using a support vector machine algorithm to identify groups of genes that could predict the Pi status of the crop. These groups of diagnostic genes were tested using field grown potatoes that had either been fertilised or unfertilised. A group of 200 genes could correctly predict the Pi status of field grown potatoes. Conclusions This paper provides a proof-of-concept demonstration for using microarrays and class prediction tools to predict the Pi status of a field grown potato crop. There is potential to develop this technology for other biotic and abiotic stresses in field grown crops. Ultimately, a better understanding of crop stresses may improve our management of the crop, improving the sustainability of agriculture. PMID:21935429

Virulence and extended-spectrum β-lactamase encoding genes in Escherichia coli recovered from chicken meat intended for hospitalized human consumption.

PubMed

Younis, Gamal A; Elkenany, Rasha M; Fouda, Mohamed A; Mostafa, Noura F

2017-10-01

This study describes the prevalence of Escherichia coli in frozen chicken meat intended for human consumption with emphasis on their virulence determinants through detection of the virulence genes and recognition of the extended-spectrum β-lactamase (ESBL) encoding genes ( bla OXA and bla TEM genes). A total of 120 frozen chicken meat samples were investigated for isolation of E. coli . All isolates were subjected to biochemical and serological tests. Eight serotypes isolated from samples were analyzed for the presence of various virulence genes ( stx1, stx2 , and eae A genes) using multiplex polymerase chain reaction (PCR) technique. Moreover, the strains were evaluated for the ESBL encoding genes ( bla TEM and bla OXA ). Overall, 11.66% (14/120) chicken meat samples carried E. coli according to cultural and biochemical properties. The most predominant serotypes were O78 and O128: H2 (21.5%, each), followed by O121: H7 and O44: H18. Molecular method detected that 2 strains (25%) harbored stx1 , 3 strains (37.5%) stx2 , and 3 strains (37.5%) both stx1 and stx2 , while 1 (12.5%) strain carried eae A gene. Particularly, only O26 serotype had all tested virulence genes ( stx1, stx2, and eae A ). The results revealed that all examined 8 serotypes were Shiga toxin-producing E. coli (STEC). The ESBL encoding genes ( bla TEM and bla OXA ) of STEC were detected in 4 (50%) isolates by multiplex PCR. The overall incidence of bla TEM and bla OXA genes was 3 (37.5%) and 2 (25%) isolates. The present study indicates the prevalence of virulent and ESBL-producing E. coli in frozen chicken meat intended for hospitalized human consumption due to poor hygienic measures and irregular use of antibiotics. Therefore, the basic instructions regarding good hygienic measures should be adapted to limit public health hazard.

Dietary adaptation of FADS genes in Europe varied across time and geography.

PubMed

Ye, Kaixiong; Gao, Feng; Wang, David; Bar-Yosef, Ofer; Keinan, Alon

2017-05-26

Fatty acid desaturase (FADS) genes encode rate-limiting enzymes for the biosynthesis of omega-6 and omega-3 long-chain polyunsaturated fatty acids (LCPUFAs). This biosynthesis is essential for individuals subsisting on LCPUFA-poor diets (for example, plant-based). Positive selection on FADS genes has been reported in multiple populations, but its cause and pattern in Europeans remain unknown. Here we demonstrate, using ancient and modern DNA, that positive selection acted on the same FADS variants both before and after the advent of farming in Europe, but on opposite (that is, alternative) alleles. Recent selection in farmers also varied geographically, with the strongest signal in southern Europe. These varying selection patterns concur with anthropological evidence of varying diets, and with the association of farming-adaptive alleles with higher FADS1 expression and thus enhanced LCPUFA biosynthesis. Genome-wide association studies reveal that farming-adaptive alleles not only increase LCPUFAs, but also affect other lipid levels and protect against several inflammatory diseases.

Nucleotide sequences of two genomic DNAs encoding peroxidase of Arabidopsis thaliana.

PubMed

Intapruk, C; Higashimura, N; Yamamoto, K; Okada, N; Shinmyo, A; Takano, M

1991-02-15

The peroxidase (EC 1.11.1.7)-encoding gene of Arabidopsis thaliana was screened from a genomic library using a cDNA encoding a neutral isozyme of horseradish, Armoracia rusticana, peroxidase (HRP) as a probe, and two positive clones were isolated. From the comparison with the sequences of the HRP-encoding genes, we concluded that two clones contained peroxidase-encoding genes, and they were named prxCa and prxEa. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, GT and AG, at the 5' and 3' ends, respectively. The lengths of each putative exon of the prxEa gene were the same as those of the HRP-basic-isozyme-encoding gene, prxC3, and coded for 349 amino acids (aa) with a sequence homology of 89% to that encoded by prxC3. The prxCa gene was very close to the HRP-neutral-isozyme-encoding gene, prxC1b, and coded for 354 aa with 91% homology to that encoded by prxC1b. The aa sequence homology was 64% between the two peroxidases encoded by prxCa and prxEa.

Differential regulation of mnp2, a new manganese peroxidase-encoding gene from the ligninolytic fungus Trametes versicolor PRL 572.

PubMed

Johansson, Tomas; Nyman, Per Olof; Cullen, Daniel

2002-04-01

A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO(4) to cultures increased mnp2 transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of T. versicolor, mnp1, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in T. versicolor are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the mnp1 and mnp2 polypeptides to have internal homology.

Differential Regulation of mnp2, a New Manganese Peroxidase-Encoding Gene from the Ligninolytic Fungus Trametes versicolor PRL 572

PubMed Central

Johansson, Tomas; Nyman, Per Olof; Cullen, Daniel

2002-01-01

A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO4 to cultures increased mnp2 transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of T. versicolor, mnp1, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in T. versicolor are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the mnp1 and mnp2 polypeptides to have internal homology. PMID:11916737

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

PubMed

Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

2014-03-18

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

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/.

Strong positive selection and recombination drive the antigenic variation of the PilE protein of the human pathogen Neisseria meningitidis.

PubMed

Andrews, T Daniel; Gojobori, Takashi

2004-01-01

The PilE protein is the major component of the Neisseria meningitidis pilus, which is encoded by the pilE/pilS locus that includes an expressed gene and eight homologous silent fragments. The silent gene fragments have been shown to recombine through gene conversion with the expressed gene and thereby provide a means by which novel antigenic variants of the PilE protein can be generated. We have analyzed the evolutionary rate of the pilE gene using the nucleotide sequence of two complete pilE/pilS loci. The very high rate of evolution displayed by the PilE protein appears driven by both recombination and positive selection. Within the semivariable region of the pilE and pilS genes, recombination appears to occur within multiple small sequence blocks that lie between conserved sequence elements. Within the hypervariable region, positive selection was identified from comparison of the silent and expressed genes. The unusual gene conversion mechanism that operates at the pilE/pilS locus is a strategy employed by N. meningitidis to enhance mutation of certain regions of the PilE protein. The silent copies of the gene effectively allow "parallelized" evolution of pilE, thus enabling the encoded protein to rapidly explore a large area of sequence space in an effort to find novel antigenic variants.

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

PubMed

de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Vilchez-Vargas, Ramiro; Pieper, Dietmar H

2016-01-01

Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Phylogenomics databases for facilitating functional genomics in rice.

PubMed

Jung, Ki-Hong; Cao, Peijian; Sharma, Rita; Jain, Rashmi; Ronald, Pamela C

2015-12-01

The completion of whole genome sequence of rice (Oryza sativa) has significantly accelerated functional genomics studies. Prior to the release of the sequence, only a few genes were assigned a function each year. Since sequencing was completed in 2005, the rate has exponentially increased. As of 2014, 1,021 genes have been described and added to the collection at The Overview of functionally characterized Genes in Rice online database (OGRO). Despite this progress, that number is still very low compared with the total number of genes estimated in the rice genome. One limitation to progress is the presence of functional redundancy among members of the same rice gene family, which covers 51.6 % of all non-transposable element-encoding genes. There remain a significant portion or rice genes that are not functionally redundant, as reflected in the recovery of loss-of-function mutants. To more accurately analyze functional redundancy in the rice genome, we have developed a phylogenomics databases for six large gene families in rice, including those for glycosyltransferases, glycoside hydrolases, kinases, transcription factors, transporters, and cytochrome P450 monooxygenases. In this review, we introduce key features and applications of these databases. We expect that they will serve as a very useful guide in the post-genomics era of research.

A system-level model for the microbial regulatory genome.

PubMed

Brooks, Aaron N; Reiss, David J; Allard, Antoine; Wu, Wei-Ju; Salvanha, Diego M; Plaisier, Christopher L; Chandrasekaran, Sriram; Pan, Min; Kaur, Amardeep; Baliga, Nitin S

2014-07-15

Microbes can tailor transcriptional responses to diverse environmental challenges despite having streamlined genomes and a limited number of regulators. Here, we present data-driven models that capture the dynamic interplay of the environment and genome-encoded regulatory programs of two types of prokaryotes: Escherichia coli (a bacterium) and Halobacterium salinarum (an archaeon). The models reveal how the genome-wide distributions of cis-acting gene regulatory elements and the conditional influences of transcription factors at each of those elements encode programs for eliciting a wide array of environment-specific responses. We demonstrate how these programs partition transcriptional regulation of genes within regulons and operons to re-organize gene-gene functional associations in each environment. The models capture fitness-relevant co-regulation by different transcriptional control mechanisms acting across the entire genome, to define a generalized, system-level organizing principle for prokaryotic gene regulatory networks that goes well beyond existing paradigms of gene regulation. An online resource (http://egrin2.systemsbiology.net) has been developed to facilitate multiscale exploration of conditional gene regulation in the two prokaryotes. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme.

PubMed

Anderson, Olin D; Coleman-Derr, Devin; Gu, Yong Q; Heath, Sekou

2010-06-16

Among the dietary essential amino acids, the most severely limiting in the cereals is lysine. Since cereals make up half of the human diet, lysine limitation has quality/nutritional consequences. The breakdown of lysine is controlled mainly by the catabolic bifunctional enzyme lysine ketoglutarate reductase - saccharopine dehydrogenase (LKR/SDH). The LKR/SDH gene has been reported to produce transcripts for the bifunctional enzyme and separate monofunctional transcripts. In addition to lysine metabolism, this gene has been implicated in a number of metabolic and developmental pathways, which along with its production of multiple transcript types and complex exon/intron structure suggest an important node in plant metabolism. Understanding more about the LKR/SDH gene is thus interesting both from applied standpoint and for basic plant metabolism. The current report describes a wheat genomic fragment containing an LKR/SDH gene and adjacent genes. The wheat LKR/SDH genomic segment was found to originate from the A-genome of wheat, and EST analysis indicates all three LKR/SDH genes in hexaploid wheat are transcriptionally active. A comparison of a set of plant LKR/SDH genes suggests regions of greater sequence conservation likely related to critical enzymatic functions and metabolic controls. Although most plants contain only a single LKR/SDH gene per genome, poplar contains at least two functional bifunctional genes in addition to a monofunctional LKR gene. Analysis of ESTs finds evidence for monofunctional LKR transcripts in switchgrass, and monofunctional SDH transcripts in wheat, Brachypodium, and poplar. The analysis of a wheat LKR/SDH gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes show lineage-specific differences between monocots and dicots, and findings suggest variation in activity of LKR/SDH genes among plants. Although most plant genomes seem to contain a single conserved LKR/SDH gene per genome, poplar possesses multiple contiguous genes. A preponderance of SDH transcripts suggests the LKR region may be more rate-limiting. Only switchgrass has EST evidence for LKR monofunctional transcripts. Evidence for monofunctional SDH transcripts shows a novel intron in wheat, Brachypodium, and poplar.

Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase.

PubMed

Pateau, Victoire; Razafimandimby, Bienvenue; Vandeputte, Patrick; Thornton, Christopher R; Guillemette, Thomas; Bouchara, Jean-Philippe; Giraud, Sandrine

2018-02-01

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species.

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.

Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.

PubMed

Lam, L T; Pickeral, O K; Peng, A C; Rosenwald, A; Hurt, E M; Giltnane, J M; Averett, L M; Zhao, H; Davis, R E; Sathyamoorthy, M; Wahl, L M; Harris, E D; Mikovits, J A; Monks, A P; Hollingshead, M G; Sausville, E A; Staudt, L M

2001-01-01

Flavopiridol, a flavonoid currently in cancer clinical trials, inhibits cyclin-dependent kinases (CDKs) by competitively blocking their ATP-binding pocket. However, the mechanism of action of flavopiridol as an anti-cancer agent has not been fully elucidated. Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly, in contrast to two other CDK inhibitors, roscovitine and 9-nitropaullone. The gene expression profile of flavopiridol closely resembled the profiles of two transcription inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), suggesting that flavopiridol inhibits transcription globally. We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates. In particular, genes encoding apoptosis regulators frequently had very short half-lives, as did several genes encoding key cell-cycle regulators. Strikingly, genes that were transcriptionally inducible were disproportionately represented in the class of genes with rapid mRNA turnover. The present genomic-scale measurement of mRNA turnover uncovered a regulatory logic that links gene function with mRNA half-life. The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes. In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets.

PubMed

Brooks, Lauren E; Ul-Hasan, Sabah; Chan, Benjamin K; Sistrom, Mark J

2018-01-01

Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection-representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments-specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date. Our study evaluates an often-underappreciated aspect of resistance-the impact of evolutionary selection. We evaluate selection on all annotated efflux genes in all sequenced ESKAPE pathogens, providing critical context for and insight into current and future development of efflux-targeting treatments for resistant bacterial infections.

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets

PubMed Central

Ul-Hasan, Sabah; Chan, Benjamin K.; Sistrom, Mark J.

2018-01-01

ABSTRACT Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection—representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments—specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date. Our study evaluates an often-underappreciated aspect of resistance—the impact of evolutionary selection. We evaluate selection on all annotated efflux genes in all sequenced ESKAPE pathogens, providing critical context for and insight into current and future development of efflux-targeting treatments for resistant bacterial infections. PMID:29719870

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production.

PubMed

Jo, Suah; Yoon, Jinkyung; Lee, Sun-Mi; Um, Youngsoon; Han, Sung Ok; Woo, Han Min

2017-09-20

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization. Copyright © 2017 Elsevier B.V. All rights reserved.

The Thoc1 Encoded Ribonucleoprotein Is Required for Myeloid Progenitor Cell Homeostasis in the Adult Mouse

PubMed Central

Chinnam, Meenalakshmi; Povinelli, Benjamin J.; Fisher, Daniel T.; Golding, Michelle; Appenheimer, Michelle M.; Nemeth, Michael J.; Evans, Sharon; Goodrich, David W.

2014-01-01

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover. PMID:24830368

The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

PubMed

Pitzonka, Laura; Ullas, Sumana; Chinnam, Meenalakshmi; Povinelli, Benjamin J; Fisher, Daniel T; Golding, Michelle; Appenheimer, Michelle M; Nemeth, Michael J; Evans, Sharon; Goodrich, David W

2014-01-01

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

The FBPase Encoding Gene glpX Is Required for Gluconeogenesis, Bacterial Proliferation and Division In Vivo of Mycobacterium marinum

PubMed Central

Lyu, Liangdong; Wang, Chuan; Li, Yang; Gao, Qian; Yang, Chen

2016-01-01

Lipids have been identified as important carbon sources for Mycobacterium tuberculosis (Mtb) to utilize in vivo. Thus gluconeogenesis bears a key role for Mtb to survive and replicate in host. A rate-limiting enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (FBPase) is encoded by the gene glpX. The functions of glpX were studied in M. marinum, a closely related species to Mtb. The glpX deletion strain (ΔglpX) displayed altered gluconeogenesis, attenuated virulence, and altered bacterial proliferation. Metabolic profiles indicate an accumulation of the FBPase substrate, fructose 1, 6-bisphosphate (FBP) and altered gluconeogenic flux when ΔglpX is cultivated in a gluconeogenic carbon substrate, acetate. In both macrophages and zebrafish, the proliferation of ΔglpX was halted, resulting in dramatically attenuated virulence. Intracellular ΔglpX exhibited an elongated morphology, which was also observed when ΔglpX was grown in a gluconeogenic carbon source. This elongated morphology is also supported by the observation of unseparated multi-nucleoid cell, indicating that a complete mycobacterial division in vivo is correlated with intact gluconeogenesis. Together, our results indicate that glpX has essential functions in gluconeogenesis, and plays an indispensable role in bacterial proliferation in vivo and virulence of M. marinum. PMID:27233038

The FBPase Encoding Gene glpX Is Required for Gluconeogenesis, Bacterial Proliferation and Division In Vivo of Mycobacterium marinum.

PubMed

Tong, Jingfeng; Meng, Lu; Wang, Xinwei; Liu, Lixia; Lyu, Liangdong; Wang, Chuan; Li, Yang; Gao, Qian; Yang, Chen; Niu, Chen

2016-01-01

Lipids have been identified as important carbon sources for Mycobacterium tuberculosis (Mtb) to utilize in vivo. Thus gluconeogenesis bears a key role for Mtb to survive and replicate in host. A rate-limiting enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (FBPase) is encoded by the gene glpX. The functions of glpX were studied in M. marinum, a closely related species to Mtb. The glpX deletion strain (ΔglpX) displayed altered gluconeogenesis, attenuated virulence, and altered bacterial proliferation. Metabolic profiles indicate an accumulation of the FBPase substrate, fructose 1, 6-bisphosphate (FBP) and altered gluconeogenic flux when ΔglpX is cultivated in a gluconeogenic carbon substrate, acetate. In both macrophages and zebrafish, the proliferation of ΔglpX was halted, resulting in dramatically attenuated virulence. Intracellular ΔglpX exhibited an elongated morphology, which was also observed when ΔglpX was grown in a gluconeogenic carbon source. This elongated morphology is also supported by the observation of unseparated multi-nucleoid cell, indicating that a complete mycobacterial division in vivo is correlated with intact gluconeogenesis. Together, our results indicate that glpX has essential functions in gluconeogenesis, and plays an indispensable role in bacterial proliferation in vivo and virulence of M. marinum.

Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis.

PubMed Central

Li, S J; Cronan, J E

1993-01-01

Acetyl coenzyme A (CoA) carboxylase catalyzes the synthesis of malonyl-CoA, the first intermediate of fatty acid synthesis. The Escherichia coli enzyme is encoded by four subunits located at three different positions on the E. coli chromosome. The accBC genes lie in a small operon at min 72, whereas accA and accD are located at min 4.3 and 50, respectively. We examined the expression of the genes that encode the E. coli acetyl-CoA carboxylase subunits (accA, accBC, and accD) under a variety of growth conditions by quantitative Northern (RNA) blot analysis. We found a direct correlation between the levels of transcription of the acc genes and the rate of cellular growth. Consistent results were also obtained upon nutritional upshift and downshift experiments and upon dilution of stationary-phase cultures into fresh media. We also determined the 5' end of the accA and accD mRNAs by primer extension and did transcriptional fusion analysis of the previously reported accBC promoter. Several interesting features were found in the promoter regions of these genes, including a bent DNA sequence and an open reading frame within the unusually long leader mRNA of the accBC operon, potential stem-loop structures in the accA and accD mRNA leader regions, and a stretch of GC-rich sequences followed by AT-rich sequences common to all three promoters. In addition, both accA and accD are located in complex gene clusters. For example, the accA promoter was localized within the upstream polC gene (which encodes the DNA polymerase III catalytic subunit), suggesting that additional regulatory mechanisms exist. Images PMID:7678242

Complete genome sequencing and analysis of a Lancefield group G Streptococcus dysgalactiae subsp. equisimilis strain causing streptococcal toxic shock syndrome (STSS)

PubMed Central

2011-01-01

Background Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes invasive streptococcal infections, including streptococcal toxic shock syndrome (STSS), as does Lancefield group A Streptococcus pyogenes (GAS). We sequenced the entire genome of SDSE strain GGS_124 isolated from a patient with STSS. Results We found that GGS_124 consisted of a circular genome of 2,106,340 bp. Comparative analyses among bacterial genomes indicated that GGS_124 was most closely related to GAS. GGS_124 and GAS, but not other streptococci, shared a number of virulence factor genes, including genes encoding streptolysin O, NADase, and streptokinase A, distantly related to SIC (DRS), suggesting the importance of these factors in the development of invasive disease. GGS_124 contained 3 prophages, with one containing a virulence factor gene for streptodornase. All 3 prophages were significantly similar to GAS prophages that carry virulence factor genes, indicating that these prophages had transferred these genes between pathogens. SDSE was found to contain a gene encoding a superantigen, streptococcal exotoxin type G, but lacked several genes present in GAS that encode virulence factors, such as other superantigens, cysteine protease speB, and hyaluronan synthase operon hasABC. Similar to GGS_124, the SDSE strains contained larger numbers of clustered, regularly interspaced, short palindromic repeats (CRISPR) spacers than did GAS, suggesting that horizontal gene transfer via streptococcal phages between SDSE and GAS is somewhat restricted, although they share phage species. Conclusion Genome wide comparisons of SDSE with GAS indicate that SDSE is closely and quantitatively related to GAS. SDSE, however, lacks several virulence factors of GAS, including superantigens, SPE-B and the hasABC operon. CRISPR spacers may limit the horizontal transfer of phage encoded GAS virulence genes into SDSE. These findings may provide clues for dissecting the pathological roles of the virulence factors in SDSE and GAS that cause STSS. PMID:21223537

Complete genome sequencing and analysis of a Lancefield group G Streptococcus dysgalactiae subsp. equisimilis strain causing streptococcal toxic shock syndrome (STSS).

PubMed

Shimomura, Yumi; Okumura, Kayo; Murayama, Somay Yamagata; Yagi, Junji; Ubukata, Kimiko; Kirikae, Teruo; Miyoshi-Akiyama, Tohru

2011-01-11

Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes invasive streptococcal infections, including streptococcal toxic shock syndrome (STSS), as does Lancefield group A Streptococcus pyogenes (GAS). We sequenced the entire genome of SDSE strain GGS_124 isolated from a patient with STSS. We found that GGS_124 consisted of a circular genome of 2,106,340 bp. Comparative analyses among bacterial genomes indicated that GGS_124 was most closely related to GAS. GGS_124 and GAS, but not other streptococci, shared a number of virulence factor genes, including genes encoding streptolysin O, NADase, and streptokinase A, distantly related to SIC (DRS), suggesting the importance of these factors in the development of invasive disease. GGS_124 contained 3 prophages, with one containing a virulence factor gene for streptodornase. All 3 prophages were significantly similar to GAS prophages that carry virulence factor genes, indicating that these prophages had transferred these genes between pathogens. SDSE was found to contain a gene encoding a superantigen, streptococcal exotoxin type G, but lacked several genes present in GAS that encode virulence factors, such as other superantigens, cysteine protease speB, and hyaluronan synthase operon hasABC. Similar to GGS_124, the SDSE strains contained larger numbers of clustered, regularly interspaced, short palindromic repeats (CRISPR) spacers than did GAS, suggesting that horizontal gene transfer via streptococcal phages between SDSE and GAS is somewhat restricted, although they share phage species. Genome wide comparisons of SDSE with GAS indicate that SDSE is closely and quantitatively related to GAS. SDSE, however, lacks several virulence factors of GAS, including superantigens, SPE-B and the hasABC operon. CRISPR spacers may limit the horizontal transfer of phage encoded GAS virulence genes into SDSE. These findings may provide clues for dissecting the pathological roles of the virulence factors in SDSE and GAS that cause STSS.

Genetic programs can be compressed and autonomously decompressed in live cells

NASA Astrophysics Data System (ADS)

Lapique, Nicolas; Benenson, Yaakov

2018-04-01

Fundamental computer science concepts have inspired novel information-processing molecular systems in test tubes1-13 and genetically encoded circuits in live cells14-21. Recent research has shown that digital information storage in DNA, implemented using deep sequencing and conventional software, can approach the maximum Shannon information capacity22 of two bits per nucleotide23. In nature, DNA is used to store genetic programs, but the information content of the encoding rarely approaches this maximum24. We hypothesize that the biological function of a genetic program can be preserved while reducing the length of its DNA encoding and increasing the information content per nucleotide. Here we support this hypothesis by describing an experimental procedure for compressing a genetic program and its subsequent autonomous decompression and execution in human cells. As a test-bed we choose an RNAi cell classifier circuit25 that comprises redundant DNA sequences and is therefore amenable for compression, as are many other complex gene circuits15,18,26-28. In one example, we implement a compressed encoding of a ten-gene four-input AND gate circuit using only four genetic constructs. The compression principles applied to gene circuits can enable fitting complex genetic programs into DNA delivery vehicles with limited cargo capacity, and storing compressed and biologically inert programs in vivo for on-demand activation.

Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon “Candidatus Nitrosotalea devanaterra”

PubMed Central

Sayavedra-Soto, Luis A.; Gallois, Nicolas; Schouten, Stefan; Stein, Lisa Y.; Prosser, James I.; Nicol, Graeme W.

2016-01-01

Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer “Candidatus Nitrosotalea devanaterra” provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of “Ca. Nitrosotalea devanaterra” reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that “Ca. Nitrosotalea devanaterra” contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography–mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of “Ca. Nitrosotalea devanaterra” were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization. PMID:26896134

Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon "Candidatus Nitrosotalea devanaterra".

PubMed

Lehtovirta-Morley, Laura E; Sayavedra-Soto, Luis A; Gallois, Nicolas; Schouten, Stefan; Stein, Lisa Y; Prosser, James I; Nicol, Graeme W

2016-05-01

Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer "Candidatus Nitrosotalea devanaterra" provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of "Ca Nitrosotalea devanaterra" reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that "Ca Nitrosotalea devanaterra" contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of "Ca Nitrosotalea devanaterra" were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization. Copyright © 2016 Lehtovirta-Morley et al.

A Novel Plasmid-Based Microarray Screen Identifies Suppressors of rrp6Δ in Saccharomyces cerevisiae▿†

PubMed Central

Abruzzi, Katharine; Denome, Sylvia; Olsen, Jens Raabjerg; Assenholt, Jannie; Haaning, Line Lindegaard; Jensen, Torben Heick; Rosbash, Michael

2007-01-01

Genetic screens in Saccharomyces cerevisiae provide novel information about interacting genes and pathways. We screened for high-copy-number suppressors of a strain with the gene encoding the nuclear exosome component Rrp6p deleted, with either a traditional plate screen for suppressors of rrp6Δ temperature sensitivity or a novel microarray enhancer/suppressor screening (MES) strategy. MES combines DNA microarray technology with high-copy-number plasmid expression in liquid media. The plate screen and MES identified overlapping, but also different, suppressor genes. Only MES identified the novel mRNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6Δ strains at 37°C. Nab6p binds poly(A)+ RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis are growth rate limiting in rrp6Δ strains. Microarray analyses of gene expression in rrp6Δ strains and a number of suppressor strains support this hypothesis. PMID:17101774

Induction of 9-cis-epoxycarotenoid dioxygenase in Arabidopsis thaliana seeds enhances seed dormancy

PubMed Central

Martínez-Andújar, Cristina; Ordiz, M. Isabel; Huang, Zhonglian; Nonogaki, Mariko; Beachy, Roger N.; Nonogaki, Hiroyuki

2011-01-01

Full understanding of mechanisms that control seed dormancy and germination remains elusive. Whereas it has been proposed that translational control plays a predominant role in germination, other studies suggest the importance of specific gene expression patterns in imbibed seeds. Transgenic plants were developed to permit conditional expression of a gene encoding 9-cis-epoxycarotenoid dioxygenase 6 (NCED6), a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, using the ecdysone receptor-based plant gene switch system and the ligand methoxyfenozide. Induction of NCED6 during imbibition increased ABA levels more than 20-fold and was sufficient to prevent seed germination. Germination suppression was prevented by fluridone, an inhibitor of ABA biosynthesis. In another study, induction of the NCED6 gene in transgenic seeds of nondormant mutants tt3 and tt4 reestablished seed dormancy. Furthermore, inducing expression of NCED6 during seed development suppressed vivipary, precocious germination of developing seeds. These results indicate that expression of a hormone metabolism gene in seeds can be a sole determinant of dormancy. This study opens the possibility of developing a robust technology to suppress or promote seed germination through engineering pathways of hormone metabolism. PMID:21969557

Induction of 9-cis-epoxycarotenoid dioxygenase in Arabidopsis thaliana seeds enhances seed dormancy.

PubMed

Martínez-Andújar, Cristina; Ordiz, M Isabel; Huang, Zhonglian; Nonogaki, Mariko; Beachy, Roger N; Nonogaki, Hiroyuki

2011-10-11

Full understanding of mechanisms that control seed dormancy and germination remains elusive. Whereas it has been proposed that translational control plays a predominant role in germination, other studies suggest the importance of specific gene expression patterns in imbibed seeds. Transgenic plants were developed to permit conditional expression of a gene encoding 9-cis-epoxycarotenoid dioxygenase 6 (NCED6), a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, using the ecdysone receptor-based plant gene switch system and the ligand methoxyfenozide. Induction of NCED6 during imbibition increased ABA levels more than 20-fold and was sufficient to prevent seed germination. Germination suppression was prevented by fluridone, an inhibitor of ABA biosynthesis. In another study, induction of the NCED6 gene in transgenic seeds of nondormant mutants tt3 and tt4 reestablished seed dormancy. Furthermore, inducing expression of NCED6 during seed development suppressed vivipary, precocious germination of developing seeds. These results indicate that expression of a hormone metabolism gene in seeds can be a sole determinant of dormancy. This study opens the possibility of developing a robust technology to suppress or promote seed germination through engineering pathways of hormone metabolism.

I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

PubMed

Fenina, Myriam; Simon-Chazottes, Dominique; Vandormael-Pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-Tannoudji, Michel; Bernard, Bruno A; Panthier, Jean-Jacques

2012-01-01

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

funRNA: a fungi-centered genomics platform for genes encoding key components of RNAi.

PubMed

Choi, Jaeyoung; Kim, Ki-Tae; Jeon, Jongbum; Wu, Jiayao; Song, Hyeunjeong; Asiegbu, Fred O; Lee, Yong-Hwan

2014-01-01

RNA interference (RNAi) is involved in genome defense as well as diverse cellular, developmental, and physiological processes. Key components of RNAi are Argonaute, Dicer, and RNA-dependent RNA polymerase (RdRP), which have been functionally characterized mainly in model organisms. The key components are believed to exist throughout eukaryotes; however, there is no systematic platform for archiving and dissecting these important gene families. In addition, few fungi have been studied to date, limiting our understanding of RNAi in fungi. Here we present funRNA http://funrna.riceblast.snu.ac.kr/, a fungal kingdom-wide comparative genomics platform for putative genes encoding Argonaute, Dicer, and RdRP. To identify and archive genes encoding the abovementioned key components, protein domain profiles were determined from reference sequences obtained from UniProtKB/SwissProt. The domain profiles were searched using fungal, metazoan, and plant genomes, as well as bacterial and archaeal genomes. 1,163, 442, and 678 genes encoding Argonaute, Dicer, and RdRP, respectively, were predicted. Based on the identification results, active site variation of Argonaute, diversification of Dicer, and sequence analysis of RdRP were discussed in a fungus-oriented manner. funRNA provides results from diverse bioinformatics programs and job submission forms for BLAST, BLASTMatrix, and ClustalW. Furthermore, sequence collections created in funRNA are synced with several gene family analysis portals and databases, offering further analysis opportunities. funRNA provides identification results from a broad taxonomic range and diverse analysis functions, and could be used in diverse comparative and evolutionary studies. It could serve as a versatile genomics workbench for key components of RNAi.

Association of candidate genes with drought tolerance traits in diverse perennial ryegrass accessions

PubMed Central

Jiang, Yiwei

2013-01-01

Drought is a major environmental stress limiting growth of perennial grasses in temperate regions. Plant drought tolerance is a complex trait that is controlled by multiple genes. Candidate gene association mapping provides a powerful tool for dissection of complex traits. Candidate gene association mapping of drought tolerance traits was conducted in 192 diverse perennial ryegrass (Lolium perenne L.) accessions from 43 countries. The panel showed significant variations in leaf wilting, leaf water content, canopy and air temperature difference, and chlorophyll fluorescence under well-watered and drought conditions across six environments. Analysis of 109 simple sequence repeat markers revealed five population structures in the mapping panel. A total of 2520 expression-based sequence readings were obtained for a set of candidate genes involved in antioxidant metabolism, dehydration, water movement across membranes, and signal transduction, from which 346 single nucleotide polymorphisms were identified. Significant associations were identified between a putative LpLEA3 encoding late embryogenesis abundant group 3 protein and a putative LpFeSOD encoding iron superoxide dismutase and leaf water content, as well as between a putative LpCyt Cu-ZnSOD encoding cytosolic copper-zinc superoxide dismutase and chlorophyll fluorescence under drought conditions. Four of these identified significantly associated single nucleotide polymorphisms from these three genes were also translated to amino acid substitutions in different genotypes. These results indicate that allelic variation in these genes may affect whole-plant response to drought stress in perennial ryegrass. PMID:23386684

piRNA-mediated transposon regulation and the germ-line mutation rate in Drosophila melanogaster males.

PubMed

Simmons, Michael J; Peterson, Mark P; Thorp, Michael W; Buschette, Jared T; DiPrima, Stephanie N; Harter, Christine L; Skolnick, Matthew J

2015-03-01

Transposons, especially retrotransposons, are abundant in the genome of Drosophila melanogaster. These mobile elements are regulated by small RNAs that interact with the Piwi family of proteins-the piwi-interacting or piRNAs. The Piwi proteins are encoded by the genes argonaute3 (ago3), aubergine (aub), and piwi. Heterochromatin Protein 1 (HP1), a chromatin-organizing protein encoded by the Suppressor of variegation 205 [Su(var)205] gene, also plays a role in this regulation. To assess the mutational impact of weakening the system for transposon regulation, we measured the frequency of recessive X-linked lethal mutations occurring in the germ lines of males from stocks that were heterozygous for mutant alleles of the ago3, aub, piwi, or Su(var)205 genes. These mutant alleles are expected to deplete the wild-type proteins encoded by these genes by as much as 50%. The mutant alleles of piwi and Su(var)205 significantly increased the X-linked lethal mutation frequency, whereas the mutant alleles of ago3 did not. An increased mutation frequency was also observed in males from one of two mutant aub stocks, but this increase may not have been due to the aub mutant. The increased mutation frequency caused by depleting Piwi or HP1suggests that chromatin-organizing proteins play important roles in minimizing the germ-line mutation rate, possibly by stabilizing the structure of the heterochromatin in which many transposons are situated. Copyright © 2015 Elsevier B.V. All rights reserved.

Gene Encoding the Hydrolase for the Product of the meta-Cleavage Reaction in Testosterone Degradation by Comamonas testosteroni

PubMed Central

Horinouchi, Masae; Hayashi, Toshiaki; Koshino, Hiroyuki; Yamamoto, Takako; Kudo, Toshiaki

2003-01-01

In a previous study we isolated the meta-cleavage enzyme gene, tesB, that encodes an enzyme that carries out a meta-cleavage reaction in the breakdown of testosterone by Comamonas testeroni TA441 (M. Horinouchi et al., Microbiology 147:3367-3375, 2001). Here we report the isolation of a gene, tesD, that encodes a hydrolase which acts on the product of the meta-cleavage reaction. We isolated tesD by using a Tn5 mutant of TA441 that showed limited growth on testosterone. TesD exhibited ca. 40% identity in amino acid sequence with BphDs, known hydrolases of biphenyl degradation in Pseudomonas spp. The TesD-disrupted mutant showed limited growth on testosterone, and the culture shows an intense yellow color. High-pressure liquid chromatography analysis of the culture of TesD-disrupted mutant incubated with testosterone detected five major intermediate compounds, one of which, showing yellow color under neutral conditions, was considered to be the product of the meta-cleavage reaction. The methylation product was analyzed and identified as methyl-4,5-9,10-diseco-3-methoxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oate, indicating that the substrate of TesD in testosterone degradation is 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid. 4,5-9,10-Diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid was transformed by Escherichia coli-expressed TesD. Downstream of tesD, we identified tesE, F, and G, which encode for enzymes that degrade one of the products of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid converted by TesD. PMID:12676694

Preclinical and clinical experience in vascular gene therapy: advantages over conservative/standard therapy.

PubMed

Nikol, S; Huehns, T Y

2001-04-01

No systemic pharmacological treatment has been shown to convincingly reduce the incidence of restenosis after angioplasty or increase the formation of collaterals in ischemic tissue in patients. The lack of success of many pharmaceutical agents in reducing restenosis rates or in inducing angiogenesis post-angioplasty and following stent implantation has encouraged the development of new technological treatment approaches. Gene therapy is a novel strategy with the potential to prevent some of the sequelae after arterial injury, particularly cell proliferation, and to induce growth of new vessels or remodeling of pre-existing vessel branches, which may help patients with critical ischemia. Gene therapy strategies have the advantage of minimizing systemic side effects and may have a long-term effect as the encoded protein is released. Most clinical trials investigating gene therapy for vascular disease have been uncontrolled phase I and IIa trials. Gene therapy into vessels with the genes for growth factors has been demonstrated to be feasible and efficient. Local drug delivery devices have been used in combination with gene therapy in several trials to maximize safety and efficiency. Data from experimental animal work indicates that gene therapy may modify intimal hyperplasia after arterial injury, but there are few clinical trials on restenosis in patients. Preliminary clinical results show only limited success in altering restenosis rates. In vitro and experimental in vivo investigations into gene therapy for angiogenesis demonstrate increased formation of collaterals and functional improvement of limb ischemia. There is some evidence of increased collateral formation and clinical improvement in patients with critical limb ischemia. Results of placebo-controlled and double-blind trials of gene therapy for vascular disease are awaited.

Improvement of the Redox Balance Increases l-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

PubMed Central

Hasegawa, Satoshi; Uematsu, Kimio; Natsuma, Yumi; Suda, Masako; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki

2012-01-01

Production of l-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the l-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall l-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of l-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for l-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD+ ratio significantly decreased, and glucose consumption and l-valine production drastically improved. Moreover, l-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD+ ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for l-valine production under oxygen deprivation conditions. The l-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM l-valine after 24 h with a yield of 0.63 mol mol of glucose−1, and the l-valine productivity reached 1,940 mM after 48 h. PMID:22138982

Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions.

PubMed

Hasegawa, Satoshi; Uematsu, Kimio; Natsuma, Yumi; Suda, Masako; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

2012-02-01

Production of L-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the L-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall L-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of L-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for L-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD(+) ratio significantly decreased, and glucose consumption and L-valine production drastically improved. Moreover, L-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD(+) ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for L-valine production under oxygen deprivation conditions. The L-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM L-valine after 24 h with a yield of 0.63 mol mol of glucose(-1), and the L-valine productivity reached 1,940 mM after 48 h.

DNA microarray analysis of the cyanotroph Pseudomonas pseudoalcaligenes CECT5344 in response to nitrogen starvation, cyanide and a jewelry wastewater.

PubMed

Luque-Almagro, V M; Escribano, M P; Manso, I; Sáez, L P; Cabello, P; Moreno-Vivián, C; Roldán, M D

2015-11-20

Pseudomonas pseudoalcaligenes CECT5344 is an alkaliphilic bacterium that can use cyanide as nitrogen source for growth, becoming a suitable candidate to be applied in biological treatment of cyanide-containing wastewaters. The assessment of the whole genome sequence of the strain CECT5344 has allowed the generation of DNA microarrays to analyze the response to different nitrogen sources. The mRNA of P. pseudoalcaligenes CECT5344 cells grown under nitrogen limiting conditions showed considerable changes when compared against the transcripts from cells grown with ammonium; up-regulated genes were, among others, the glnK gene encoding the nitrogen regulatory protein PII, the two-component ntrBC system involved in global nitrogen regulation, and the ammonium transporter-encoding amtB gene. The protein coding transcripts of P. pseudoalcaligenes CECT5344 cells grown with sodium cyanide or an industrial jewelry wastewater that contains high concentration of cyanide and metals like iron, copper and zinc, were also compared against the transcripts of cells grown with ammonium as nitrogen source. This analysis revealed the induction by cyanide and the cyanide-rich wastewater of four nitrilase-encoding genes, including the nitC gene that is essential for cyanide assimilation, the cyanase cynS gene involved in cyanate assimilation, the cioAB genes required for the cyanide-insensitive respiration, and the ahpC gene coding for an alkyl-hydroperoxide reductase that could be related with iron homeostasis and oxidative stress. The nitC and cynS genes were also induced in cells grown under nitrogen starvation conditions. In cells grown with the jewelry wastewater, a malate quinone:oxidoreductase mqoB gene and several genes coding for metal extrusion systems were specifically induced. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Dynamic Maternal Gradients Control Timing and Shift-Rates for Drosophila Gap Gene Expression

PubMed Central

Verd, Berta; Crombach, Anton

2017-01-01

Pattern formation during development is a highly dynamic process. In spite of this, few experimental and modelling approaches take into account the explicit time-dependence of the rules governing regulatory systems. We address this problem by studying dynamic morphogen interpretation by the gap gene network in Drosophila melanogaster. Gap genes are involved in segment determination during early embryogenesis. They are activated by maternal morphogen gradients encoded by bicoid (bcd) and caudal (cad). These gradients decay at the same time-scale as the establishment of the antero-posterior gap gene pattern. We use a reverse-engineering approach, based on data-driven regulatory models called gene circuits, to isolate and characterise the explicitly time-dependent effects of changing morphogen concentrations on gap gene regulation. To achieve this, we simulate the system in the presence and absence of dynamic gradient decay. Comparison between these simulations reveals that maternal morphogen decay controls the timing and limits the rate of gap gene expression. In the anterior of the embyro, it affects peak expression and leads to the establishment of smooth spatial boundaries between gap domains. In the posterior of the embryo, it causes a progressive slow-down in the rate of gap domain shifts, which is necessary to correctly position domain boundaries and to stabilise the spatial gap gene expression pattern. We use a newly developed method for the analysis of transient dynamics in non-autonomous (time-variable) systems to understand the regulatory causes of these effects. By providing a rigorous mechanistic explanation for the role of maternal gradient decay in gap gene regulation, our study demonstrates that such analyses are feasible and reveal important aspects of dynamic gene regulation which would have been missed by a traditional steady-state approach. More generally, it highlights the importance of transient dynamics for understanding complex regulatory processes in development. PMID:28158178

Dynamic Maternal Gradients Control Timing and Shift-Rates for Drosophila Gap Gene Expression.

PubMed

Verd, Berta; Crombach, Anton; Jaeger, Johannes

2017-02-01

Pattern formation during development is a highly dynamic process. In spite of this, few experimental and modelling approaches take into account the explicit time-dependence of the rules governing regulatory systems. We address this problem by studying dynamic morphogen interpretation by the gap gene network in Drosophila melanogaster. Gap genes are involved in segment determination during early embryogenesis. They are activated by maternal morphogen gradients encoded by bicoid (bcd) and caudal (cad). These gradients decay at the same time-scale as the establishment of the antero-posterior gap gene pattern. We use a reverse-engineering approach, based on data-driven regulatory models called gene circuits, to isolate and characterise the explicitly time-dependent effects of changing morphogen concentrations on gap gene regulation. To achieve this, we simulate the system in the presence and absence of dynamic gradient decay. Comparison between these simulations reveals that maternal morphogen decay controls the timing and limits the rate of gap gene expression. In the anterior of the embyro, it affects peak expression and leads to the establishment of smooth spatial boundaries between gap domains. In the posterior of the embryo, it causes a progressive slow-down in the rate of gap domain shifts, which is necessary to correctly position domain boundaries and to stabilise the spatial gap gene expression pattern. We use a newly developed method for the analysis of transient dynamics in non-autonomous (time-variable) systems to understand the regulatory causes of these effects. By providing a rigorous mechanistic explanation for the role of maternal gradient decay in gap gene regulation, our study demonstrates that such analyses are feasible and reveal important aspects of dynamic gene regulation which would have been missed by a traditional steady-state approach. More generally, it highlights the importance of transient dynamics for understanding complex regulatory processes in development.

Evidence of translation efficiency adaptation of the coding regions of the bacteriophage lambda.

PubMed

Goz, Eli; Mioduser, Oriah; Diament, Alon; Tuller, Tamir

2017-08-01

Deciphering the way gene expression regulatory aspects are encoded in viral genomes is a challenging mission with ramifications related to all biomedical disciplines. Here, we aimed to understand how the evolution shapes the bacteriophage lambda genes by performing a high resolution analysis of ribosomal profiling data and gene expression related synonymous/silent information encoded in bacteriophage coding regions.We demonstrated evidence of selection for distinct compositions of synonymous codons in early and late viral genes related to the adaptation of translation efficiency to different bacteriophage developmental stages. Specifically, we showed that evolution of viral coding regions is driven, among others, by selection for codons with higher decoding rates; during the initial/progressive stages of infection the decoding rates in early/late genes were found to be superior to those in late/early genes, respectively. Moreover, we argued that selection for translation efficiency could be partially explained by adaptation to Escherichia coli tRNA pool and the fact that it can change during the bacteriophage life cycle.An analysis of additional aspects related to the expression of viral genes, such as mRNA folding and more complex/longer regulatory signals in the coding regions, is also reported. The reported conclusions are likely to be relevant also to additional viruses. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Diversity of Antisense and Other Non-Coding RNAs in Archaea Revealed by Comparative Small RNA Sequencing in Four Pyrobaculum Species

PubMed Central

Bernick, David L.; Dennis, Patrick P.; Lui, Lauren M.; Lowe, Todd M.

2012-01-01

A great diversity of small, non-coding RNA (ncRNA) molecules with roles in gene regulation and RNA processing have been intensely studied in eukaryotic and bacterial model organisms, yet our knowledge of possible parallel roles for small RNAs (sRNA) in archaea is limited. We employed RNA-seq to identify novel sRNA across multiple species of the hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By comparing transcriptional data collected in parallel among four species, we were able to identify conserved RNA genes fitting into known and novel families. Among our findings, we highlight three novel cis-antisense sRNAs encoded opposite to key regulatory (ferric uptake regulator), metabolic (triose-phosphate isomerase), and core transcriptional apparatus genes (transcription factor B). We also found a large increase in the number of conserved C/D box sRNA genes over what had been previously recognized; many of these genes are encoded antisense to protein coding genes. The conserved opposition to orthologous genes across the Pyrobaculum genus suggests similarities to other cis-antisense regulatory systems. Furthermore, the genus-specific nature of these sRNAs indicates they are relatively recent, stable adaptations. PMID:22783241

Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae.

PubMed

Elbing, Karin; Ståhlberg, Anders; Hohmann, Stefan; Gustafsson, Lena

2004-12-01

The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation. Glucose control of SUC2 expression is mediated by the Snf1-Mig1 pathway. Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes. Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates. Remarkably, transient Mig1-dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates. This suggests that Mig1-mediated repression can be overruled by factors mediating induction via a low glucose signal. At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes. Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1-dependent and -independent mechanisms that can now be dissected and resolved as early and late/sustained responses.

Mollusk genes encoding lysine tRNA (UUU) contain introns.

PubMed

Matsuo, M; Abe, Y; Saruta, Y; Okada, N

1995-11-20

New intron-containing genes encoding tRNAs were discovered when genomic DNA isolated from various animal species was amplified by the polymerase chain reaction (PCR) with primers based on sequences of rabbit tRNA(Lys). From sequencing analysis of the products of PCR, we found that introns are present in several genes encoding tRNA(Lys) in mollusks, such as Loligo bleekeri (squid) and Octopus vulgaris (octopus). These introns were specific to genes encoding tRNA(Lys)(CUU) and were not present in genes encoding tRNA(Lys)(CUU). In addition, the sequences of the introns were different from one another. To confirm the results of our initial experiments, we isolated and sequenced genes encoding tRNA(Lys)(CUU) and tRNA(Lys)(UUU). The gene for tRNA(Lys)(UUU) from squid contained an intron, whose sequence was the same as that identified by PCR, and the gene formed a cluster with a corresponding pseudogene. Several DNA regions of 2.1 kb containing this cluster appeared to be tandemly arrayed in the squid genome. By contrast, the gene encoding tRNA(Lys)(CUU) did not contain an intron, as shown also by PCR. The tRNA(Lys)(UUU) that corresponded to the analyzed gene was isolated and characterized. The present study provides the first example of an intron-containing gene encoding a tRNA in mollusks and suggests the universality of introns in such genes in higher eukaryotes.

Detection and Diversity Evaluation of Tetracycline Resistance Genes in Grassland-Based Production Systems in Colombia, South America

PubMed Central

Santamaría, Johanna; López, Liliana; Soto, Carlos Yesid

2011-01-01

Grassland-based production systems use ∼26% of land surface on earth. However, there are no evaluations of these systems as a source of antibiotic pollution. This study was conducted to evaluate the presence, diversity, and distribution of tetracycline resistance genes in the grasslands of the Colombian Andes, where administration of antibiotics to animals is limited to treat disease and growth promoters are not included in animals’ diet. Animal (ruminal fluid and feces) and environmental (soil and water) samples were collected from different dairy cattle farms and evaluated by PCR for the genes tet(M), tet(O), tetB(P), tet(Q), tet(W), tet(S), tet(T), otr(A), which encode ribosomal protection proteins (RPPs), and the genes tet(A), tet(B), tet(D), tet(H), tet(J), and tet(Z), encoding efflux pumps. A wide distribution and high frequency for genes tet(W) and tet(Q) were found in both sample types. Genes tet(O) and tetB(P), detected in high frequencies in feces, were detected in low frequencies or not detected at all in the environment. Other genes encoding RPPs, such as tet(M), tet(S), and tet(T), were detected at very low frequencies and restricted distributions. Genes encoding efflux pumps were not common in this region, and only two of them, tet(B) and tet(Z), were detected. DGGE–PCR followed by comparative sequence analysis of tet(W) and tet(Q) showed that the sequences detected in animals did not differ from those coming from soil and water. Finally, the farms sampled in this study showed more than 50% similarity in relation to the tet genes detected. In conclusion, there was a remarkable presence of tet genes in these production systems and, although not all genes detected in animal reservoirs were detected in the environment, there is a predominant distribution of tet(W) and tet(Q) in both animal and environmental reservoirs. Sequence similarity analysis suggests the transmission of these genes from animals to the environment. PMID:22174707

Human AZU-1 gene, variants thereof and expressed gene products

DOEpatents

Chen, Huei-Mei; Bissell, Mina

2004-06-22

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

Microaerobic glycerol formation in Saccharomyces cerevisiae.

PubMed

Costenoble, R; Valadi, H; Gustafsson, L; Niklasson, C; Franzén, C J

2000-12-01

The yeast Saccharomyces cerevisiae produces large amounts of glycerol as an osmoregulator during hyperosmotic stress and as a redox sink at low oxygen availability. NAD(+)-dependent glycerol-3-phosphate dehydrogenase in S. cerevisiae is present in two isoforms, coded for by two different genes, GPD1 and GPD2. Mutants for either one or both of these genes were investigated under carefully controlled static and dynamic conditions in continuous cultures at low oxygen transfer rates. Our results show that S. cerevisiae controls the production of glycerol in response to hypoxic conditions by regulating the expression of several genes. At high demand for NADH reoxidation, a strong induction was seen not only of the GPD2 gene, but also of GPP1, encoding one of the molecular forms of glycerol-3-phosphatase. Induction of the GPP1 gene appears to play a decisive role at elevated growth rates. At low demand for NADH reoxidation via glycerol formation, the GPD1, GPD2, GPP1, and GPP2 genes were all expressed at basal levels. The dynamics of the gene induction and the glycerol formation at low demand for NADH reoxidation point to an important role of the Gpd1p; deletion of the GPD1 gene strongly altered the expression patterns of the GPD2 and GPP1 genes under such conditions. Furthermore, our results indicate that GCY1 and DAK1, tentatively encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, may be involved in the redox regulation of S. cerevisiae. Copyright 2000 John Wiley & Sons, Ltd.

The mitochondrial gene encoding ribosomal protein S12 has been translocated to the nuclear genome in Oenothera.

PubMed Central

Grohmann, L; Brennicke, A; Schuster, W

1992-01-01

The Oenothera mitochondrial genome contains only a gene fragment for ribosomal protein S12 (rps12), while other plants encode a functional gene in the mitochondrion. The complete Oenothera rps12 gene is located in the nucleus. The transit sequence necessary to target this protein to the mitochondrion is encoded by a 5'-extension of the open reading frame. Comparison of the amino acid sequence encoded by the nuclear gene with the polypeptides encoded by edited mitochondrial cDNA and genomic sequences of other plants suggests that gene transfer between mitochondrion and nucleus started from edited mitochondrial RNA molecules. Mechanisms and requirements of gene transfer and activation are discussed. Images PMID:1454526

Restricted VH gene usage and generation of antibody diversity in rabbit.

PubMed

Knight, K L

1992-01-01

The presence of VHa allotypic specificities on nearly all rabbit Ig molecules has perplexed immunologists for many years. How could these allotypic specificities be inherited as if controlled by alleles if the germline has hundreds of VHa allotype-encoding genes and if most of these genes are used in VDJ gene rearrangements. I review recent data indicating that the allelic inheritance of the VHa allotypes can be explained by preferential utilization of the D-proximal VH gene VH1 in VDJ gene rearrangements. The preferential usage of one VH gene, however, limits the contribution of combinatorial joining of multiple VH, D and JH gene segments to the generation of antibody diversity. The roles of somatic gene conversion and somatic mutation in generating antibody diversity are discussed. Further, the limited usage of germline VH genes in normal, allotype-suppressed and the mutant Alicia rabbit as well as the molecular basis of latent allotypes and VH/CH recombinants is reviewed.

Growth of Aerobic Ripening Bacteria at the Cheese Surface Is Limited by the Availability of Iron

PubMed Central

Back, Alexandre; Irlinger, Françoise

2012-01-01

The microflora on the surface of smear-ripened cheeses is composed of various species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. The objective of the present study was to show that iron availability is a limiting factor in the growth of typical aerobic ripening bacteria in cheese. For that purpose, we investigated the effect of iron or siderophore addition in model cheeses that were coinoculated with a yeast and a ripening bacterium. Both iron and the siderophore desferrioxamine B stimulated the growth of ripening bacteria belonging to the genera Arthrobacter, Corynebacterium, and Brevibacterium. The extent of stimulation was strain dependent, and generally, the effect of desferrioxamine B was greater than that of iron. Measurements of the expression of genes related to the metabolism of iron by Arthrobacter arilaitensis Re117 by real-time reverse transcription-PCR showed that these genes were transcribed during growth in cheese. The addition of desferrioxamine B increased the expression of two genes encoding iron-siderophore ABC transport binding proteins. The addition of iron decreased the expression of siderophore biosynthesis genes and of part of the genes encoding iron-siderophore ABC transport components. It was concluded that iron availability is a limiting factor in the growth of typical cheese surface bacteria. The selection of strains with efficient iron acquisition systems may be useful for the development of defined-strain surface cultures. Furthermore, the importance of iron metabolism in the microbial ecology of cheeses should be investigated since it may result in positive or negative microbial interactions. PMID:22367081

Evaluation of fosfomycin activity against ESBL producing Enterobacteriaceae isolated from Iran.

PubMed

Kazemian, Hossein

2018-05-16

Rising rates of antimicrobial resistance among Enterobacteriaceae limit the use of reliably active forms of available drugs. The aim of this study was to investigate the prevalence of fosfomycin (US6794490B2) resistance gene among ESBL producing isolates in Iran. We tested 355 isolates of Enterobacteriacea collected from various clinical samples including urine, wounds, blood and other sources during June 2016 to July 2017. Antibiotic sensitivity and extended spectrum beta lactamase (ESBL) production were tested using agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. ESBL genes (blaTEM, bla SHV,bla CTX-M), plasmid-encoded fosfomycin resistance genes (fosA, fosB, fosA3 and fosC2) and chromosomal mutations (murA, glpT, uhpT) were detected by polymerase chain reaction (PCR). In this study, 151 of the 355 isolates were ESBL-positive. blaCTX-M (77%) was the most common gene followed by blaSHV (70%) and blaTEM (58%), either alone or in combination. Eighty nine percent (132/151) of the ESBL-positive isolates were MDR. Antimicrobial susceptibility rates were higher for fosfomycin (92.8%) and imipenem (35.5%) among ESBL-positive isolates. None of the ESBL- positive isolates harbored any mutations or plasmid-mediated fosfomycin resistance determinants. In conclusion, fosfomycin showed good antimicrobial activity against multidrug resistance ESBL- positive Enterobacteriaceae. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Tissue-specific impact of FADS cluster variants on FADS1 and FADS2 gene expression.

PubMed

Reynolds, Lindsay M; Howard, Timothy D; Ruczinski, Ingo; Kanchan, Kanika; Seeds, Michael C; Mathias, Rasika A; Chilton, Floyd H

2018-01-01

Omega-6 (n-6) and omega-3 (n-3) long (≥ 20 carbon) chain polyunsaturated fatty acids (LC-PUFAs) play a critical role in human health and disease. Biosynthesis of LC-PUFAs from dietary 18 carbon PUFAs in tissues such as the liver is highly associated with genetic variation within the fatty acid desaturase (FADS) gene cluster, containing FADS1 and FADS2 that encode the rate-limiting desaturation enzymes in the LC-PUFA biosynthesis pathway. However, the molecular mechanisms by which FADS genetic variants affect LC-PUFA biosynthesis, and in which tissues, are unclear. The current study examined associations between common single nucleotide polymorphisms (SNPs) within the FADS gene cluster and FADS1 and FADS2 gene expression in 44 different human tissues (sample sizes ranging 70-361) from the Genotype-Tissue Expression (GTEx) Project. FADS1 and FADS2 expression were detected in all 44 tissues. Significant cis-eQTLs (within 1 megabase of each gene, False Discovery Rate, FDR<0.05, as defined by GTEx) were identified in 12 tissues for FADS1 gene expression and 23 tissues for FADS2 gene expression. Six tissues had significant (FDR< 0.05) eQTLs associated with both FADS1 and FADS2 (including artery, esophagus, heart, muscle, nerve, and thyroid). Interestingly, the identified eQTLs were consistently found to be associated in opposite directions for FADS1 and FADS2 expression. Taken together, findings from this study suggest common SNPs within the FADS gene cluster impact the transcription of FADS1 and FADS2 in numerous tissues and raise important questions about how the inverse expression of these two genes impact intermediate molecular (such a LC-PUFA and LC-PUFA-containing glycerolipid levels) and ultimately clinical phenotypes associated with inflammatory diseases and brain health.

Pnp gene modification for improved xylose utilization in Zymomonas

DOEpatents

Caimi, Perry G G; Qi, Min; Tao, Luan; Viitanen, Paul V; Yang, Jianjun

2014-12-16

The endogenous pnp gene encoding polynucleotide phosphorylase in the Zymomonas genome was identified as a target for modification to provide improved xylose utilizing cells for ethanol production. The cells are in addition genetically modified to have increased expression of ribose-5-phosphate isomerase (RPI) activity, as compared to cells without this genetic modification, and are not limited in xylose isomerase activity in the absence of the pnp modification.

Aldouronate utilization in Paenibacillus sp. strain JDR-2: Physiological and enzymatic evidence for coupling of extracellular depolymerization and intracellular metabolism.

PubMed

Nong, Guang; Rice, John D; Chow, Virginia; Preston, James F

2009-07-01

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 alpha-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/beta-xylosidase (XynB). Here we show that this Paenibacillus sp. strain is able to utilize methylglucuronoxylose (MeGAX(1)), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX(3)), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAX(n)). The average rates of utilization of MeGAX(n), MeGAX(1), and MeGAX(3) were 149.8, 59.4, and 54.3 microg xylose equivalents.ml(-1).h(-1), respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX(1) and MeGAX(3), releasing 4-O-methyl-d-glucuronate alpha-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX(3), to xylose and xylobiose. The ability to utilize MeGAX(1) provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAX(n) than that of MeGAX(3) indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the aguA and xynA2 genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals.

The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme.

PubMed

Teste, M A; Enjalbert, B; Parrou, J L; François, J M

2000-12-01

The YPR184w gene encodes a 1536-amino acid protein that is 34-39% identical to the mammal, Drosophila melanogaster and Caenorhabditis elegans glycogen debranching enzyme. The N-terminal part of the protein possesses the four conserved sequences of the alpha-amylase superfamily, while the C-terminal part displays 50% similarity with the C-terminal of other eukaryotic glycogen debranching enzymes. Reliable measurement of alpha-1,4-glucanotransferase and alpha-1, 6-glucosidase activity of the yeast debranching enzyme was determined in strains overexpressing YPR184w. The alpha-1, 4-glucanotransferase activity of a partially purified preparation of debranching enzyme preferentially transferred maltosyl units than maltotriosyl. Deletion of YPR184w prevents glycogen degradation, whereas overexpression had no effect on the rate of glycogen breakdown. In response to stress and growth conditions, the transcriptional control of YPR184w gene, renamed GDB1 (for Glycogen DeBranching gene), is strictly identical to that of other genes involved in glycogen metabolism.

A complete mitochondrial genome of wheat (Triticum aestivum cv. Chinese Yumai), and fast evolving mitochondrial genes in higher plants.

PubMed

Cui, Peng; Liu, Huitao; Lin, Qiang; Ding, Feng; Zhuo, Guoyin; Hu, Songnian; Liu, Dongcheng; Yang, Wenlong; Zhan, Kehui; Zhang, Aimin; Yu, Jun

2009-12-01

Plant mitochondrial genomes, encoding necessary proteins involved in the system of energy production, play an important role in the development and reproduction of the plant. They occupy a specific evolutionary pattern relative to their nuclear counterparts. Here, we determined the winter wheat (Triticum aestivum cv. Chinese Yumai) mitochondrial genome in a length of 452 and 526 bp by shotgun sequencing its BAC library. It contains 202 genes, including 35 known protein-coding genes, three rRNA and 17 tRNA genes, as well as 149 open reading frames (ORFs; greater than 300 bp in length). The sequence is almost identical to the previously reported sequence of the spring wheat (T. aestivum cv. Chinese Spring); we only identified seven SNPs (three transitions and four transversions) and 10 indels (insertions and deletions) between the two independently acquired sequences, and all variations were found in non-coding regions. This result confirmed the accuracy of the previously reported mitochondrial sequence of the Chinese Spring wheat. The nucleotide frequency and codon usage of wheat are common among the lineage of higher plant with a high AT-content of 58%. Molecular evolutionary analysis demonstrated that plant mitochondrial genomes evolved at different rates, which may correlate with substantial variations in metabolic rate and generation time among plant lineages. In addition, through the estimation of the ratio of non-synonymous to synonymous substitution rates between orthologous mitochondrion-encoded genes of higher plants, we found an accelerated evolutionary rate that seems to be the result of relaxed selection.

Physiology and Genetics of Biogenic Methane-Production from Acetate

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

Sowers, Kevin R

Biomass conversion catalyzed by methanogenic consortia is a widely available, renewable resource for both energy production and waste treatment. The efficiency of this process is directly dependent upon the interaction of three metabolically distinct groups of microorganisms; the fermentative and acetogenic Bacteria and the methanogenic Archaea. One of the rate limiting steps in the degradation of soluble organic matter is the dismutation of acetate, a predominant intermediate in the process, which accounts for 70 % or more of the methane produced by the methanogens. Acetate utilization is controlled by regulation of expression of carbon monoxide dehydrogensase (COdh), which catalyzes themore » dismutation of acetate. However, physiological and molecular factors that control differential substrate utilization have not been identified in these Archaea. Our laboratory has identified sequence elements near the promoter of the gene (cdh) encoding for COdh and we have confirmed that these sequences have a role in the in vivo expression of cdh. The current proposal focuses on identifying the regulatory components that interact with DNA and RNA elements, and identifying the mechanisms used to control cdh expression. We will determine whether expression is controlled at the level of transcription or if it is mediated by coordinate interaction of transcription initiation with other processes such as transcription elongation rate and differential mRNA stability. Utilizing recently sequenced methanosarcinal genomes and a DNA microarray currently under development genes that encode regulatory proteins and transcription factors will be identified and function confirmed by gene disruption and subsequent screening on different substrates. Functional interactions will be determined in vivo by assaying the effects of gene dosage and site-directed mutagenesis of the regulatory gene on the expression of a cdh::lacZ operon fusion. Results of this study will reveal whether this critical catabolic pathway is controlled by mechanisms similar to those employed by the Bacteria and Eukarya, or by a regulatory paradigm that is unique to the Archaea. The mechanism(s) revealed by this investigation will provide insight into the regulatory strategies employed by the aceticlastic methanogenic Archaea to efficiently direct carbon and electron flow in anaerobic consortia during fermentative processes.« less

GH51 Arabinofuranosidase and Its Role in the Methylglucuronoarabinoxylan Utilization System in Paenibacillus sp. Strain JDR-2

PubMed Central

Sawhney, Neha

2014-01-01

Methylglucuronoarabinoxylan (MeGAXn) from agricultural residues and energy crops is a significant yet underutilized biomass resource for production of biofuels and chemicals. Mild thermochemical pretreatment of bagasse yields MeGAXn requiring saccharifying enzymes for conversion to fermentable sugars. A xylanolytic bacterium, Paenibacillus sp. strain JDR-2, produces an extracellular cell-associated GH10 endoxylanse (XynA1) which efficiently depolymerizes methylglucuronoxylan (MeGXn) from hardwoods coupled with assimilation of oligosaccharides for further processing by intracellular GH67 α-glucuronidase, GH10 endoxylanase, and GH43 β-xylosidase. This process has been ascribed to genes that comprise a xylan utilization regulon that encodes XynA1 and includes a gene cluster encoding transcriptional regulators, ABC transporters, and intracellular enzymes that convert assimilated oligosaccharides to fermentable sugars. Here we show that Paenibacillus sp. JDR-2 utilized MeGAXn without accumulation of oligosaccharides in the medium. The Paenibacillus sp. JDR-2 growth rate on MeGAXn was 3.1-fold greater than that on oligosaccharides generated from MeGAXn by XynA1. Candidate genes encoding GH51 arabinofuranosidases with potential roles were identified. Following growth on MeGAXn, quantitative reverse transcription-PCR identified a cluster of genes encoding a GH51 arabinofuranosidase (AbfB) and transcriptional regulators which were coordinately expressed along with the genes comprising the xylan utilization regulon. The action of XynA1 on MeGAXn generated arabinoxylobiose, arabinoxylotriose, xylobiose, xylotriose, and methylglucuronoxylotriose. Recombinant AbfB processed arabinoxylooligosaccharides to xylooligosaccharides and arabinose. MeGAXn processing by Paenibacillus sp. JDR-2 may be achieved by extracellular depolymerization by XynA1 coupled to assimilation of oligosaccharides and further processing by intracellular enzymes, including AbfB. Paenibacillus sp. JDR-2 provides a GH10/GH67 system complemented with genes encoding intracellular GH51 arabinofuranosidases for efficient utilization of MeGAXn. PMID:25063665

Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

PubMed

Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

2013-07-01

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

Comparative genomic analysis of Helicobacter pylori from Malaysia identifies three distinct lineages suggestive of differential evolution

PubMed Central

Kumar, Narender; Mariappan, Vanitha; Baddam, Ramani; Lankapalli, Aditya K.; Shaik, Sabiha; Goh, Khean-Lee; Loke, Mun Fai; Perkins, Tim; Benghezal, Mohammed; Hasnain, Seyed E.; Vadivelu, Jamuna; Marshall, Barry J.; Ahmed, Niyaz

2015-01-01

The discordant prevalence of Helicobacter pylori and its related diseases, for a long time, fostered certain enigmatic situations observed in the countries of the southern world. Variation in H. pylori infection rates and disease outcomes among different populations in multi-ethnic Malaysia provides a unique opportunity to understand dynamics of host–pathogen interaction and genome evolution. In this study, we extensively analyzed and compared genomes of 27 Malaysian H. pylori isolates and identified three major phylogeographic lineages: hspEastAsia, hpEurope and hpSouthIndia. The analysis of the virulence genes within the core genome, however, revealed a comparable pathogenic potential of the strains. In addition, we identified four genes limited to strains of East-Asian lineage. Our analyses identified a few strain-specific genes encoding restriction modification systems and outlined 311 core genes possibly under differential evolutionary constraints, among the strains representing different ethnic groups. The cagA and vacA genes also showed variations in accordance with the host genetic background of the strains. Moreover, restriction modification genes were found to be significantly enriched in East-Asian strains. An understanding of these variations in the genome content would provide significant insights into various adaptive and host modulation strategies harnessed by H. pylori to effectively persist in a host-specific manner. PMID:25452339

Molecular cloning and characterization of two genes for the biotin carboxylase and carboxyltransferase subunits of acetyl coenzyme A carboxylase in Myxococcus xanthus.

PubMed

Kimura, Y; Miyake, R; Tokumasu, Y; Sato, M

2000-10-01

We have cloned a DNA fragment from a genomic library of Myxococcus xanthus using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the accB and accA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene was strikingly similar to biotin carboxylase subunits of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylase. The putative motifs for ATP binding, CO(2) fixation, and biotin binding were found in AccA. The accB gene was located upstream of the accA gene, and they formed a two-gene operon. The protein (AccB) encoded by the accB gene showed high degrees of sequence similarity with carboxyltransferase subunits of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA decarboxylase. Carboxybiotin-binding and acyl-CoA-binding domains, which are conserved in several carboxyltransferase subunits of acyl-CoA carboxylases, were found in AccB. An accA disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type strain. Western blot analysis indicated that the product of the accA gene was a biotinylated protein that was expressed during the exponential growth phase. Based on these results, we propose that this M. xanthus acetyl-CoA carboxylase consists of two subunits, which are encoded by the accB and accA genes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.

Phylogenetically marking the limits of the genus Fusarium for post-Article 59 usage

USDA-ARS?s Scientific Manuscript database

Fusarium (Hypocreales, Nectriaceae) is one of the most important and systematically challenging groups of mycotoxigenic, plant pathogenic, and human pathogenic fungi. We conducted maximum likelihood (ML), maximum parsimony (MP) and Bayesian (B) analyses on partial nucleotide sequences of genes encod...

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

Genetically encoded reporters for hyperpolarized xenon magnetic resonance imaging

NASA Astrophysics Data System (ADS)

Shapiro, Mikhail G.; Ramirez, R. Matthew; Sperling, Lindsay J.; Sun, George; Sun, Jinny; Pines, Alexander; Schaffer, David V.; Bajaj, Vikram S.

2014-07-01

Magnetic resonance imaging (MRI) enables high-resolution non-invasive observation of the anatomy and function of intact organisms. However, previous MRI reporters of key biological processes tied to gene expression have been limited by the inherently low molecular sensitivity of conventional 1H MRI. This limitation could be overcome through the use of hyperpolarized nuclei, such as in the noble gas xenon, but previous reporters acting on such nuclei have been synthetic. Here, we introduce the first genetically encoded reporters for hyperpolarized 129Xe MRI. These expressible reporters are based on gas vesicles (GVs), gas-binding protein nanostructures expressed by certain buoyant microorganisms. We show that GVs are capable of chemical exchange saturation transfer interactions with xenon, which enables chemically amplified GV detection at picomolar concentrations (a 100- to 10,000-fold improvement over comparable constructs for 1H MRI). We demonstrate the use of GVs as heterologously expressed indicators of gene expression and chemically targeted exogenous labels in MRI experiments performed on living cells.

Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation

PubMed Central

2013-01-01

Background Lyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. “bavariensis” (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. “finlandensis” (1). Results Robust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases. Conclusions Intra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues. PMID:24112474

Regulation of Aerobic Energy Metabolism in Podospora anserina by Two Paralogous Genes Encoding Structurally Different c-Subunits of ATP Synthase.

PubMed

Sellem, Carole H; di Rago, Jean-Paul; Lasserre, Jean-Paul; Ackerman, Sharon H; Sainsard-Chanet, Annie

2016-07-01

Most of the ATP in living cells is produced by an F-type ATP synthase. This enzyme uses the energy of a transmembrane electrochemical proton gradient to synthesize ATP from ADP and inorganic phosphate. Proton movements across the membrane domain (FO) of the ATP synthase drive the rotation of a ring of 8-15 c-subunits, which induces conformational changes in the catalytic part (F1) of the enzyme that ultimately promote ATP synthesis. Two paralogous nuclear genes, called Atp9-5 and Atp9-7, encode structurally different c-subunits in the filamentous fungus Podospora anserina. We have in this study identified differences in the expression pattern for the two genes that correlate with the mitotic activity of cells in vegetative mycelia: Atp9-7 is transcriptionally active in non-proliferating (stationary) cells while Atp9-5 is expressed in the cells at the extremity (apex) of filaments that divide and are responsible for mycelium growth. When active, the Atp9-5 gene sustains a much higher rate of c-subunit synthesis than Atp9-7. We further show that the ATP9-7 and ATP9-5 proteins have antagonist effects on the longevity of P. anserina. Finally, we provide evidence that the ATP9-5 protein sustains a higher rate of mitochondrial ATP synthesis and yield in ATP molecules per electron transferred to oxygen than the c-subunit encoded by Atp9-7. These findings reveal that the c-subunit genes play a key role in the modulation of ATP synthase production and activity along the life cycle of P. anserina. Such a degree of sophistication for regulating aerobic energy metabolism has not been described before.

Saccharomyces cerevisiae ribosomal protein L37 is encoded by duplicate genes that are differentially expressed.

PubMed

Tornow, J; Santangelo, G M

1994-06-01

A duplicate copy of the RPL37A gene (encoding ribosomal protein L37) was cloned and sequenced. The coding region of RPL37B is very similar to that of RPL37A, with only one conservative amino-acid difference. However, the intron and flanking sequences of the two genes are extremely dissimilar. Disruption experiments indicate that the two loci are not functionally equivalent: disruption of RPL37B was insignificant, but disruption of RPL37A severely impaired the growth rate of the cell. When both RPL37 loci are disrupted, the cell is unable to grow at all, indicating that rpL37 is an essential protein. The functional disparity between the two RPL37 loci could be explained by differential gene expression. The results of two experiments support this idea: gene fusion of RPL37A to a reporter gene resulted in six-fold higher mRNA levels than was generated by the same reporter gene fused to RPL37B, and a modest increase in gene dosage of RPL37B overcame the lack of a functional RPL37A gene.

Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells.

PubMed

Mariscal, Ana M; Kakizawa, Shigeyuki; Hsu, Jonathan Y; Tanaka, Kazuki; González-González, Luis; Broto, Alicia; Querol, Enrique; Lluch-Senar, Maria; Piñero-Lambea, Carlos; Sun, Lijie; Weyman, Philip D; Wise, Kim S; Merryman, Chuck; Tse, Gavin; Moore, Adam J; Hutchison, Clyde A; Smith, Hamilton O; Tomita, Masaru; Venter, J Craig; Glass, John I; Piñol, Jaume; Suzuki, Yo

2018-05-22

Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.

Characterization, Function, and Transcriptional Profiling Analysis of 3-Hydroxy-3-methylglutaryl-CoA Synthase Gene (GbHMGS1) towards Stresses and Exogenous Hormone Treatments in Ginkgo biloba.

PubMed

Meng, Xiangxiang; Song, Qiling; Ye, Jiabao; Wang, Lanlan; Xu, Feng

2017-10-12

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is one of the rate-limiting enzymes in the mevalonate pathway as it catalyzes the condensation of acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA. In this study, A HMGS gene (designated as GbHMGS1 ) was cloned from Ginkgo biloba for the first time. GbHMGS1 contained a 1422-bp open-reading frame encoding 474 amino acids. Comparative and bioinformatics analysis revealed that GbHMGS1 was extensively homologous to HMGSs from other plant species. Phylogenetic analysis indicated that the GbHMGS1 belonged to the plant HMGS superfamily, sharing a common evolutionary ancestor with other HMGSs, and had a further relationship with other gymnosperm species. The yeast complement assay of GbHMGS1 in HMGS -deficient Saccharomyces cerevisiae strain YSC6274 demonstrated that GbHMGS1 gene encodes a functional HMGS enzyme. The recombinant protein of GbHMGS1 was successfully expressed in E. coli . The in vitro enzyme activity assay showed that the k cat and K m values of GbHMGS1 were 195.4 min -1 and 689 μM, respectively. GbHMGS1 was constitutively expressed in all tested tissues, including the roots, stems, leaves, female flowers, male flowers and fruits. The transcript accumulation for GbHMGS1 was highest in the leaves. Expression profiling analyses revealed that GbHMGS1 expression was induced by abiotic stresses (ultraviolet B and cold) and hormone treatments (salicylic acid, methyl jasmonate, and ethephon) in G. biloba , indicating that GbHMGS1 gene was involved in the response to environmental stresses and plant hormones.

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis.

PubMed

Spangler, Jacob B; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression.

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis

PubMed Central

Spangler, Jacob B.; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

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.

The organization of the fuc regulon specifying L-fucose dissimilation in Escherichia coli K12 as determined by gene cloning.

PubMed

Chen, Y M; Zhu, Y; Lin, E C

1987-12-01

In Escherichia coli the six known genes specifying the utilization of L-fucose as carbon and energy source cluster at 60.2 min and constitute a regulon. These genes include fucP (encoding L-fucose permease), fucI (encoding L-fucose isomerase), fucK (encoding L-fuculose kinase), fucA (encoding L-fuculose 1-phosphate aldolase), fucO (encoding L-1,2-propanediol oxidoreductase), and fucR (encoding the regulatory protein). In this study the fuc genes were cloned and their positions on the chromosome were established by restriction endonuclease and complementation analyses. Clockwise, the gene order is: fucO-fucA-fucP-fucI-fucK-fucR. The operons comprising the structural genes and the direction of transcription were determined by complementation analysis and Southern blot hybridization. The fucPIK and fucA operons are transcribed clockwise. The fucO operon is transcribed counterclockwise. The fucR gene product activates the three structural operons in trans.

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.

Map-based cloning and characterization of the novel yellow-green leaf gene ys83 in rice (Oryza sativa).

PubMed

Ma, Xiaozhi; Sun, Xiaoqiu; Li, Chunmei; Huan, Rui; Sun, Changhui; Wang, Yang; Xiao, Fuliang; Wang, Qian; Chen, Purui; Ma, Furong; Zhang, Kuan; Wang, Pingrong; Deng, Xiaojian

2017-02-01

Leaf-color mutants have been extensively studied in rice, and many corresponding genes have been identified up to now. However, leaf-color mutation mechanisms are diverse and still need further research through identification of novel genes. In the present paper, we isolated a leaf-color mutant, ys83, in rice (Oryza sativa). The mutant displayed a yellow-green leaf phenotype at seedling stage, and then slowly turned into light-green leaf from late tillering stage. In its yellow leaves, photosynthetic pigment contents significantly decreased and the chloroplast development was retarded. The mutant phenotype was controlled by a recessive mutation in a nuclear gene on the short arm of rice chromosome 2. Map-based cloning and sequencing analysis suggested that the candidate gene was YS83 (LOC_Os02g05890) encoding a protein containing 165 amino acid residues. Gene YS83 was expressed in a wide range of tissues, and its encoded protein was targeted to the chloroplast. In the mutant, a T-to-A substitution occurred in coding sequence of gene YS83, which caused a premature translation of its encoded product. By introduction of the wild-type gene, the ys83 mutant recovered to normal green-leaf phenotype. Taken together, we successfully identified a novel yellow-green leaf gene YS83. In addition, number of productive panicles per plant and number of spikelets per panicle only reduced by 6.7% and 7.6%, respectively, meanwhile its seed setting rate and 1000-grain weight (seed size) were not significantly affected in the mutant, so leaf-color mutant gene ys83 could be used as a trait marker gene in commercial hybrid rice production. Copyright © 2016. Published by Elsevier Masson SAS.

High-level generation of polyclonal antibodies by genetic immunization.

PubMed

Chambers, Ross S; Johnston, Stephen Albert

2003-09-01

Antibodies are important tools for investigating the proteome, but current methods for producing them have become a rate-limiting step. A primary obstacle in most methods for generating antibodies or antibody-like molecules is the requirement for at least microgram quantities of purified protein. We have developed a technology for producing antibodies using genetic immunization. Genetic immunization-based antibody production offers several advantages, including high throughput and high specificity. Moreover, antibodies produced from genetically immunized animals are more likely to recognize the native protein. Here we show that a genetic immunization-based system can be used to efficiently raise useful antibodies to a wide range of antigens. We accomplished this by linking the antigen gene to various elements that enhance antigenicity and by codelivering plasmids encoding genetic adjuvants. Our system, which was tested by immunizing mice with >130 antigens, has shown a final success rate of 84%.

Gene and Protein Expression in Response to Different Growth Temperatures and Oxygen Availability in Burkholderia thailandensis

PubMed Central

Peano, Clelia; Chiaramonte, Fabrizio; Motta, Sara; Pietrelli, Alessandro; Jaillon, Sebastien; Rossi, Elio; Consolandi, Clarissa; Champion, Olivia L.; Michell, Stephen L.; Freddi, Luca; Falciola, Luigi; Basilico, Fabrizio; Garlanda, Cecilia; Mauri, Pierluigi; De Bellis, Gianluca; Landini, Paolo

2014-01-01

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors. PMID:24671187

Characterization of Putative Iron Responsive Genes as Species-Specific Indicators of Iron Stress in Thalassiosiroid Diatoms

PubMed Central

Whitney, LeAnn P.; Lins, Jeremy J.; Hughes, Margaret P.; Wells, Mark L.; Chappell, P. Dreux; Jenkins, Bethany D.

2011-01-01

Iron (Fe) availability restricts diatom growth and primary production in large areas of the oceans. It is a challenge to assess the bulk Fe nutritional health of natural diatom populations, since species can differ in their physiological and molecular responses to Fe limitation. We assayed expression of selected genes in diatoms from the Thalassiosira genus to assess their potential utility as species-specific molecular markers to indicate Fe status in natural diatom assemblages. In this study, we compared the expression of the photosynthetic genes encoding ferredoxin (a Fe-requiring protein) and flavodoxin (a Fe-free protein) in culture experiments with Fe replete and Fe stressed Thalassiosira pseudonana (CCMP 1335) isolated from coastal waters and Thalassiosira weissflogii (CCMP 1010) isolated from the open ocean. In T. pseudonana, expression of flavodoxin and ferredoxin genes were not sensitive to Fe status but were found to display diel periodicities. In T. weissflogii, expression of flavodoxin was highly responsive to iron levels and was only detectable when cultures were Fe limited. Flavodoxin genes have been duplicated in most diatoms with available genome data and we show that T. pseudonana has lost its copy related to the Fe-responsive copy in T. weissflogii. We also examined the expression of genes for a putative high affinity, copper (Cu)-dependent Fe uptake system in T. pseudonana. Our results indicate that genes encoding putative Cu transporters, a multi-Cu oxidase, and a Fe reductase are not linked to Fe status. The expression of a second putative Fe reductase increased in Fe limited cultures, but this gene was also highly expressed in Fe replete cultures, indicating it may not be a useful marker in the field. Our findings highlight that Fe metabolism may differ among diatoms even within a genus and show a need to validate responses in different species as part of the development pipeline for genetic markers of Fe status in field populations. PMID:22275908

Quantitative Detection of the nosZ Gene, Encoding Nitrous Oxide Reductase, and Comparison of the Abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils

PubMed Central

Henry, S.; Bru, D.; Stres, B.; Hallet, S.; Philippot, L.

2006-01-01

Nitrous oxide (N2O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N2O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 101 and 102 target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 105 to 107 target copies g−1 of dry soil, whereas genes for 16S rRNA were found at 108 to 109 target copies g−1 of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils. PMID:16885263

Driver gene classification reveals a substantial overrepresentation of tumor suppressors among very large chromatin-regulating proteins.

PubMed

Waks, Zeev; Weissbrod, Omer; Carmeli, Boaz; Norel, Raquel; Utro, Filippo; Goldschmidt, Yaara

2016-12-23

Compiling a comprehensive list of cancer driver genes is imperative for oncology diagnostics and drug development. While driver genes are typically discovered by analysis of tumor genomes, infrequently mutated driver genes often evade detection due to limited sample sizes. Here, we address sample size limitations by integrating tumor genomics data with a wide spectrum of gene-specific properties to search for rare drivers, functionally classify them, and detect features characteristic of driver genes. We show that our approach, CAnceR geNe similarity-based Annotator and Finder (CARNAF), enables detection of potentially novel drivers that eluded over a dozen pan-cancer/multi-tumor type studies. In particular, feature analysis reveals a highly concentrated pool of known and putative tumor suppressors among the <1% of genes that encode very large, chromatin-regulating proteins. Thus, our study highlights the need for deeper characterization of very large, epigenetic regulators in the context of cancer causality.

Temporal dynamics of encoding, storage and reallocation of visual working memory

PubMed Central

Bays, Paul M; Gorgoraptis, Nikos; Wee, Natalie; Marshall, Louise; Husain, Masud

2012-01-01

The process of encoding a visual scene into working memory has previously been studied using binary measures of recall. Here we examine the temporal evolution of memory resolution, based on observers’ ability to reproduce the orientations of objects presented in brief, masked displays. Recall precision was accurately described by the interaction of two independent constraints: an encoding limit that determines the maximum rate at which information can be transferred into memory, and a separate storage limit that determines the maximum fidelity with which information can be maintained. Recall variability decreased incrementally with time, consistent with a parallel encoding process in which visual information from multiple objects accumulates simultaneously in working memory. No evidence was observed for a limit on the number of items stored. Cueing one display item with a brief flash led to rapid development of a recall advantage for that item. This advantage was short-lived if the cue was simply a salient visual event, but was maintained if it indicated an object of particular relevance to the task. These cueing effects were observed even for items that had already been encoded into memory, indicating that limited memory resources can be rapidly reallocated to prioritize salient or goal-relevant information. PMID:21911739

Temporal dynamics of encoding, storage, and reallocation of visual working memory.

PubMed

Bays, Paul M; Gorgoraptis, Nikos; Wee, Natalie; Marshall, Louise; Husain, Masud

2011-09-12

The process of encoding a visual scene into working memory has previously been studied using binary measures of recall. Here, we examine the temporal evolution of memory resolution, based on observers' ability to reproduce the orientations of objects presented in brief, masked displays. Recall precision was accurately described by the interaction of two independent constraints: an encoding limit that determines the maximum rate at which information can be transferred into memory and a separate storage limit that determines the maximum fidelity with which information can be maintained. Recall variability decreased incrementally with time, consistent with a parallel encoding process in which visual information from multiple objects accumulates simultaneously in working memory. No evidence was observed for a limit on the number of items stored. Cuing one display item with a brief flash led to rapid development of a recall advantage for that item. This advantage was short-lived if the cue was simply a salient visual event but was maintained if it indicated an object of particular relevance to the task. These cuing effects were observed even for items that had already been encoded into memory, indicating that limited memory resources can be rapidly reallocated to prioritize salient or goal-relevant information.

Genomic and functional characterisation of IncX3 plasmids encoding blaSHV-12 in Escherichia coli from human and animal origin.

PubMed

Liakopoulos, Apostolos; van der Goot, Jeanet; Bossers, Alex; Betts, Jonathan; Brouwer, Michael S M; Kant, Arie; Smith, Hilde; Ceccarelli, Daniela; Mevius, Dik

2018-05-16

The bla SHV-12 β-lactamase gene is one of the most prevalent genes conferring resistance to extended-spectrum β-lactams in Enterobacteriaceae disseminating within and between reservoirs, mostly via plasmid-mediated horizontal gene transfer. Yet, studies regarding the biology of plasmids encoding bla SHV-12 are very limited. In this study, we revealed the emergence of IncX3 plasmids alongside IncI1α/γ in bla SHV-12 in animal-related Escherichia coli isolates. Four representative bla SHV-12 -encoding IncX3 plasmids were selected for genome sequencing and further genetic and functional characterization. We report here the first complete sequences of IncX3 plasmids of animal origin and show that IncX3 plasmids exhibit remarkable synteny in their backbone, while the major differences lie in their bla SHV-12 -flanking region. Our findings indicate that plasmids of this subgroup are conjugative and highly stable, while they exert no fitness cost on their bacterial host. These favourable features might have contributed to the emergence of IncX3 amongst SHV-12-producing E. coli in the Netherlands, highlighting the epidemic potential of these plasmids.

Mitochondrial-nuclear interactions and accelerated compensatory evolution: evidence from the primate cytochrome C oxidase complex.

PubMed

Osada, Naoki; Akashi, Hiroshi

2012-01-01

Accelerated rates of mitochondrial protein evolution have been proposed to reflect Darwinian coadaptation for efficient energy production for mammalian flight and brain activity. However, several features of mammalian mtDNA (absence of recombination, small effective population size, and high mutation rate) promote genome degradation through the accumulation of weakly deleterious mutations. Here, we present evidence for "compensatory" adaptive substitutions in nuclear DNA- (nDNA) encoded mitochondrial proteins to prevent fitness decline in primate mitochondrial protein complexes. We show that high mutation rate and small effective population size, key features of primate mitochondrial genomes, can accelerate compensatory adaptive evolution in nDNA-encoded genes. We combine phylogenetic information and the 3D structure of the cytochrome c oxidase (COX) complex to test for accelerated compensatory changes among interacting sites. Physical interactions among mtDNA- and nDNA-encoded components are critical in COX evolution; amino acids in close physical proximity in the 3D structure show a strong tendency for correlated evolution among lineages. Only nuclear-encoded components of COX show evidence for positive selection and adaptive nDNA-encoded changes tend to follow mtDNA-encoded amino acid changes at nearby sites in the 3D structure. This bias in the temporal order of substitutions supports compensatory weak selection as a major factor in accelerated primate COX evolution.

[Genetic instability of probiotic characteristics in the Bifidobacterium longum subsp. longum B379M strain during cultivation and maintenance].

PubMed

Averina, O V; Nezametdinova, V Z; Alekseeva, M G; Danilenko, V N

2012-11-01

The stability of inheriting several genes in the Russian commercial strain Bifidobacterium longum subsp. longum B379M during cultivation and maintenance under laboratory conditions has been studied. The examined genes code for probiotic characteristics, such as utilization of several sugars (lacA2 gene, encoding beta-galactosidase; ara gene, encoding arabinosidase; and galA gene, encoding arabinogalactan endo-beta-galactosidase); synthesis of bacteriocins (lans gene, encoding lanthionine synthetase); and mobile gene tet(W), conferring resistance to the antibiotic tetracycline. The other gene families studied include the genes responsible for signal transduction and adaptation to stress conditions in the majority of bacteria (serine/threonine protein kinases and the toxin-antitoxin systems of MazEF and RelBE types) and transcription regulators (genes encoding WhiB family proteins). Genomic DNA was analyzed by PCR using specially selected primers. A loss of the genes galA and tet(W) has been shown. It is proposed to expand the requirements on probiotic strains, namely, to control retention of the key probiotic genes using molecular biological methods.

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, Eric E.; Roessler, Paul G.

1999-01-01

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

Human Genomic Signatures of Brain Oscillations During Memory Encoding.

PubMed

Berto, Stefano; Wang, Guang-Zhong; Germi, James; Lega, Bradley C; Konopka, Genevieve

2018-05-01

Memory encoding is an essential step for all learning. However, the genetic and molecular mechanisms underlying human memory encoding remain poorly understood, and how this molecular framework permits the emergence of specific patterns of brain oscillations observed during mnemonic processing is unknown. Here, we directly compare intracranial electroencephalography recordings from the neocortex in individuals performing an episodic memory task with human gene expression from the same areas. We identify genes correlated with oscillatory memory effects across 6 frequency bands. These genes are enriched for autism-related genes and have preferential expression in neurons, in particular genes encoding synaptic proteins and ion channels, supporting the idea that the genes regulating voltage gradients are involved in the modulation of oscillatory patterns during successful memory encoding across brain areas. Memory-related genes are distinct from those correlated with other forms of cognitive processing and resting state fMRI. These data are the first to identify correlations between gene expression and active human brain states as well as provide a molecular window into memory encoding oscillations in the human brain.

Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation.

PubMed

Tao, Xiang; Fang, Yang; Xiao, Yao; Jin, Yan-Ling; Ma, Xin-Rong; Zhao, Yun; He, Kai-Ze; Zhao, Hai; Wang, Hai-Yan

2013-05-08

Duckweed can thrive on anthropogenic wastewater and produce tremendous biomass production. Due to its relatively high starch and low lignin percentage, duckweed is a good candidate for bioethanol fermentation. Previous studies have observed that water devoid of nutrients is good for starch accumulation, but its molecular mechanism remains unrevealed. This study globally analyzed the response to nutrient starvation in order to investigate the starch accumulation in duckweed (Landoltia punctata). L. punctata was transferred from nutrient-rich solution to distilled water and sampled at different time points. Physiological measurements demonstrated that the activity of ADP-glucose pyrophosphorylase, the key enzyme of starch synthesis, as well as the starch percentage in duckweed, increased continuously under nutrient starvation. Samples collected at 0 h, 2 h and 24 h time points respectively were used for comparative gene expression analysis using RNA-Seq. A comprehensive transcriptome, comprising of 74,797 contigs, was constructed by a de novo assembly of the RNA-Seq reads. Gene expression profiling results showed that the expression of some transcripts encoding key enzymes involved in starch biosynthesis was up-regulated, while the expression of transcripts encoding enzymes involved in starch consumption were down-regulated, the expression of some photosynthesis-related transcripts were down-regulated during the first 24 h, and the expression of some transporter transcripts were up-regulated within the first 2 h. Very interestingly, most transcripts encoding key enzymes involved in flavonoid biosynthesis were highly expressed regardless of starvation, while transcripts encoding laccase, the last rate-limiting enzyme of lignifications, exhibited very low expression abundance in all three samples. Our study provides a comprehensive expression profiling of L. punctata under nutrient starvation, which indicates that nutrient starvation down-regulated the global metabolic status, redirects metabolic flux of fixed CO2 into starch synthesis branch resulting in starch accumulation in L. punctata.

Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation

PubMed Central

2013-01-01

Background Duckweed can thrive on anthropogenic wastewater and produce tremendous biomass production. Due to its relatively high starch and low lignin percentage, duckweed is a good candidate for bioethanol fermentation. Previous studies have observed that water devoid of nutrients is good for starch accumulation, but its molecular mechanism remains unrevealed. Results This study globally analyzed the response to nutrient starvation in order to investigate the starch accumulation in duckweed (Landoltia punctata). L. punctata was transferred from nutrient-rich solution to distilled water and sampled at different time points. Physiological measurements demonstrated that the activity of ADP-glucose pyrophosphorylase, the key enzyme of starch synthesis, as well as the starch percentage in duckweed, increased continuously under nutrient starvation. Samples collected at 0 h, 2 h and 24 h time points respectively were used for comparative gene expression analysis using RNA-Seq. A comprehensive transcriptome, comprising of 74,797 contigs, was constructed by a de novo assembly of the RNA-Seq reads. Gene expression profiling results showed that the expression of some transcripts encoding key enzymes involved in starch biosynthesis was up-regulated, while the expression of transcripts encoding enzymes involved in starch consumption were down-regulated, the expression of some photosynthesis-related transcripts were down-regulated during the first 24 h, and the expression of some transporter transcripts were up-regulated within the first 2 h. Very interestingly, most transcripts encoding key enzymes involved in flavonoid biosynthesis were highly expressed regardless of starvation, while transcripts encoding laccase, the last rate-limiting enzyme of lignifications, exhibited very low expression abundance in all three samples. Conclusion Our study provides a comprehensive expression profiling of L. punctata under nutrient starvation, which indicates that nutrient starvation down-regulated the global metabolic status, redirects metabolic flux of fixed CO2 into starch synthesis branch resulting in starch accumulation in L. punctata. PMID:23651472

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

Neisseria gonorrhoeae Modulates Iron-Limiting Innate Immune Defenses in Macrophages

PubMed Central

Zughaier, Susu M.; Kandler, Justin L.; Shafer, William M.

2014-01-01

Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted infection termed gonorrhea. The gonococcus can survive extracellularly and intracellularly, but in both environments the bacteria must acquire iron from host proteins for survival. However, upon infection the host uses a defensive response by limiting the bioavailability of iron by a number of mechanisms including the enhanced expression of hepcidin, the master iron-regulating hormone, which reduces iron uptake from the gut and retains iron in macrophages. The host also secretes the antibacterial protein NGAL, which sequesters bacterial siderophores and therefore inhibits bacterial growth. To learn whether intracellular gonococci can subvert this defensive response, we examined expression of host genes that encode proteins involved in modulating levels of intracellular iron. We found that N. gonorrhoeae can survive in association (tightly adherent and intracellular) with monocytes and macrophages and upregulates a panel of its iron-responsive genes in this environment. We also found that gonococcal infection of human monocytes or murine macrophages resulted in the upregulation of hepcidin, NGAL, and NRAMP1 as well as downregulation of the expression of the gene encoding the short chain 3-hydroxybutyrate dehydrogenase (BDH2); BDH2 catalyzes the production of the mammalian siderophore 2,5-DHBA involved in chelating and detoxifying iron. Based on these findings, we propose that N. gonorrhoeae can subvert the iron-limiting innate immune defenses to facilitate iron acquisition and intracellular survival. PMID:24489950

The SUD1 gene encodes a putative E3 ubiquitin ligase and is a positive regulator of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in Arabidopsis.

PubMed

Doblas, Verónica G; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M; Botella, Miguel A

2013-02-01

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum-associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

The Global Regulators GacA and ςS Form Part of a Cascade That Controls Alginate Production in Azotobacter vinelandii

PubMed Central

Castañeda, Miguel; Sánchez, Judith; Moreno, Soledad; Núñez, Cinthia; Espín, Guadalupe

2001-01-01

Transcription of the Azotobacter vinelandii algD gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme of alginate synthesis), starts from three sites: p1, p2, and p3. The sensor kinase GacS, a member of the two-component regulatory system, is required for transcription of algD from its three sites during the stationary phase. Here we show that algD is expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between the exponential growth phase and stationary phase. We constructed A. vinelandii strains that carried mutations in gacA encoding the cognate response regulator of GacS and in rpoS coding for the stationary-phase ςS factor. The gacA mutation impaired alginate production and transcription of algD from its three promoters. Transcription of rpoS was also abolished by the gacA mutation. The rpoS mutation impaired transcription of algD from the p1 promoter and increased it from the p2 ςE promoter. The results of this study provide evidence for the predominant role of GacA in a regulatory cascade controlling alginate production and gene expression during the stationary phase in A. vinelandii. PMID:11698366

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis[C][W

PubMed Central

Doblas, Verónica G.; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M.; Botella, Miguel A.

2013-01-01

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals. PMID:23404890

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.

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

Chan, Chi Ho; Levar, Caleb E.; Zacharoff, Lori

Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellarmore » regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented Δ imcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. Lastly, these tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.« less

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, E.E.; Roessler, P.G.

1999-07-27

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

JPEG 2000 Encoding with Perceptual Distortion Control

NASA Technical Reports Server (NTRS)

Watson, Andrew B.; Liu, Zhen; Karam, Lina J.

2008-01-01

An alternative approach has been devised for encoding image data in compliance with JPEG 2000, the most recent still-image data-compression standard of the Joint Photographic Experts Group. Heretofore, JPEG 2000 encoding has been implemented by several related schemes classified as rate-based distortion-minimization encoding. In each of these schemes, the end user specifies a desired bit rate and the encoding algorithm strives to attain that rate while minimizing a mean squared error (MSE). While rate-based distortion minimization is appropriate for transmitting data over a limited-bandwidth channel, it is not the best approach for applications in which the perceptual quality of reconstructed images is a major consideration. A better approach for such applications is the present alternative one, denoted perceptual distortion control, in which the encoding algorithm strives to compress data to the lowest bit rate that yields at least a specified level of perceptual image quality. Some additional background information on JPEG 2000 is prerequisite to a meaningful summary of JPEG encoding with perceptual distortion control. The JPEG 2000 encoding process includes two subprocesses known as tier-1 and tier-2 coding. In order to minimize the MSE for the desired bit rate, a rate-distortion- optimization subprocess is introduced between the tier-1 and tier-2 subprocesses. In tier-1 coding, each coding block is independently bit-plane coded from the most-significant-bit (MSB) plane to the least-significant-bit (LSB) plane, using three coding passes (except for the MSB plane, which is coded using only one "clean up" coding pass). For M bit planes, this subprocess involves a total number of (3M - 2) coding passes. An embedded bit stream is then generated for each coding block. Information on the reduction in distortion and the increase in the bit rate associated with each coding pass is collected. This information is then used in a rate-control procedure to determine the contribution of each coding block to the output compressed bit stream.

Overexpression of OsSAP16 Regulates Photosynthesis and the Expression of a Broad Range of Stress Response Genes in Rice (Oryza sativa L.).

PubMed

Wang, Fei; Coe, Robert A; Karki, Shanta; Wanchana, Samart; Thakur, Vivek; Henry, Amelia; Lin, Hsiang-Chun; Huang, Jianliang; Peng, Shaobing; Quick, William Paul

2016-01-01

This study set out to identify and characterize transcription factors regulating photosynthesis in rice. Screening populations of rice T-DNA activation lines led to the identification of a T-DNA mutant with an increase in intrinsic water use efficiency (iWUE) under well-watered conditions. Flanking sequence analysis showed that the T-DNA construct was located upstream of LOC_Os07g38240 (OsSAP16) encoding for a stress-associated protein (SAP). A second mutant identified with activation in the same gene exhibited the same phenotype; expression of OsSAP16 was shown to be enhanced in both lines. There were no differences in stomatal development or morphology in either of these mutants, although overexpression of OsSAP16 reduced stomatal conductance. This phenotype limited CO2 uptake and the rate of photosynthesis, which resulted in the accumulation of less biomass in the two mutants. Whole transcriptome analysis showed that overexpression of OsSAP16 led to global changes in gene expression consistent with the function of zinc-finger transcription factors. These results show that the gene is involved in modulating the response of rice to drought stress through regulation of the expression of a set of stress-associated genes.

Overexpression of OsSAP16 Regulates Photosynthesis and the Expression of a Broad Range of Stress Response Genes in Rice (Oryza sativa L.)

PubMed Central

Wang, Fei; Coe, Robert A.; Karki, Shanta; Wanchana, Samart; Thakur, Vivek; Henry, Amelia; Lin, Hsiang-Chun; Huang, Jianliang; Peng, Shaobing; Quick, William Paul

2016-01-01

This study set out to identify and characterize transcription factors regulating photosynthesis in rice. Screening populations of rice T-DNA activation lines led to the identification of a T-DNA mutant with an increase in intrinsic water use efficiency (iWUE) under well-watered conditions. Flanking sequence analysis showed that the T-DNA construct was located upstream of LOC_Os07g38240 (OsSAP16) encoding for a stress-associated protein (SAP). A second mutant identified with activation in the same gene exhibited the same phenotype; expression of OsSAP16 was shown to be enhanced in both lines. There were no differences in stomatal development or morphology in either of these mutants, although overexpression of OsSAP16 reduced stomatal conductance. This phenotype limited CO2 uptake and the rate of photosynthesis, which resulted in the accumulation of less biomass in the two mutants. Whole transcriptome analysis showed that overexpression of OsSAP16 led to global changes in gene expression consistent with the function of zinc-finger transcription factors. These results show that the gene is involved in modulating the response of rice to drought stress through regulation of the expression of a set of stress-associated genes. PMID:27303811

Power-rate-distortion analysis for wireless video communication under energy constraint

NASA Astrophysics Data System (ADS)

He, Zhihai; Liang, Yongfang; Ahmad, Ishfaq

2004-01-01

In video coding and streaming over wireless communication network, the power-demanding video encoding operates on the mobile devices with limited energy supply. To analyze, control, and optimize the rate-distortion (R-D) behavior of the wireless video communication system under the energy constraint, we need to develop a power-rate-distortion (P-R-D) analysis framework, which extends the traditional R-D analysis by including another dimension, the power consumption. Specifically, in this paper, we analyze the encoding mechanism of typical video encoding systems and develop a parametric video encoding architecture which is fully scalable in computational complexity. Using dynamic voltage scaling (DVS), a hardware technology recently developed in CMOS circuits design, the complexity scalability can be translated into the power consumption scalability of the video encoder. We investigate the rate-distortion behaviors of the complexity control parameters and establish an analytic framework to explore the P-R-D behavior of the video encoding system. Both theoretically and experimentally, we show that, using this P-R-D model, the encoding system is able to automatically adjust its complexity control parameters to match the available energy supply of the mobile device while maximizing the picture quality. The P-R-D model provides a theoretical guideline for system design and performance optimization in wireless video communication under energy constraint, especially over the wireless video sensor network.

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

Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons.

PubMed Central

Lloyd-Jones, G; Lau, P C

1997-01-01

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria. PMID:9251217

Enterotoxin-encoding genes in Staphylococcus spp. from bulk goat milk.

PubMed

Lyra, Daniele G; Sousa, Francisca G C; Borges, Maria F; Givisiez, Patrícia E N; Queiroga, Rita C R E; Souza, Evandro L; Gebreyes, Wondwossen A; Oliveira, Celso J B

2013-02-01

Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates.

Gene expansion shapes genome architecture in the human pathogen Lichtheimia corymbifera: an evolutionary genomics analysis in the ancient terrestrial mucorales (Mucoromycotina).

PubMed

Schwartze, Volker U; Winter, Sascha; Shelest, Ekaterina; Marcet-Houben, Marina; Horn, Fabian; Wehner, Stefanie; Linde, Jörg; Valiante, Vito; Sammeth, Michael; Riege, Konstantin; Nowrousian, Minou; Kaerger, Kerstin; Jacobsen, Ilse D; Marz, Manja; Brakhage, Axel A; Gabaldón, Toni; Böcker, Sebastian; Voigt, Kerstin

2014-08-01

Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1-4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparison to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae.

Gene Expansion Shapes Genome Architecture in the Human Pathogen Lichtheimia corymbifera: An Evolutionary Genomics Analysis in the Ancient Terrestrial Mucorales (Mucoromycotina)

PubMed Central

Wehner, Stefanie; Linde, Jörg; Valiante, Vito; Sammeth, Michael; Riege, Konstantin; Nowrousian, Minou; Kaerger, Kerstin; Jacobsen, Ilse D.; Marz, Manja; Brakhage, Axel A.; Gabaldón, Toni; Böcker, Sebastian; Voigt, Kerstin

2014-01-01

Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1–4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparision to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae. PMID:25121733

Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia

PubMed Central

Lozada, Mariana; Riva Mercadal, Juan P; Guerrero, Leandro D; Di Marzio, Walter D; Ferrero, Marcela A; Dionisi, Hebe M

2008-01-01

Background Polycyclic aromatic hydrocarbons (PAHs), widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination. Results Genes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs) were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (r = 0.834, p < 0.05). Overall, eight different ARHD gene types were detected in the sediments. In five of them, their deduced amino acid sequences formed deeply rooted branches with previously described ARHD peptide sequences, exhibiting less than 70% identity to them. They contain consensus sequences of the Rieske type [2Fe-2S] cluster binding site, suggesting that these gene fragments encode for ARHDs. On the other hand, three gene types were closely related to previously described ARHDs: archetypical nahAc-like genes, phnAc-like genes as identified in Alcaligenes faecalis AFK2, and phnA1-like genes from marine PAH-degraders from the genus Cycloclasticus. Conclusion These results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information can be used to study the geographical distribution and ecological significance of bacterial populations carrying these genes, and to design molecular assays to monitor the progress and effectiveness of remediation technologies. PMID:18366740

Pheno- and Genotyping of Hopanoid Production in Acidobacteria

PubMed Central

Damsté, Jaap S. Sinninghe; Rijpstra, W. Irene C.; Dedysh, Svetlana N.; Foesel, Bärbel U.; Villanueva, Laura

2017-01-01

Hopanoids are pentacyclic triterpenoid lipids synthesized by different bacterial groups. Methylated hopanoids were believed to be exclusively synthesized by cyanobacteria and aerobic methanotrophs until the genes encoding for the methylation at the C-2 and C-3 position (hpnP and hpnR) were found to be widespread in the bacterial domain, invalidating their use as specific biomarkers. These genes have been detected in the genome of the Acidobacterium “Ca. Koribacter versatilis,” but our knowledge of the synthesis of hopanoids and the presence of genes of their biosynthetic pathway in other member of the Acidobacteria is limited. We analyzed 38 different strains of seven Acidobacteria subdivisions (SDs 1, 3, 4, 6, 8, 10, and 23) for the presence of C30 hopenes and C30+ bacteriohopane polyols (BHPs) using the Rohmer reaction. BHPs and/or C30 hopenes were detected in all strains of SD1 and SD3 but not in SD4 (excepting Chloracidobacterium thermophilum), 6, 8, 10, and 23. This is in good agreement with the presence of genes required for hopanoid biosynthesis in the 31 available whole genomes of cultivated Acidobacteria. All genomes encode the enzymes involved in the non-mevalonate pathway ultimately leading to farnesyl diphosphate but only SD1 and 3 Acidobacteria and C. thermophilum encode all three enzymes required for the synthesis of squalene, its cyclization (shc), and addition and modification of the extended side chain (hpnG, hpnH, hpnI, hpnJ, hpnO). In almost all strains, only tetrafunctionalized BHPs were detected; three strains contained variable relative abundances (up to 45%) of pentafunctionalized BHPs. Only “Ca. K. versatilis” contained methylated hopanoids (i.e., 2,3-dimethyl bishomohopanol), although in low (<10%) amounts. These genes are not present in any other Acidobacterium, consistent with the absence of methylated BHPs in the other examined strains. These data are in agreement with the scattered occurrence of methylated BHPs in other bacterial phyla such as the Alpha-, Beta-, and Gammaproteobacteria and the Cyanobacteria, limiting their biomarker potential. Metagenomes of Acidobacteria were also examined for the presence of genes required for hopanoid biosynthesis. The complete pathway for BHP biosynthesis was evident in SD2 Acidobacteria and a group phylogenetically related to SD1 and SD3, in line with the limited occurrence of BHPs in acidobacterial cultures. PMID:28642737

Absolute Position Encoders With Vertical Image Binning

NASA Technical Reports Server (NTRS)

Leviton, Douglas B.

2005-01-01

Improved optoelectronic patternrecognition encoders that measure rotary and linear 1-dimensional positions at conversion rates (numbers of readings per unit time) exceeding 20 kHz have been invented. Heretofore, optoelectronic pattern-recognition absoluteposition encoders have been limited to conversion rates <15 Hz -- too low for emerging industrial applications in which conversion rates ranging from 1 kHz to as much as 100 kHz are required. The high conversion rates of the improved encoders are made possible, in part, by use of vertically compressible or binnable (as described below) scale patterns in combination with modified readout sequences of the image sensors [charge-coupled devices (CCDs)] used to read the scale patterns. The modified readout sequences and the processing of the images thus read out are amenable to implementation by use of modern, high-speed, ultra-compact microprocessors and digital signal processors or field-programmable gate arrays. This combination of improvements makes it possible to greatly increase conversion rates through substantial reductions in all three components of conversion time: exposure time, image-readout time, and image-processing time.

Genes encoding intrinsic disorder in Eukaryota have high GC content

PubMed Central

Peng, Zhenling; Uversky, Vladimir N.

2016-01-01

ABSTRACT We analyze a correlation between the GC content in genes of 12 eukaryotic species and the level of intrinsic disorder in their corresponding proteins. Comprehensive computational analysis has revealed that the disordered regions in eukaryotes are encoded by the GC-enriched gene regions and that this enrichment is correlated with the amount of disorder and is present across proteins and species characterized by varying amounts of disorder. The GC enrichment is a result of higher rate of amino acid coded by GC-rich codons in the disordered regions. Individual amino acids have the same GC-content profile between different species. Eukaryotic proteins with the disordered regions encoded by the GC-enriched gene segments carry out important biological functions including interactions with RNAs, DNAs, nucleotides, binding of calcium and metal ions, are involved in transcription, transport, cell division and certain signaling pathways, and are localized primarily in nucleus, cytosol and cytoplasm. We also investigate a possible relationship between GC content, intrinsic disorder and protein evolution. Analysis of a devised “age” of amino acids, their disorder-promoting capacity and the GC-enrichment of their codons suggests that the early amino acids are mostly disorder-promoting and their codons are GC-rich while most of late amino acids are mostly order-promoting. PMID:28232902

Identification of Fitness Determinants during Energy-Limited Growth Arrest in Pseudomonas aeruginosa

PubMed Central

Basta, David W.; Bergkessel, Megan

2017-01-01

ABSTRACT Microbial growth arrest can be triggered by diverse factors, one of which is energy limitation due to scarcity of electron donors or acceptors. Genes that govern fitness during energy-limited growth arrest and the extent to which they overlap between different types of energy limitation are poorly defined. In this study, we exploited the fact that Pseudomonas aeruginosa can remain viable over several weeks when limited for organic carbon (pyruvate) as an electron donor or oxygen as an electron acceptor. ATP values were reduced under both types of limitation, yet more severely in the absence of oxygen. Using transposon-insertion sequencing (Tn-seq), we identified fitness determinants in these two energy-limited states. Multiple genes encoding general functions like transcriptional regulation and energy generation were required for fitness during carbon or oxygen limitation, yet many specific genes, and thus specific activities, differed in their relevance between these states. For instance, the global regulator RpoS was required during both types of energy limitation, while other global regulators such as DksA and LasR were required only during carbon or oxygen limitation, respectively. Similarly, certain ribosomal and tRNA modifications were specifically required during oxygen limitation. We validated fitness defects during energy limitation using independently generated mutants of genes detected in our screen. Mutants in distinct functional categories exhibited different fitness dynamics: regulatory genes generally manifested a phenotype early, whereas genes involved in cell wall metabolism were required later. Together, these results provide a new window into how P. aeruginosa survives growth arrest. PMID:29184024

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.

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.

Enhancing the natural folate level in wine using bioengineering and stabilization strategies.

PubMed

Liu, Yazheng; Walkey, Christopher J; Green, Timothy J; van Vuuren, Hennie J J; Kitts, David D

2016-03-01

Folate deficiency is linked to many diseases, some of which may have higher probability in individuals with alcohol-induced alterations in one-carbon metabolism. Our study shows that folate content in commercial wine is not related to white or red varieties, but associated with the yeast that is used to produce the wine. The stability of folate in these wines, once opened for consumption, did not correlate with total phenolic or sulfite content. In addition, we employed yeast bioengineering to fortify wine with folate. We confirmed by overexpression that FOL2 was the key gene encoding the rate-limiting step of folate biosynthesis in wine yeast. In this study, we also show that overexpression of other folate biosynthesis genes, including ABZ1, ABZ2, DFR1, FOL1 and FOL3, had no effect on folate levels in wine. Ensuring stability of the increased natural folate in all wines was achieved by the addition of ascorbate. Copyright © 2015 Elsevier Ltd. All rights reserved.

Putative recombination events and evolutionary history of five economically important viruses of fruit trees based on coat protein-encoding gene sequence analysis.

PubMed

Boulila, Moncef

2010-06-01

To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

The master regulator PhoP coordinates phosphate and nitrogen metabolism, respiration, cell differentiation and antibiotic biosynthesis: comparison in Streptomyces coelicolor and Streptomyces avermitilis.

PubMed

Martín, Juan F; Rodríguez-García, Antonio; Liras, Paloma

2017-05-01

Phosphate limitation is important for production of antibiotics and other secondary metabolites in Streptomyces. Phosphate control is mediated by the two-component system PhoR-PhoP. Following phosphate depletion, PhoP stimulates expression of genes involved in scavenging, transport and mobilization of phosphate, and represses the utilization of nitrogen sources. PhoP reduces expression of genes for aerobic respiration and activates nitrate respiration genes. PhoP activates genes for teichuronic acid formation and reduces expression of genes for phosphate-rich teichoic acid biosynthesis. In Streptomyces coelicolor, PhoP repressed several differentiation and pleiotropic regulatory genes, which affects development and indirectly antibiotic biosynthesis. A new bioinformatics analysis of the putative PhoP-binding sequences in Streptomyces avermitilis was made. Many sequences in S. avermitilis genome showed high weight values and were classified according to the available genetic information. These genes encode phosphate scavenging proteins, phosphate transporters and nitrogen metabolism genes. Among of the genes highlighted in the new studies was aveR, located in the avermectin gene cluster, encoding a LAL-type regulator, and afsS, which is regulated by PhoP and AfsR. The sequence logo for S. avermitilis PHO boxes is similar to that of S. coelicolor, with differences in the weight value for specific nucleotides in the sequence.

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

The tyrosine B10 hydroxyl is crucial for oxygen avidity of Ascaris hemoglobin.

PubMed

Kloek, A P; Yang, J; Mathews, F S; Frieden, C; Goldberg, D E

1994-01-28

The parasitic nematode Ascaris suum has a gene encoding a two-domain hemoglobin with remarkable oxygen avidity. The strong interaction with oxygen is a consequence of a particularly slow oxygen off-rate. The single polypeptide chain consists of two domains, each of which can be expressed separately in Escherichia coli as a globin-like protein exhibiting oxygen binding characteristics comparable with the native molecule. Site-directed mutagenesis was performed on the gene segment encoding domain one. The E7 position, involved in forming a hydrogen bond with the liganded oxygen in vertebrate globins, is a glutamine in both Ascaris domains. Conversion of this residue to leucine or alanine produced a hemoglobin variant with an oxygen off-rate 5- or 60-fold faster than that of unaltered domain one. Replacement of the tyrosine B10 with either phenylalanine or leucine (as found in vertebrate globins) yielded hemoglobin mutants with oxygen off-rates 280- or 570-fold faster, approaching rates found with vertebrate myoglobins. The data suggest that the distal glutamine hydrogen bonds with the liganded oxygen and that the tyrosine B10 hydroxyl contributes an additional hydrogen bond that appears substantially responsible for the extreme oxygen avidity of Ascaris hemoglobin.

Regulatory systems for hypoxia-inducible gene expression in ischemic heart disease gene therapy.

PubMed

Kim, Hyun Ah; Rhim, Taiyoun; Lee, Minhyung

2011-07-18

Ischemic heart diseases are caused by narrowed coronary arteries that decrease the blood supply to the myocardium. In the ischemic myocardium, hypoxia-responsive genes are up-regulated by hypoxia-inducible factor-1 (HIF-1). Gene therapy for ischemic heart diseases uses genes encoding angiogenic growth factors and anti-apoptotic proteins as therapeutic genes. These genes increase blood supply into the myocardium by angiogenesis and protect cardiomyocytes from cell death. However, non-specific expression of these genes in normal tissues may be harmful, since growth factors and anti-apoptotic proteins may induce tumor growth. Therefore, tight gene regulation is required to limit gene expression to ischemic tissues, to avoid unwanted side effects. For this purpose, various gene expression strategies have been developed for ischemic-specific gene expression. Transcriptional, post-transcriptional, and post-translational regulatory strategies have been developed and evaluated in ischemic heart disease animal models. The regulatory systems can limit therapeutic gene expression to ischemic tissues and increase the efficiency of gene therapy. In this review, recent progresses in ischemic-specific gene expression systems are presented, and their applications to ischemic heart diseases are discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

Sequence- and Structure-Based Functional Annotation and Assessment of Metabolic Transporters in Aspergillus oryzae: A Representative Case Study

PubMed Central

Raethong, Nachon; Wong-ekkabut, Jirasak; Laoteng, Kobkul; Vongsangnak, Wanwipa

2016-01-01

Aspergillus oryzae is widely used for the industrial production of enzymes. In A. oryzae metabolism, transporters appear to play crucial roles in controlling the flux of molecules for energy generation, nutrients delivery, and waste elimination in the cell. While the A. oryzae genome sequence is available, transporter annotation remains limited and thus the connectivity of metabolic networks is incomplete. In this study, we developed a metabolic annotation strategy to understand the relationship between the sequence, structure, and function for annotation of A. oryzae metabolic transporters. Sequence-based analysis with manual curation showed that 58 genes of 12,096 total genes in the A. oryzae genome encoded metabolic transporters. Under consensus integrative databases, 55 unambiguous metabolic transporter genes were distributed into channels and pores (7 genes), electrochemical potential-driven transporters (33 genes), and primary active transporters (15 genes). To reveal the transporter functional role, a combination of homology modeling and molecular dynamics simulation was implemented to assess the relationship between sequence to structure and structure to function. As in the energy metabolism of A. oryzae, the H+-ATPase encoded by the AO090005000842 gene was selected as a representative case study of multilevel linkage annotation. Our developed strategy can be used for enhancing metabolic network reconstruction. PMID:27274991

Sequence- and Structure-Based Functional Annotation and Assessment of Metabolic Transporters in Aspergillus oryzae: A Representative Case Study.

PubMed

Raethong, Nachon; Wong-Ekkabut, Jirasak; Laoteng, Kobkul; Vongsangnak, Wanwipa

2016-01-01

Aspergillus oryzae is widely used for the industrial production of enzymes. In A. oryzae metabolism, transporters appear to play crucial roles in controlling the flux of molecules for energy generation, nutrients delivery, and waste elimination in the cell. While the A. oryzae genome sequence is available, transporter annotation remains limited and thus the connectivity of metabolic networks is incomplete. In this study, we developed a metabolic annotation strategy to understand the relationship between the sequence, structure, and function for annotation of A. oryzae metabolic transporters. Sequence-based analysis with manual curation showed that 58 genes of 12,096 total genes in the A. oryzae genome encoded metabolic transporters. Under consensus integrative databases, 55 unambiguous metabolic transporter genes were distributed into channels and pores (7 genes), electrochemical potential-driven transporters (33 genes), and primary active transporters (15 genes). To reveal the transporter functional role, a combination of homology modeling and molecular dynamics simulation was implemented to assess the relationship between sequence to structure and structure to function. As in the energy metabolism of A. oryzae, the H(+)-ATPase encoded by the AO090005000842 gene was selected as a representative case study of multilevel linkage annotation. Our developed strategy can be used for enhancing metabolic network reconstruction.

Molecular Characterization and Expression of a Phytase Gene from the Thermophilic Fungus Thermomyces lanuginosus

PubMed Central

Berka, Randy M.; Rey, Michael W.; Brown, Kimberly M.; Byun, Tony; Klotz, Alan V.

1998-01-01

The phyA gene encoding an extracellular phytase from the thermophilic fungus Thermomyces lanuginosus was cloned and heterologously expressed, and the recombinant gene product was biochemically characterized. The phyA gene encodes a primary translation product (PhyA) of 475 amino acids (aa) which includes a putative signal peptide (23 aa) and propeptide (10 aa). The deduced amino acid sequence of PhyA has limited sequence identity (ca. 47%) with Aspergillus niger phytase. The phyA gene was inserted into an expression vector under transcriptional control of the Fusarium oxysporum trypsin gene promoter and used to transform a Fusarium venenatum recipient strain. The secreted recombinant phytase protein was enzymatically active between pHs 3 and 7.5, with a specific activity of 110 μmol of inorganic phosphate released per min per mg of protein at pH 6 and 37°C. The Thermomyces phytase retained activity at assay temperatures up to 75°C and demonstrated superior catalytic efficiency to any known fungal phytase at 65°C (the temperature optimum). Comparison of this new Thermomyces catalyst with the well-known Aspergillus niger phytase reveals other favorable properties for the enzyme derived from the thermophilic gene donor, including catalytic activity over an expanded pH range. PMID:9797301

Heterogeneous genetic diversity pattern in Plasmodium vivax genes encoding merozoite surface proteins (MSP) -7E, -7F and -7L.

PubMed

Garzón-Ospina, Diego; Forero-Rodríguez, Johanna; Patarroyo, Manuel A

2014-12-13

The msp-7 gene has become differentially expanded in the Plasmodium genus; Plasmodium vivax has the highest copy number of this gene, several of which encode antigenic proteins in merozoites. DNA sequences from thirty-six Colombian clinical isolates from P. vivax (pv) msp-7E, -7F and -7L genes were analysed for characterizing and studying the genetic diversity of these pvmsp-7 members which are expressed during the intra-erythrocyte stage; natural selection signals producing the variation pattern so observed were evaluated. The pvmsp-7E gene was highly polymorphic compared to pvmsp-7F and pvmsp-7L which were seen to have limited genetic diversity; pvmsp-7E polymorphism was seen to have been maintained by different types of positive selection. Even though these copies seemed to be species-specific duplications, a search in the Plasmodium cynomolgi genome (P. vivax sister taxon) showed that both species shared the whole msp-7 repertoire. This led to exploring the long-term effect of natural selection by comparing the orthologous sequences which led to finding signatures for lineage-specific positive selection. The results confirmed that the P. vivax msp-7 family has a heterogeneous genetic diversity pattern; some members are highly conserved whilst others are highly diverse. The results suggested that the 3'-end of these genes encode MSP-7 proteins' functional region whilst the central region of pvmsp-7E has evolved rapidly. The lineage-specific positive selection signals found suggested that mutations occurring in msp-7s genes during host switch may have succeeded in adapting the ancestral P. vivax parasite population to humans.

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

PubMed

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01). Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.

PubMed

Milne, N; Luttik, M A H; Cueto Rojas, H F; Wahl, A; van Maris, A J A; Pronk, J T; Daran, J M

2015-07-01

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

A Novel GDP-d-glucose Phosphorylase Involved in Quality Control of the Nucleoside Diphosphate Sugar Pool in Caenorhabditis elegans and Mammals*

PubMed Central

Adler, Lital N.; Gomez, Tara A.; Clarke, Steven G.; Linster, Carole L.

2011-01-01

The plant VTC2 gene encodes GDP-l-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-d-glucose to GDP and d-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-d-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-d-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-d-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-d-glucose in the C10F3.4 mutant worms, suggesting that the GDP-d-glucose phosphorylase may function to remove GDP-d-glucose formed by GDP-d-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological d-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals. PMID:21507950

A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

PubMed

Adler, Lital N; Gomez, Tara A; Clarke, Steven G; Linster, Carole L

2011-06-17

The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-D-glucose to GDP and D-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-D-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-D-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-D-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-D-glucose in the C10F3.4 mutant worms, suggesting that the GDP-D-glucose phosphorylase may function to remove GDP-D-glucose formed by GDP-D-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological D-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals.

Whole genome sequence analysis of Geitlerinema sp. FC II unveils competitive edge of the strain in marine cultivation system for biofuel production.

PubMed

Batchu, Navish Kumar; Khater, Shradha; Patil, Sonal; Nagle, Vinod; Das, Gautam; Bhadra, Bhaskar; Sapre, Ajit; Dasgupta, Santanu

2018-03-05

A filamentous cyanobacteria, Geitlerinema sp. FC II, was isolated from marine algae culture pond at Reliance Industries Limited (RIL), India. The 6.7 Mb draft genome of FC II encodes for 6697 protein coding genes. Analysis of the whole genome sequence revealed presence of nif gene cluster, supporting its capability to fix atmospheric nitrogen. FC II genome contains two variants of sulfide:quinone oxidoreductases (SQR), which is a crucial elector donor in cyanobacterial metabolic processes. FC II is characterized by the presence of multiple CRISPR- Cas (Clustered Regularly Interspaced Short Palindrome Repeats - CRISPR associated proteins) clusters, multiple variants of genes encoding photosystem reaction centres, biosynthetic gene clusters of alkane, polyketides and non-ribosomal peptides. Presence of these pathways will help FC II in gaining an ecological advantage over other strains for biomass production in large scale cultivation system. Hence, FC II may be used for production of biofuel and other industrially important metabolites. Copyright © 2018 Elsevier Inc. All rights reserved.

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during L-tryptophan production in Escherichia coli.

PubMed

Liu, Lina; Chen, Sheng; Wu, Jing

2017-10-01

Escherichia coli FB-04(pta1), a recombinant L-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (L-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key L-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher L-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, L-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g -1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.

Multiplex cDNA quantification method that facilitates the standardization of gene expression data

PubMed Central

Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

2011-01-01

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

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

A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

NASA Technical Reports Server (NTRS)

Lu, C.; Fedoroff, N.

2000-01-01

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

Recombinant antibodies encoded by IGHV1-69 react with pUL32, a phosphoprotein of cytomegalovirus and B-cell superantigen

PubMed Central

Steininger, Christoph; Widhopf, George F.; Ghia, Emanuela M.; Morello, Christopher S.; Vanura, Katrina; Sanders, Rebecca; Spector, Deborah; Guiney, Don; Jäger, Ulrich

2012-01-01

Leukemia cells from patients with chronic lymphocytic leukemia (CLL) express a highly restricted immunoglobulin heavy variable chain (IGHV) repertoire, suggesting that a limited set of antigens reacts with leukemic cells. Here, we evaluated the reactivity of a panel of different CLL recombinant antibodies (rAbs) encoded by the most commonly expressed IGHV genes with a panel of selected viral and bacterial pathogens. Six different CLL rAbs encoded by IGHV1-69 or IGHV3-21, but not a CLL rAb encoded by IGHV4-39 genes, reacted with a single protein of human cytomegalovirus (CMV). The CMV protein was identified as the large structural phosphoprotein pUL32. In contrast, none of the CLL rAbs bound to any other structure of CMV, adenovirus serotype 2, Salmonella enterica serovar Typhimurium, or of cells used for propagation of these microorganisms. Monoclonal antibodies or humanized rAbs of irrelevant specificity to pUL32 did not react with any of the proteins present in the different lysates. Still, rAbs encoded by a germ line IGHV1-69 51p1 allele from CMV-seropositive and -negative adults also reacted with pUL32. The observed reactivity of multiple different CLL rAbs and natural antibodies from CMV-seronegative adults with pUL32 is consistent with the properties of a superantigen. PMID:22234695

Comparative genomic analysis of Helicobacter pylori from Malaysia identifies three distinct lineages suggestive of differential evolution.

PubMed

Kumar, Narender; Mariappan, Vanitha; Baddam, Ramani; Lankapalli, Aditya K; Shaik, Sabiha; Goh, Khean-Lee; Loke, Mun Fai; Perkins, Tim; Benghezal, Mohammed; Hasnain, Seyed E; Vadivelu, Jamuna; Marshall, Barry J; Ahmed, Niyaz

2015-01-01

The discordant prevalence of Helicobacter pylori and its related diseases, for a long time, fostered certain enigmatic situations observed in the countries of the southern world. Variation in H. pylori infection rates and disease outcomes among different populations in multi-ethnic Malaysia provides a unique opportunity to understand dynamics of host-pathogen interaction and genome evolution. In this study, we extensively analyzed and compared genomes of 27 Malaysian H. pylori isolates and identified three major phylogeographic lineages: hspEastAsia, hpEurope and hpSouthIndia. The analysis of the virulence genes within the core genome, however, revealed a comparable pathogenic potential of the strains. In addition, we identified four genes limited to strains of East-Asian lineage. Our analyses identified a few strain-specific genes encoding restriction modification systems and outlined 311 core genes possibly under differential evolutionary constraints, among the strains representing different ethnic groups. The cagA and vacA genes also showed variations in accordance with the host genetic background of the strains. Moreover, restriction modification genes were found to be significantly enriched in East-Asian strains. An understanding of these variations in the genome content would provide significant insights into various adaptive and host modulation strategies harnessed by H. pylori to effectively persist in a host-specific manner. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Plant rhabdoviruses.

PubMed

Redinbaugh, M G; Hogenhout, S A

2005-01-01

This chapter provides an overview of plant rhabdovirus structure and taxonomy, genome structure, protein function, and insect and plant infection. It is focused on recent research and unique aspects of rhabdovirus biology. Plant rhabdoviruses are transmitted by aphid, leafhopper or planthopper vectors, and the viruses replicate in both their insect and plant hosts. The two plant rhabdovirus genera, Nucleorhabdovirus and Cytorhabdovirus, can be distinguished on the basis of their intracellular site of morphogenesis in plant cells. All plant rhabdoviruses carry analogs of the five core genes: the nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G) and large or polymerase (L). However, compared to vesiculoviruses that are composed of the five core genes, all plant rhabdoviruses encode more than these five genes, at least one of which is inserted between the P and M genes in the rhabdoviral genome. Interestingly, while these extra genes are not similar among plant rhabdoviruses, two encode proteins with similarity to the 30K superfamily of plant virus movement proteins. Analysis of nucleorhabdoviral protein sequences revealed nuclear localization signals for the N, P, M and L proteins, consistent with virus replication and morphogenesis of these viruses in the nucleus. Plant and insect factors that limit virus infection and transmission are discussed.

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.

Fine mapping of Restorer-of-fertility in pepper (Capsicum annuum L.) identified a candidate gene encoding a pentatricopeptide repeat (PPR)-containing protein.

PubMed

Jo, Yeong Deuk; Ha, Yeaseong; Lee, Joung-Ho; Park, Minkyu; Bergsma, Alex C; Choi, Hong-Il; Goritschnig, Sandra; Kloosterman, Bjorn; van Dijk, Peter J; Choi, Doil; Kang, Byoung-Cheorl

2016-10-01

Using fine mapping techniques, the genomic region co-segregating with Restorer - of - fertility ( Rf ) in pepper was delimited to a region of 821 kb in length. A PPR gene in this region, CaPPR6 , was identified as a strong candidate for Rf based on expression pattern and characteristics of encoding sequence. Cytoplasmic-genic male sterility (CGMS) has been used for the efficient production of hybrid seeds in peppers (Capsicum annuum L.). Although the mitochondrial candidate genes that might be responsible for cytoplasmic male sterility (CMS) have been identified, the nuclear Restorer-of-fertility (Rf) gene has not been isolated. To identify the genomic region co-segregating with Rf in pepper, we performed fine mapping using an Rf-segregating population consisting of 1068 F2 individuals, based on BSA-AFLP and a comparative mapping approach. Through six cycles of chromosome walking, the co-segregating region harboring the Rf locus was delimited to be within 821 kb of sequence. Prediction of expressed genes in this region based on transcription analysis revealed four candidate genes. Among these, CaPPR6 encodes a pentatricopeptide repeat (PPR) protein with PPR motifs that are repeated 14 times. Characterization of the CaPPR6 protein sequence, based on alignment with other homologs, showed that CaPPR6 is a typical Rf-like (RFL) gene reported to have undergone diversifying selection during evolution. A marker developed from a sequence near CaPPR6 showed a higher prediction rate of the Rf phenotype than those of previously developed markers when applied to a panel of breeding lines of diverse origin. These results suggest that CaPPR6 is a strong candidate for the Rf gene in pepper.

Trichoderma genes

DOEpatents

Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

2012-06-19

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

The rice blast resistance gene Ptr encodes an atypical protein required for broad spectrum disease resistance

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. We identified a novel, broad-spectrum rice blast R gene, Ptr, encoding a non-NLR protein with four Armadillo repeats. Ptr was originally identified by fast neutron mutagenesis as a ...

Rare variants analysis of cutaneous malignant melanoma genes in Parkinson's disease.

PubMed

Lubbe, S J; Escott-Price, V; Brice, A; Gasser, T; Pittman, A M; Bras, J; Hardy, J; Heutink, P; Wood, N M; Singleton, A B; Grosset, D G; Carroll, C B; Law, M H; Demenais, F; Iles, M M; Bishop, D T; Newton-Bishop, J; Williams, N M; Morris, H R

2016-12-01

A shared genetic susceptibility between cutaneous malignant melanoma (CMM) and Parkinson's disease (PD) has been suggested. We investigated this by assessing the contribution of rare variants in genes involved in CMM to PD risk. We studied rare variation across 29 CMM risk genes using high-quality genotype data in 6875 PD cases and 6065 controls and sought to replicate findings using whole-exome sequencing data from a second independent cohort totaling 1255 PD cases and 473 controls. No statistically significant enrichment of rare variants across all genes, per gene, or for any individual variant was detected in either cohort. There were nonsignificant trends toward different carrier frequencies between PD cases and controls, under different inheritance models, in the following CMM risk genes: BAP1, DCC, ERBB4, KIT, MAPK2, MITF, PTEN, and TP53. The very rare TYR p.V275F variant, which is a pathogenic allele for recessive albinism, was more common in PD cases than controls in 3 independent cohorts. Tyrosinase, encoded by TYR, is the rate-limiting enzyme for the production of neuromelanin, and has a role in the production of dopamine. These results suggest a possible role for another gene in the dopamine-biosynthetic pathway in susceptibility to neurodegenerative Parkinsonism, but further studies in larger PD cohorts are needed to accurately determine the role of these genes/variants in disease pathogenesis. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Modulation by geraniol of gene expression involved in lipid metabolism leading to a reduction of serum-cholesterol and triglyceride levels.

PubMed

Galle, Marianela; Kladniew, Boris Rodenak; Castro, María Agustina; Villegas, Sandra Montero; Lacunza, Ezequiel; Polo, Mónica; de Bravo, Margarita García; Crespo, Rosana

2015-07-15

Geraniol (G) is a natural isoprenoid present in the essential oils of several aromatic plants, with various biochemical and pharmacologic properties. Nevertheless, the mechanisms of action of G on cellular metabolism are largely unknown. We propose that G could be a potential agent for the treatment of hyperlipidemia that could contribute to the prevention of cardiovascular disease. The aim of the present study was to advance our understanding of its mechanism of action on cholesterol and TG metabolism. NIH mice received supplemented diets containing 25, 50, and 75 mmol G/kg chow. After a 3-week treatment, serum total-cholesterol and triglyceride levels were measured by commercial kits and lipid biosynthesis determined by the [(14)C] acetate incorporated into fatty acids plus nonsaponifiable and total hepatic lipids of the mice. The activity of the mRNA encoding HMGCR-the rate-limiting step in cholesterol biosynthesis-along with the enzyme levels and catalysis were assessed by real-time RT-PCR, Western blotting, and HMG-CoA-conversion assays, respectively. In-silico analysis of several genes involved in lipid metabolism and regulated by G in cultured cells was also performed. Finally, the mRNA levels encoded by the genes for the low-density-lipoprotein receptor (LDLR), the sterol-regulatory-element-binding transcription factor (SREBF2), the very-low-density-lipoprotein receptor (VLDLR), and the acetyl-CoA carboxylase (ACACA) were determined by real-time RT-PCR. Plasma total-cholesterol and triglyceride levels plus hepatic fatty-acid, total-lipid, and nonsaponifiable-lipid biosynthesis were significantly reduced by feeding with G. Even though an up-regulation of the mRNA encoding HMGCR occurred in the G treated mouse livers, the protein levels and specific activity of the enzyme were both inhibited. G also enhanced the mRNAs encoding the LDL and VLDL receptors and reduced ACACA mRNA, without altering the transcription of the mRNA encoding the SREBF2. The following mechanisms may have mediated the decrease in plasma lipids levels in mice: a down-regulation of hepatocyte-cholesterol synthesis occurred as a result of decreased HMGCR protein levels and catalytic activity; the levels of LDLR mRNA became elevated, thus suggesting an increase in the uptake of serum LDL, especially by the liver; and TG synthesis became reduced very likely because of a decrease in fatty-acid synthesis. Copyright © 2015 Elsevier GmbH. All rights reserved.

Phosphate transporters in marine phytoplankton and their viruses: cross-domain commonalities in viral-host gene exchanges.

PubMed

Monier, Adam; Welsh, Rory M; Gentemann, Chelle; Weinstock, George; Sodergren, Erica; Armbrust, E Virginia; Eisen, Jonathan A; Worden, Alexandra Z

2012-01-01

Phosphate (PO(4)) is an important limiting nutrient in marine environments. Marine cyanobacteria scavenge PO(4) using the high-affinity periplasmic phosphate binding protein PstS. The pstS gene has recently been identified in genomes of cyanobacterial viruses as well. Here, we analyse genes encoding transporters in genomes from viruses that infect eukaryotic phytoplankton. We identified inorganic PO(4) transporter-encoding genes from the PHO4 superfamily in several virus genomes, along with other transporter-encoding genes. Homologues of the viral pho4 genes were also identified in genome sequences from the genera that these viruses infect. Genome sequences were available from host genera of all the phytoplankton viruses analysed except the host genus Bathycoccus. Pho4 was recovered from Bathycoccus by sequencing a targeted metagenome from an uncultured Atlantic Ocean population. Phylogenetic reconstruction showed that pho4 genes from pelagophytes, haptophytes and infecting viruses were more closely related to homologues in prasinophytes than to those in what, at the species level, are considered to be closer relatives (e.g. diatoms). We also identified PHO4 superfamily members in ocean metagenomes, including new metagenomes from the Pacific Ocean. The environmental sequences grouped with pelagophytes, haptophytes, prasinophytes and viruses as well as bacteria. The analyses suggest that multiple independent pho4 gene transfer events have occurred between marine viruses and both eukaryotic and bacterial hosts. Additionally, pho4 genes were identified in available genomes from viruses that infect marine eukaryotes but not those that infect terrestrial hosts. Commonalities in marine host-virus gene exchanges indicate that manipulation of host-PO(4) uptake is an important adaptation for viral proliferation in marine systems. Our findings suggest that PO(4) -availability may not serve as a simple bottom-up control of marine phytoplankton. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Enhancement of  l-phenylalanine production in Escherichia coli by heterologous expression of Vitreoscilla hemoglobin.

PubMed

Wu, Wei-Bin; Guo, Xiao-Lei; Zhang, Ming-Liang; Huang, Qing-Gen; Qi, Feng; Huang, Jian-Zhong

2018-05-01

l-Phenylalanine is an important amino acid that is widely used in the production of food flavors and pharmaceuticals. Generally, l-phenylalanine production by engineered Escherichia coli requires a high rate of oxygen supply. However, the coexpression of Vitreoscilla hemoglobin gene (vgb), driven bya tac promoter, with the genes encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase (aroF) and feedback-resistant chorismate mutase/prephenate dehydratase (pheA fbr ), led to increased productivity and decreased demand for aeration by E. coli CICC10245. Shake-flask studies showed that vgb-expressing strains displayed higher rates of oxygen uptake, and l-phenylalanine production under standard aeration conditions was increased. In the aerobic fermentation process, cell growth, l-phenylalanine production, and glucose consumption by the recombinant E. coli strain PAPV, which harbored aroF, pheA fbr , and tac-vgb genes, were increased compared to that in the strain harboring only aroF and pheA fbr (E. coli strain PAP), especially under oxygen-limited conditions. The vgb-expressing strain PAPV produced 21.9% more biomass and 16.6% more l-phenylalanine, while consuming only approximately 5% more glucose after 48 H of fermentation. This study demonstrates a method to enhance the l-phenylalanine production by E. coli using less intensive and thus more economical aeration conditions. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Proteobactin and a yersiniabactin-related siderophore mediate iron acquisition in Proteus mirabilis

PubMed Central

Himpsl, Stephanie D.; Pearson, Melanie M.; Arewång, Carl J.; Nusca, Tyler D.; Sherman, David H.; Mobley, Harry L. T.

2010-01-01

Proteus mirabilis causes complicated urinary tract infections (UTI). While the urinary tract is an iron-limiting environment, iron acquisition remains poorly characterized for this uropathogen. Microarray analysis of P. mirabilis HI4320 cultured under iron limitation identified 45 significantly up-regulated genes (P ≤ 0.05) that represent 21 putative iron-regulated systems. Two gene clusters, PMI0229-0239 and PMI2596–2605, encode putative siderophore systems. PMI0229-0239 encodes a nonribosomal peptide synthetase (NRPS)-independent siderophore (NIS) system for producing a novel siderophore, proteobactin. PMI2596-2605 are contained within the high-pathogenicity island, originally described in Yersinia pestis, and encodes proteins with apparent homology and organization to those involved in yersiniabactin production and uptake. Cross-feeding and biochemical analysis shows that P. mirabilis is unable to utilize or produce yersiniabactin, suggesting that this yersiniabactin-related locus is functionally distinct. Only disruption of both systems resulted in an in vitro iron-chelating defect; demonstrating production and iron-chelating activity for both siderophores. These findings clearly show that proteobactin and the yersiniabactin-related siderophore function as iron acquisition systems. Despite the activity of both siderophores, only mutants lacking the yersiniabactin-related siderophore reduce fitness in vivo. The fitness requirement for the yersiniabactin-related siderophore during UTI shows, for the first time, the importance of siderophore production in vivo for P. mirabilis. PMID:20923418

l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

PubMed Central

2014-01-01

The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C. PMID:24443973

Aldouronate Utilization in Paenibacillus sp. Strain JDR-2: Physiological and Enzymatic Evidence for Coupling of Extracellular Depolymerization and Intracellular Metabolism ▿

PubMed Central

Nong, Guang; Rice, John D.; Chow, Virginia; Preston, James F.

2009-01-01

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 α-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/β-xylosidase (XynB). Here we show that this Paenibacillus sp. strain is able to utilize methylglucuronoxylose (MeGAX1), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX3), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAXn). The average rates of utilization of MeGAXn, MeGAX1, and MeGAX3 were 149.8, 59.4, and 54.3 μg xylose equivalents·ml−1·h−1, respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX1 and MeGAX3, releasing 4-O-methyl-d-glucuronate α-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX3, to xylose and xylobiose. The ability to utilize MeGAX1 provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAXn than that of MeGAX3 indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the aguA and xynA2 genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals. PMID:19395566

Selenium Pretreatment Alleviated LPS-Induced Immunological Stress Via Upregulation of Several Selenoprotein Encoding Genes in Murine RAW264.7 Cells.

PubMed

Wang, Longqiong; Jing, Jinzhong; Yan, Hui; Tang, Jiayong; Jia, Gang; Liu, Guangmang; Chen, Xiaoling; Tian, Gang; Cai, Jingyi; Shang, Haiying; Zhao, Hua

2018-04-18

This study was conducted to profile selenoprotein encoding genes in mouse RAW264.7 cells upon lipopolysaccharide (LPS) challenge and integrate their roles into immunological regulation in response to selenium (Se) pretreatment. LPS was used to develop immunological stress in macrophages. Cells were pretreated with different levels of Se (0, 0.5, 1.0, 1.5, 2.0 μmol Se/L) for 2 h, followed by LPS (100 ng/mL) stimulation for another 3 h. The mRNA expression of 24 selenoprotein encoding genes and 9 inflammation-related genes were investigated. The results showed that LPS (100 ng/mL) effectively induced immunological stress in RAW264.7 cells with induced inflammation cytokines, IL-6 and TNF-α, mRNA expression, and cellular secretion. LPS increased (P < 0.05) mRNA profiles of 9 inflammation-related genes in cells, while short-time Se pretreatment modestly reversed (P < 0.05) the LPS-induced upregulation of 7 genes (COX-2, ICAM-1, IL-1β, IL-6, IL-10, iNOS, and MCP-1) and further increased (P < 0.05) expression of IFN-β and TNF-α in stressed cells. Meanwhile, LPS decreased (P < 0.05) mRNA levels of 18 selenoprotein encoding genes and upregulated mRNA levels of TXNRD1 and TXNRD3 in cells. Se pretreatment recovered (P < 0.05) expression of 3 selenoprotein encoding genes (GPX1, SELENOH, and SELENOW) in a dose-dependent manner and increased (P < 0.05) expression of another 5 selenoprotein encoding genes (SELENOK, SELENOM, SELENOS, SELENOT, and TXNRD2) only at a high level (2.0 μmol Se/L). Taken together, LPS-induced immunological stress in RAW264.7 cells accompanied with the global downregulation of selenoprotein encoding genes and Se pretreatment alleviated immunological stress via upregulation of a subset of selenoprotein encoding genes.

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

Generation of Trichoderma atroviride mutants with constitutively activated G protein signaling by using geneticin resistance as selection marker.

PubMed

Gruber, Sabine; Omann, Markus; Rodrìguez, Carolina Escobar; Radebner, Theresa; Zeilinger, Susanne

2012-11-17

Species of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, but also widely used as biocontrol agents (BCAs) in agriculture. In the latter function Trichoderma species stimulate plant growth, induce plant defense and directly antagonize plant pathogenic fungi through their mycoparasitic capabilities. The recent release of the genome sequences of four mycoparasitic Trichoderma species now forms the basis for large-scale genetic manipulations of these important BCAs. Thus far, only a limited number of dominant selection markers, including Hygromycin B resistance (hph) and the acetamidase-encoding amdS gene, have been available for transformation of Trichoderma spp. For more extensive functional genomics studies the utilization of additional dominant markers will be essential. We established the Escherichia coli neomycin phosphotransferase II-encoding nptII gene as a novel selectable marker for the transformation of Trichoderma atroviride conferring geneticin resistance. The nptII marker cassette was stably integrated into the fungal genome and transformants exhibited unaltered phenotypes compared to the wild-type. Co-transformation of T. atroviride with nptII and a constitutively activated version of the Gα subunit-encoding tga3 gene (tga3Q207L) resulted in a high number of mitotically stable, geneticin-resistant transformants. Further analyses revealed a co-transformation frequency of 68% with 15 transformants having additionally integrated tga3Q207L into their genome. Constitutive activation of the Tga3-mediated signaling pathway resulted in increased vegetative growth and an enhanced ability to antagonize plant pathogenic host fungi. The neomycin phosphotransferase II-encoding nptII gene from Escherichia coli proved to be a valuable tool for conferring geneticin resistance to the filamentous fungus T. atroviride thereby contributing to an enhanced genetic tractability of these important BCAs.

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.

Clostridium perfringens type A–E toxin plasmids

PubMed Central

Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

2014-01-01

Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

A hitchhiker's guide to the MADS world of plants.

PubMed

Gramzow, Lydia; Theissen, Guenter

2010-01-01

Plant life critically depends on the function of MADS-box genes encoding MADS-domain transcription factors, which are present to a limited extent in nearly all major eukaryotic groups, but constitute a large gene family in land plants. There are two types of MADS-box genes, termed type I and type II, and in plants these groups are distinguished by exon-intron and domain structure, rates of evolution, developmental function and degree of functional redundancy. The type I genes are further subdivided into three groups - M alpha, M beta and M gamma - while the type II genes are subdivided into the MIKCC and MIKC* groups. The functional diversification of MIKCC genes is closely linked to the origin of developmental and morphological novelties in the sporophytic (usually diploid) generation of seed plants, most spectacularly the floral organs and fruits of angiosperms. Functional studies suggest different specializations for the different classes of genes; whereas type I genes may preferentially contribute to female gametophyte, embryo and seed development and MIKC*-group genes to male gametophyte development, the MIKCC-group genes became essential for diverse aspects of sporophyte development. Beyond the usual transcriptional regulation, including feedback and feed-forward loops, various specialized mechanisms have evolved to control the expression of MADS-box genes, such as epigenetic control and regulation by small RNAs. In future, more data from genome projects and reverse genetic studies will allow us to understand the birth, functional diversification and death of members of this dynamic and important family of transcription factors in much more detail.

PCR screening of an African fermented pearl-millet porridge metagenome to investigate the nutritional potential of its microbiota.

PubMed

Saubade, Fabien; Humblot, Christèle; Hemery, Youna M; Guyot, Jean-Pierre

2017-03-06

Cereals are staple foods in most African countries, and many African cereal-based foods are spontaneously fermented. The nutritional quality of cereal products can be enhanced through fermentation, and traditional cereal-based fermented foods (CBFFs) are possible sources of lactic acid bacteria (LAB) with useful nutritional properties. The nutritional properties of LAB vary depending on the species and even on the strain, and the microbial composition of traditional CBFFs varies from one traditional production unit (TPU) to another. The nutritional quality of traditional CBFFs may thus vary depending on their microbial composition. As the isolation of potentially useful LAB from traditional CBFFs can be very time consuming, the aim of this study was to use PCR to assess the nutritional potential of LAB directly on the metagenomes of pearl-millet based fermented porridges (ben-saalga) from Burkina Faso. Genes encoding enzymes involved in different nutritional activities were screened in 50 metagenomes extracted from samples collected in 10 TPUs in Ouagadougou. The variability of the genetic potential was recorded. Certain genes were never detected in the metagenomes (genes involved in carotenoid synthesis) while others were frequently detected (genes involved in folate and riboflavin production, starch hydrolysis, polyphenol degradation). Highly variable microbial composition - assessed by real-time PCR - was observed among samples collected in different TPUs, but also among samples from the same TPU. The high frequency of the presence of genes did not necessarily correlate with in situ measurements of the expected products. Indeed, no significant correlation was found between the microbial variability and the variability of the genetic potential. In spite of the high rate of detection (80%) of both genes folP and folK, encoding enzymes involved in folate synthesis, the folate content in ben-saalga was rather low (median: 0.5μg/100g fresh weight basis). This work highlighted the limit of evaluating the nutritional potential of the microbiota of traditional fermented foods by the only screening of genes in metagenomes, and suggests that such a screening should be completed by a functional analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

A DEMETER-like DNA demethylase governs tomato fruit ripening.

PubMed

Liu, Ruie; How-Kit, Alexandre; Stammitti, Linda; Teyssier, Emeline; Rolin, Dominique; Mortain-Bertrand, Anne; Halle, Stefanie; Liu, Mingchun; Kong, Junhua; Wu, Chaoqun; Degraeve-Guibault, Charlotte; Chapman, Natalie H; Maucourt, Mickael; Hodgman, T Charlie; Tost, Jörg; Bouzayen, Mondher; Hong, Yiguo; Seymour, Graham B; Giovannoni, James J; Gallusci, Philippe

2015-08-25

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening- an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

PubMed Central

Lozano, Roberto; Ponce, Olga; Ramirez, Manuel; Mostajo, Nelly; Orjeda, Gisella

2012-01-01

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes. PMID:22493716

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.

Selective survival of peripheral blood lymphocytes in children with HIV-1 following delivery of an anti-HIV gene to bone marrow CD34(+) cells.

PubMed

Podsakoff, Greg M; Engel, Barbara C; Carbonaro, Denise A; Choi, Chris; Smogorzewska, Elzbieta M; Bauer, Gerhard; Selander, David; Csik, Susan; Wilson, Kathy; Betts, Michael R; Koup, Richard A; Nabel, Gary J; Bishop, Keith; King, Steven; Schmidt, Manfred; von Kalle, Christof; Church, Joseph A; Kohn, Donald B

2005-07-01

Two HIV-1-infected children on antiretroviral therapy were enrolled into a clinical study of retroviral-mediated transfer of a gene that inhibits replication of HIV-1, targeting bone marrow CD34+ hematopoietic stem/progenitor cells. Two retroviral vectors were used, one encoding a "humanized" dominant-negative REV protein (huM10) that is a potent inhibitor of HIV-1 replication and one encoding a nontranslated marker gene (FX) to serve as an internal control for the level of gene marking. Peripheral blood mononuclear cells (PBMC) containing the huM10 gene or FX gene were detected by quantitative PCR at frequencies of approximately 1/10,000 in both subjects for the first 1-3 months following re-infusion of the gene-transduced bone marrow, but then were at or below the limits of detection (<1/1,000,000) at most times over 2 years. In one patient, a reappearance of PBMC containing the huM10 gene, but not the FX gene, occurred concomitant with a rise in the HIV-1 viral load during a period of nonadherence to the antiretroviral regimen. Unique clones of gene-marked PBMC were detected by LAM-PCR during the time of elevated HIV-1 levels. These findings indicate that there was a selective survival advantage for PBMC containing the huM10 gene during the time of increased HIV-1 load.

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.

Click train encoding in primary and non-primary auditory cortex of anesthetized macaque monkeys.

PubMed

Oshurkova, E; Scheich, H; Brosch, M

2008-06-02

We studied encoding of temporally modulated sounds in 28 multiunits in the primary auditory cortical field (AI) and in 35 multiunits in the secondary auditory cortical field (caudomedial auditory cortical field, CM) by presenting periodic click trains with click rates between 1 and 300 Hz lasting for 2-4 s. We found that all multiunits increased or decreased their firing rate during the steady state portion of the click train and that all except two multiunits synchronized their firing to individual clicks in the train. Rate increases and synchronized responses were most prevalent and strongest at low click rates, as expressed by best modulation frequency, limiting frequency, percentage of responsive multiunits, and average rate response and vector strength. Synchronized responses occurred up to 100 Hz; rate response occurred up to 300 Hz. Both auditory fields responded similarly to low click rates but differed at click rates above approximately 12 Hz at which more multiunits in AI than in CM exhibited synchronized responses and increased rate responses and more multiunits in CM exhibited decreased rate responses. These findings suggest that the auditory cortex of macaque monkeys encodes temporally modulated sounds similar to the auditory cortex of other mammals. Together with other observations presented in this and other reports, our findings also suggest that AI and CM have largely overlapping sensitivities for acoustic stimulus features but encode these features differently.

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

Export of l-Isoleucine from Corynebacterium glutamicum: a Two-Gene-Encoded Member of a New Translocator Family

PubMed Central

Kennerknecht, Nicole; Sahm, Hermann; Yen, Ming-Ren; Pátek, Miroslav; Saier, Jr., Milton H.; Eggeling, Lothar

2002-01-01

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes l-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for l-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports l-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of l-leucine and l-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in α-proteobacteria but not in other prokaryotes analyzed. PMID:12081967

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.

Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins.

PubMed

Sequeira, Ana Filipa; Brás, Joana L A; Guerreiro, Catarina I P D; Vincentelli, Renaud; Fontes, Carlos M G A

2016-12-01

Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins. An automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.

Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families.

PubMed

Shah, Shiraz A; Alkhnbashi, Omer S; Behler, Juliane; Han, Wenyuan; She, Qunxin; Hess, Wolfgang R; Garrett, Roger A; Backofen, Rolf

2018-06-19

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

Spatiotemporal encoding of search strategies by prefrontal neurons.

PubMed

Chiang, Feng-Kuei; Wallis, Joni D

2018-05-08

Working memory is capacity-limited. In everyday life we rarely notice this limitation, in part because we develop behavioral strategies that help mitigate the capacity limitation. How behavioral strategies are mediated at the neural level is unclear, but a likely locus is lateral prefrontal cortex (LPFC). Neurons in LPFC play a prominent role in working memory and have been shown to encode behavioral strategies. To examine the role of LPFC in overcoming working-memory limitations, we recorded the activity of LPFC neurons in animals trained to perform a serial self-ordered search task. This task measured the ability to prospectively plan the selection of unchosen spatial search targets while retrospectively tracking which targets were previously visited. We found that individual LPFC neurons encoded the spatial location of the current search target but also encoded the spatial location of targets up to several steps away in the search sequence. Neurons were more likely to encode prospective than retrospective targets. When subjects used a behavioral strategy of stereotyped target selection, mitigating the working-memory requirements of the task, not only did the number of selection errors decrease but there was a significant reduction in the strength of spatial encoding in LFPC. These results show that LPFC neurons have spatiotemporal mnemonic fields, in that their firing rates are modulated both by the spatial location of future selection behaviors and the temporal organization of that behavior. Furthermore, the strength of this tuning can be dynamically modulated by the demands of the task.

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

Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism

PubMed Central

Marques, Wesley Leoricy; Mans, Robert; Marella, Eko Roy; Cordeiro, Rosa Lorizolla; van den Broek, Marcel; Daran, Jean-Marc G.; Pronk, Jack T.; Gombert, Andreas K.; van Maris, Antonius J.A.

2017-01-01

Abstract Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose-negative strains. A strain in which all known sucrose-transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose-hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h−1 after sequential batch cultivation. This increase was accompanied by a 3-fold increase of in vitro sucrose-hydrolysis and isomaltase activities, as well as by a 3- to 5-fold upregulation of the isomaltase-encoding genes IMA1 and IMA5. One-step Cas9-mediated deletion of all isomaltase-encoding genes (IMA1-5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose-negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport. PMID:28087672

A two-component signal transduction pathway regulates manganese homeostasis in Synechocystis 6803, a photosynthetic organism.

PubMed

Ogawa, Teruo; Bao, Ding Hui; Katoh, Hirokazu; Shibata, Mari; Pakrasi, Himadri B; Bhattacharyya-Pakrasi, Maitrayee

2002-08-09

Elemental manganese is essential for the production of molecular oxygen by cyanobacteria, plants, and algae. In the cyanobacterium Synechocystis sp. PCC 6803, transcription of the mntCAB operon, encoding a high affinity Mn transporter, occurs under Mn starvation (nm Mn) conditions but not in Mn-sufficient (microm Mn) growth medium. Using a strain in which the promoter of this operon directs the transcription of the luxAB reporter genes, we determined that inactivation of the slr0640 gene, which encodes a histidine kinase sensor protein component of a two-component signal transduction system, resulted in constitutive high levels of lux luminescence. Systematic targeted inactivation mutagenesis also identified slr1837 as the gene encoding the corresponding response regulator protein. We have named these two genes manS (manganese-sensor) and manR (manganese-regulator), respectively. A polyhistidine-tagged form of the ManS protein was localized in the Synechocystis 6803 cell membrane. Directed replacement of the conserved catalytic His-205 residue of this protein by Leu abolished its activity, although the mutated protein was present in cyanobacterial membrane. This mutant also showed suboptimal rates of Mn uptake under either Mn-starved or Mn-sufficient growth condition. These data suggest that the ManS/ManR two-component system plays a central role in the homeostasis of manganese in Synechocystis 6803 cells.

Automatic semantic encoding in verbal short-term memory: evidence from the concreteness effect.

PubMed

Campoy, Guillermo; Castellà, Judit; Provencio, Violeta; Hitch, Graham J; Baddeley, Alan D

2015-01-01

The concreteness effect in verbal short-term memory (STM) tasks is assumed to be a consequence of semantic encoding in STM, with immediate recall of concrete words benefiting from richer semantic representations. We used the concreteness effect to test the hypothesis that semantic encoding in standard verbal STM tasks is a consequence of controlled, attention-demanding mechanisms of strategic semantic retrieval and encoding. Experiment 1 analysed the effect of presentation rate, with slow presentations being assumed to benefit strategic, time-dependent semantic encoding. Experiments 2 and 3 provided a more direct test of the strategic hypothesis by introducing three different concurrent attention-demanding tasks. Although Experiment 1 showed a larger concreteness effect with slow presentations, the following two experiments yielded strong evidence against the strategic hypothesis. Limiting available attention resources by concurrent tasks reduced global memory performance, but the concreteness effect was equivalent to that found in control conditions. We conclude that semantic effects in STM result from automatic semantic encoding and provide tentative explanations for the interaction between the concreteness effect and the presentation rate.

Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp

PubMed Central

Han, Jee Eun; Tang, Kathy F. J.; Tran, Loc H.; Lightner, Donald V.

2016-01-01

The 69 kb plasmid pVPA3-1 was identified in Vibrio parahaemolyticus strain 13-028/A3 that can cause acute hepatopancreatic necrosis disease (AHPND). This disease is responsible for mass mortalities in farmed penaeid shrimp and is referred to as early mortality syndrome (EMS). The plasmid has a GC content of 45.9% with a copy number of 37 per bacterial cell as determined by comparative quantitative PCR analyses. It consists of 92 open reading frames that encode mobilization proteins, replication enzymes, transposases, virulence-associated proteins, and proteins similar to Photorhabdus insect-related (Pir) toxins. In V. parahaemolyticus, these Pir toxin-like proteins are encoded by 2 genes ( pirA- and pirB-like) located within a 3.5 kb fragment flanked with inverted repeats of a transposase-coding sequence (1 kb). The GC content of these 2 genes is only 38.2%, substantially lower than that of the rest of the plasmid, which suggests that these genes were recently acquired. Based on a proteomic analysis, the pirA-like (336 bp) and pirB-like (1317 bp) genes encode for 13 and 50 kDa proteins, respectively. In laboratory cultures of V. parahaemolyticus 13-028/A3, both proteins were secreted into the culture medium. We developed a duplex PCR diagnostic method, with a detection limit of 105 CFU ml−1 and targeting pirA- and pirB-like genes in this strain of V. parahaemolyticus. This PCR protocol can reliably detect AHPND-causing strains of V. parahaemolyticus and does not cross react with non-pathogenic strains or with other species of Vibrio isolated from shrimp ponds. PMID:25667334

Modularity of Plant Metabolic Gene Clusters: A Trio of Linked Genes That Are Collectively Required for Acylation of Triterpenes in Oat[W][OA

PubMed Central

Mugford, Sam T.; Louveau, Thomas; Melton, Rachel; Qi, Xiaoquan; Bakht, Saleha; Hill, Lionel; Tsurushima, Tetsu; Honkanen, Suvi; Rosser, Susan J.; Lomonossoff, George P.; Osbourn, Anne

2013-01-01

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis. PMID:23532069

Boron-toxicity tolerance in barley arising from efflux transporter amplification.

PubMed

Sutton, Tim; Baumann, Ute; Hayes, Julie; Collins, Nicholas C; Shi, Bu-Jun; Schnurbusch, Thorsten; Hay, Alison; Mayo, Gwenda; Pallotta, Margaret; Tester, Mark; Langridge, Peter

2007-11-30

Both limiting and toxic soil concentrations of the essential micronutrient boron represent major limitations to crop production worldwide. We identified Bot1, a BOR1 ortholog, as the gene responsible for the superior boron-toxicity tolerance of the Algerian barley landrace Sahara 3771 (Sahara). Bot1 was located at the tolerance locus by high-resolution mapping. Compared to intolerant genotypes, Sahara contains about four times as many Bot1 gene copies, produces substantially more Bot1 transcript, and encodes a Bot1 protein with a higher capacity to provide tolerance in yeast. Bot1 transcript levels identified in barley tissues are consistent with a role in limiting the net entry of boron into the root and in the disposal of boron from leaves via hydathode guttation.

GTP cyclohydrolase I gene polymorphisms are associated with endothelial dysfunction and oxidative stress in patients with type 2 diabetes mellitus.

PubMed

Wolkow, Pawel P; Kosiniak-Kamysz, Wladyslaw; Osmenda, Grzegorz; Wilk, Grzegorz; Bujak-Gizycka, Beata; Ignacak, Adam; Kanitkar, Mihir; Walus-Miarka, Malgorzata; Harrison, David G; Korbut, Ryszard; Malecki, Maciej T; Guzik, Tomasz J

2014-01-01

The genetic background of atherosclerosis in type 2 diabetes mellitus (T2DM) is complex and poorly understood. Studying genetic components of intermediate phenotypes, such as endothelial dysfunction and oxidative stress, may aid in identifying novel genetic components for atherosclerosis in diabetic patients. Five polymorphisms forming two haplotype blocks within the GTP cyclohydrolase 1 gene, encoding a rate limiting enzyme in tetrahydrobiopterin synthesis, were studied in the context of flow and nitroglycerin mediated dilation (FMD and NMD), intima-media thickness (IMT), and plasma concentrations of von Willebrand factor (vWF) and malondialdehyde (MDA). Rs841 was associated with FMD (p = 0.01), while polymorphisms Rs10483639, Rs841, Rs3783641 (which form a single haplotype) were associated with both MDA (p = 0.012, p = 0.0015 and p = 0.003, respectively) and vWF concentrations (p = 0.016, p = 0.03 and p = 0.045, respectively). In addition, polymorphism Rs8007267 was also associated with MDA (p = 0.006). Haplotype analysis confirmed the association of both haplotypes with studied variables. Genetic variation of the GCH1 gene is associated with endothelial dysfunction and oxidative stress in T2DM patients.

Modulations in primary and secondary metabolic pathways and adjustment in physiological behaviour of Withania somnifera under drought stress.

PubMed

Singh, Ruchi; Gupta, Pankhuri; Khan, Furqan; Singh, Susheel Kumar; Sanchita; Mishra, Tripti; Kumar, Anil; Dhawan, Sunita Singh; Shirke, Pramod Arvind

2018-07-01

In general medicinal plants grown under water limiting conditions show much higher concentrations of secondary metabolites in comparison to control plants. In the present study, Withania somnifera plants were subjected to water stress and data related to drought tolerance phenomenon was collected and a putative mechanistic concept considering growth responses, physiological behaviour, and metabolite content and gene expression aspects is presented. Drought induced metabolic and physiological responses as well as drastic decrease in CO 2 uptake due to stomatal limitations. As a result, the consumption of reduction equivalents (NADPH 2+ ) for CO 2 assimilation via the calvin cycle declines significantly resulting in the generation of a large oxidative stress and an oversupply of antioxidant enzymes. Drought also results in the shifting of metabolic processes towards biosynthetic activities that consume reduction equivalents. Thus, biosynthesis of reduced compounds (isoprenoids, phenols and alkaloids) is enhanced. The dynamics of various metabolites have been discussed in the light of gene expression analysis of control and drought treated leaves. Gene encoding enzymes of pathways leading to glucose, fructose and fructan production, conversion of triose phosphates to hexoses and hexose phosphorylation were up-regulated in the drought stressed leaves. The down-regulated Calvin cycle genes were co-ordinately regulated with the down-regulation of chloroplast triosephosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Expression of gene encoding Squalene Synthase (SQS) was highly upregulated under drought stress which is responsible for the diversion of carbon flux towards withanolides biosynthesis from isoprenoid pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins of Escherichia coli.

PubMed Central

Nishibuchi, M; Murakami, A; Arita, M; Jikuya, H; Takano, J; Honda, T; Miwatani, T

1989-01-01

We examined variations in the genes encoding heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) in 88 strains of Escherichia coli isolated from individuals with traveler's diarrhea to find suitable sequences for use as oligonucleotide probes. Four oligonucleotide probes of the gene encoding ST of human origin (STIb or STh), one oligonucleotide probe of the gene encoding ST of porcine origin (STIa or STp), and three oligonucleotide probes of the gene encoding LT of human origin (LTIh) were used in DNA colony hybridization tests. In 15 of 22 strains possessing the STh gene and 28 of 42 strains producing LT, the sequences of all regions tested were identical to the published sequences. One region in the STh gene examined with a 18-mer probe was relatively well conserved and was shown to be closely associated with the enterotoxicity of the E. coli strains in suckling mice. This oligonucleotide, however, hybridized with strains of Vibrio cholerae O1, V. parahaemolyticus, and Yersinia enterocolitica that gave negative results in the suckling mouse assay. PMID:2685027

Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens

DOE PAGES

Chan, Chi Ho; Levar, Caleb E.; Zacharoff, Lori; ...

2015-08-07

Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellarmore » regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented Δ imcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. Lastly, these tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.« less

Plasmid-Mediated Bioaugmentation for the Bioremediation of Contaminated Soils

PubMed Central

Garbisu, Carlos; Garaiyurrebaso, Olatz; Epelde, Lur; Grohmann, Elisabeth; Alkorta, Itziar

2017-01-01

Bioaugmentation, or the inoculation of microorganisms (e.g., bacteria harboring the required catabolic genes) into soil to enhance the rate of contaminant degradation, has great potential for the bioremediation of soils contaminated with organic compounds. Regrettably, cell bioaugmentation frequently turns into an unsuccessful initiative, owing to the rapid decrease of bacterial viability and abundance after inoculation, as well as the limited dispersal of the inoculated bacteria in the soil matrix. Genes that encode the degradation of organic compounds are often located on plasmids and, consequently, they can be spread by horizontal gene transfer into well-established, ecologically competitive, indigenous bacterial populations. Plasmid-mediated bioaugmentation aims to stimulate the spread of contaminant degradation genes among indigenous soil bacteria by the introduction of plasmids, located in donor cells, harboring such genes. But the acquisition of plasmids by recipient cells can affect the host’s fitness, a crucial aspect for the success of plasmid-mediated bioaugmentation. Besides, environmental factors (e.g., soil moisture, temperature, organic matter content) can play important roles for the transfer efficiency of catabolic plasmids, the expression of horizontally acquired genes and, finally, the contaminant degradation activity. For plasmid-mediated bioaugmentation to be reproducible, much more research is needed for a better selection of donor bacterial strains and accompanying plasmids, together with an in-depth understanding of indigenous soil bacterial populations and the environmental conditions that affect plasmid acquisition and the expression and functioning of the catabolic genes of interest. PMID:29062312

Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori.

PubMed

Wang, Pingyang; Qiu, Zhiyong; Xia, Dingguo; Tang, Shunming; Shen, Xingjia; Zhao, Qiaoling

2017-01-01

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

Cyclic stretch-induced the cytoskeleton rearrangement and gene expression of cytoskeletal regulators in human periodontal ligament cells.

PubMed

Wu, Yaqin; Zhuang, Jiabao; Zhao, Dan; Zhang, Fuqiang; Ma, Jiayin; Xu, Chun

2017-10-01

This study aimed to explore the mechanism of the stretch-induced cell realignment and cytoskeletal rearrangement by identifying several mechanoresponsive genes related to cytoskeletal regulators in human PDL cells. After the cells were stretched by 1, 10 and 20% strains for 0.5, 1, 2, 4, 6, 12 or 24 h, the changes of the morphology and content of microfilaments were recorded and calculated. Meanwhile, the expression of 84 key genes encoding cytoskeletal regulators after 6 and 24 h stretches with 20% strain was detected by using real-time PCR array. Western blot was applied to identify the protein expression level of several cytoskeletal regulators encoded by these differentially expressed genes. The confocal fluorescent staining results confirmed that stretch-induced realignment of cells and rearrangement of microfilaments. Among the 84 genes screened, one gene was up-regulated while two genes were down-regulated after 6 h stretch. Meanwhile, three genes were up-regulated while two genes were down-regulated after 24 h stretch. These genes displaying differential expression included genes regulating polymerization/depolymerization of microfilaments (CDC42EP2, FNBP1L, NCK2, PIKFYVE, WASL), polymerization/depolymerization of microtubules (STMN1), interacting between microfilaments and microtubules (MACF1), as well as a phosphatase (PPP1R12B). Among the proteins encoded by these genes, the protein expression level of Cdc42 effector protein-2 (encoded by CDC42EP2) and Stathmin-1 (encoded by STMN1) was down-regulated, while the protein expression level of N-WASP (encoded by WASL) was up-regulated. The present study confirmed the cyclic stretch-induced cellular realignment and rearrangement of microfilaments in the human PDL cells and indicated several force-sensitive genes with regard to cytoskeletal regulators.

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

Genome-Wide Architecture of Disease Resistance Genes in Lettuce

PubMed Central

Christopoulou, Marilena; Wo, Sebastian Reyes-Chin; Kozik, Alex; McHale, Leah K.; Truco, Maria-Jose; Wroblewski, Tadeusz; Michelmore, Richard W.

2015-01-01

Genome-wide motif searches identified 1134 genes in the lettuce reference genome of cv. Salinas that are potentially involved in pathogen recognition, of which 385 were predicted to encode nucleotide binding-leucine rich repeat receptor (NLR) proteins. Using a maximum-likelihood approach, we grouped the NLRs into 25 multigene families and 17 singletons. Forty-one percent of these NLR-encoding genes belong to three families, the largest being RGC16 with 62 genes in cv. Salinas. The majority of NLR-encoding genes are located in five major resistance clusters (MRCs) on chromosomes 1, 2, 3, 4, and 8 and cosegregate with multiple disease resistance phenotypes. Most MRCs contain primarily members of a single NLR gene family but a few are more complex. MRC2 spans 73 Mb and contains 61 NLRs of six different gene families that cosegregate with nine disease resistance phenotypes. MRC3, which is 25 Mb, contains 22 RGC21 genes and colocates with Dm13. A library of 33 transgenic RNA interference tester stocks was generated for functional analysis of NLR-encoding genes that cosegregated with disease resistance phenotypes in each of the MRCs. Members of four NLR-encoding families, RGC1, RGC2, RGC21, and RGC12 were shown to be required for 16 disease resistance phenotypes in lettuce. The general composition of MRCs is conserved across different genotypes; however, the specific repertoire of NLR-encoding genes varied particularly of the rapidly evolving Type I genes. These tester stocks are valuable resources for future analyses of additional resistance phenotypes. PMID:26449254

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...

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

PubMed Central

Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

Measuring semantic similarities by combining gene ontology annotations and gene co-function networks

DOE PAGES

Peng, Jiajie; Uygun, Sahra; Kim, Taehyong; ...

2015-02-14

Background: Gene Ontology (GO) has been used widely to study functional relationships between genes. The current semantic similarity measures rely only on GO annotations and GO structure. This limits the power of GO-based similarity because of the limited proportion of genes that are annotated to GO in most organisms. Results: We introduce a novel approach called NETSIM (network-based similarity measure) that incorporates information from gene co-function networks in addition to using the GO structure and annotations. Using metabolic reaction maps of yeast, Arabidopsis, and human, we demonstrate that NETSIM can improve the accuracy of GO term similarities. We also demonstratemore » that NETSIM works well even for genomes with sparser gene annotation data. We applied NETSIM on large Arabidopsis gene families such as cytochrome P450 monooxygenases to group the members functionally and show that this grouping could facilitate functional characterization of genes in these families. Conclusions: Using NETSIM as an example, we demonstrated that the performance of a semantic similarity measure could be significantly improved after incorporating genome-specific information. NETSIM incorporates both GO annotations and gene co-function network data as a priori knowledge in the model. Therefore, functional similarities of GO terms that are not explicitly encoded in GO but are relevant in a taxon-specific manner become measurable when GO annotations are limited.« less

The Repeat Sequences and Elevated Substitution Rates of the Chloroplast accD Gene in Cupressophytes

PubMed Central

Li, Jia; Su, Yingjuan; Wang, Ting

2018-01-01

The plastid accD gene encodes a subunit of the acetyl-CoA carboxylase (ACCase) enzyme. The length of accD gene has been supposed to expand in Cryptomeria japonica, Taiwania cryptomerioides, Cephalotaxus, Taxus chinensis, and Podocarpus lambertii, and the main reason for this phenomenon was the existence of tandemly repeated sequences. However, it is still unknown whether the accD gene length in other cupressophytes has expanded. Here, in order to investigate how widespread this phenomenon was, 18 accD sequences and its surrounding regions of cupressophyte were sequenced and analyzed. Together with 39 GenBank sequence data, our taxon sampling covered all the extant gymnosperm orders. The repetitive elements and substitution rates of accD among 57 gymnosperm species were analyzed, the results show: (1) Reading frame length of accD gene in 18 cupressophytes species has also expanded. (2) Many repetitive elements were identified in accD gene of cupressophyte lineages. (3) The synonymous and non-synonymous substitution rates of accD were accelerated in cupressophytes. (4) accD was located in rearrangement endpoints. These results suggested that repetitive elements may mediate the chloroplast genome rearrangement and accelerated the substitution rates. PMID:29731764

Diversity and Evolutionary History of Iron Metabolism Genes in Diatoms

PubMed Central

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

2015-01-01

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

Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

DOEpatents

Craft, David L.; Madduri, Krishna M.; Loper, John C.

2003-01-01

A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

Identification and Characterization of Genes Involved in Benzylisoquinoline Alkaloid Biosynthesis in Coptis Species

PubMed Central

He, Si-Mei; Liang, Yan-Li; Cong, Kun; Chen, Geng; Zhao, Xiu; Zhao, Qi-Ming; Zhang, Jia-Jin; Wang, Xiao; Dong, Yang; Yang, Jian-Li; Zhang, Guang-Hui; Qian, Zhi-Long; Fan, Wei; Yang, Sheng-Chao

2018-01-01

The dried rhizomes of Coptis chinensis have been extensively used in heat clearing, dampness drying, fire draining, and detoxification by virtue of their major bioactive components, benzylisoquinoline alkaloids (BIAs). However, C. teeta and C. chinensis are occasionally interchanged, and current understanding of the molecular basis of BIA biosynthesis in these two species is limited. Here, berberine, coptisine, jatrorrhizine, and palmatine were detected in two species, and showed the highest contents in the roots, while epiberberine were found only in C. chinensis. Comprehensive transcriptome analysis of the roots and leaves of C. teeta and C. chinensis, respectively, identified 53 and 52 unigenes encoding enzymes potentially involved in BIA biosynthesis. By integrating probable biosynthetic pathways for BIAs, the jatrorrhizine biosynthesis ill-informed previously was further characterized. Two genes encoding norcoclaurine/norlaudanosoline 6-O-methyltransferases (Cc6OMT1 and Cc6OMT2) and one gene encoding norcoclaurine-7OMT (Ct7OMT) catalyzed enzymatically O-methylate (S)-norcoclaurine at C6 that yield (S)-coclaurine, along with a smaller amount of O-methylation occurred at C7, thereby forming its isomer (isococlaurine). In addition, scoulerine 9-OMT (CtSOMT) was determined to show strict substrate specificity, targeting (S)-scoulerine to yield (S)-tetrahydrocolumbamine. Taken together, the integration of the transcriptome and enzyme activity assays further provides new insight into molecular mechanisms underlying BIA biosynthesis in plants and identifies candidate genes for the study of synthetic biology in microorganisms. PMID:29915609

Induction of hepatic ABC transporter expression is part of the PPARalpha-mediated fasting response in the mouse.

PubMed

Kok, Tineke; Wolters, Henk; Bloks, Vincent W; Havinga, Rick; Jansen, Peter L M; Staels, Bart; Kuipers, Folkert

2003-01-01

Fatty acids are natural ligands of the peroxisome proliferator-activated receptor alpha (PPARalpha). Synthetic ligands of this nuclear receptor, i.e., fibrates, induce the hepatic expression of the multidrug resistance 2 gene (Mdr2), encoding the canalicular phospholipid translocator, and affect hepatobiliary lipid transport. We tested whether fasting-associated fatty acid release from adipose tissues alters hepatic transporter expression and bile formation in a PPARalpha-dependent manner. A 24-hour fasting/48-hour refeeding schedule was used in wild-type and Pparalpha((-/-)) mice. Expression of genes involved in the control of bile formation was determined and related to secretion rates of biliary components. Expression of Pparalpha, farnesoid X receptor, and liver X receptor alpha genes encoding nuclear receptors that control hepatic bile salt and sterol metabolism was induced on fasting in wild-type mice only. The expression of Mdr2 was 5-fold increased in fasted wild-type mice and increased only marginally in Pparalpha((-/-)) mice, and it normalized on refeeding. Mdr2 protein levels and maximal biliary phospholipid secretion rates were clearly increased in fasted wild-type mice. Hepatic expression of the liver X receptor target genes ATP binding cassette transporter a1 (Abca1), Abcg5, and Abcg8, implicated in hepatobiliary cholesterol transport, was induced in fasted wild-type mice only. However, the maximal biliary cholesterol secretion rate was reduced by approximately 50%. Induction of Mdr2 expression and function is part of the PPARalpha-mediated fasting response in mice. Fasting also induces expression of the putative hepatobiliary cholesterol transport genes Abca1, Abcg5, and Abcg8, but, nonetheless, maximal biliary cholesterol excretion is decreased after fasting.

The Early ANTP Gene Repertoire: Insights from the Placozoan Genome

PubMed Central

Schierwater, Bernd; Kamm, Kai; Srivastava, Mansi; Rokhsar, Daniel; Rosengarten, Rafael D.; Dellaporta, Stephen L.

2008-01-01

The evolution of ANTP genes in the Metazoa has been the subject of conflicting hypotheses derived from full or partial gene sequences and genomic organization in higher animals. Whole genome sequences have recently filled in some crucial gaps for the basal metazoan phyla Cnidaria and Porifera. Here we analyze the complete genome of Trichoplax adhaerens, representing the basal metazoan phylum Placozoa, for its set of ANTP class genes. The Trichoplax genome encodes representatives of Hox/ParaHox-like, NKL, and extended Hox genes. This repertoire possibly mirrors the condition of a hypothetical cnidarian-bilaterian ancestor. The evolution of the cnidarian and bilaterian ANTP gene repertoires can be deduced by a limited number of cis-duplications of NKL and “extended Hox” genes and the presence of a single ancestral “ProtoHox” gene. PMID:18716659

Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti.

PubMed

Platero, Raúl A; Jaureguy, Melina; Battistoni, Federico J; Fabiano, Elena R

2003-01-21

Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sit B and sit D genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.

Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

PubMed

Yang, Bing-Yan; Huo, Xiu-Ai; Li, Peng-Fei; Wang, Cui-Xia; Duan, Hui-Jun

2014-08-01

Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 x 10(6) and 2.0 x 10(10) pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.

Successful aerobic bioremediation of groundwater contaminated with higher chlorinated phenols by indigenous degrader bacteria.

PubMed

Mikkonen, Anu; Yläranta, Kati; Tiirola, Marja; Dutra, Lara Ambrosio Leal; Salmi, Pauliina; Romantschuk, Martin; Copley, Shelley; Ikäheimo, Jukka; Sinkkonen, Aki

2018-07-01

The xenobiotic priority pollutant pentachlorophenol has been used as a timber preservative in a polychlorophenol bulk synthesis product containing also tetrachlorophenol and trichlorophenol. Highly soluble chlorophenol salts have leaked into groundwater, causing severe contamination of large aquifers. Natural attenuation of higher-chlorinated phenols (HCPs: pentachlorophenol + tetrachlorophenol) at historically polluted sites has been inefficient, but a 4-year full scale in situ biostimulation of a chlorophenol-contaminated aquifer by circulation and re-infiltration of aerated groundwater was remarkably successful: pentachlorophenol decreased from 400 μg L -1 to <1 μg L -1 and tetrachlorophenols from 4000 μg L -1 to <10 μg L -1 . The pcpB gene, the gene encoding pentachlorophenol hydroxylase - the first and rate-limiting enzyme in the only fully characterised aerobic HCP degradation pathway - was present in up to 10% of the indigenous bacteria already 4 months after the start of aeration. The novel quantitative PCR assay detected the pcpB gene in situ also in the chlorophenol plume of another historically polluted aquifer with no remediation history. Hotspot groundwater HCPs from this site were degraded efficiently during a 3-week microcosm incubation with one-time aeration but no other additives: from 5400 μg L -1 to 1200 μg L -1 and to 200 μg L -1 in lightly and fully aerated microcosms, respectively, coupled with up to 2400% enrichment of the pcpB gene. Accumulation of lower-chlorinated metabolites was observed in neither in situ remediation nor microcosms, supporting the assumption that HCP removal was due to the aerobic degradation pathway where the first step limits the mineralisation rate. Our results demonstrate that bacteria capable of aerobic mineralisation of xenobiotic pentachlorophenol and tetrachlorophenol can be present at long-term polluted groundwater sites, making bioremediation by simple aeration a viable and economically attractive alternative. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Carbon Dioxide Limitation-Inducible Protein, ColA, Supports the Growth of Synechococcus sp. PCC 7002.

PubMed

Shimakawa, Ginga; Watanabe, Satoru; Miyake, Chikahiro

2017-12-15

A limitation in carbon dioxide (CO₂), which occurs as a result of natural environmental variation, suppresses photosynthesis and has the potential to cause photo-oxidative damage to photosynthetic cells. Oxygenic phototrophs have strategies to alleviate photo-oxidative damage to allow life in present atmospheric CO₂ conditions. However, the mechanisms for CO₂ limitation acclimation are diverse among the various oxygenic phototrophs, and many mechanisms remain to be discovered. In this study, we found that the gene encoding a CO₂ limitation-inducible protein, ColA, is required for the cyanobacterium Synechococcus sp. PCC 7002 (S. 7002) to acclimate to limited CO₂ conditions. An S. 7002 mutant deficient in ColA (Δ colA ) showed lower chlorophyll content, based on the amount of nitrogen, than that in S. 7002 wild-type (WT) under ambient air but not high CO₂ conditions. Both thermoluminescence and protein carbonylation detected in the ambient air grown cells indicated that the lack of ColA promotes oxidative stress in S. 7002. Alterations in the photosynthetic O₂ evolution rate and relative electron transport rate in the short-term response, within an hour, to CO₂ limitation were the same between the WT and Δ colA . Conversely, these photosynthetic parameters were mostly lower in the long-term response of a few days in Δ colA than in the WT. These data suggest that ColA is required to sustain photosynthetic activity for living under ambient air in S. 7002. The unique phylogeny of ColA revealed diverse strategies to acclimate to CO₂ limitation among cyanobacteria.

Competition between conjugation and M13 phage infection in Escherichia coli in the absence of selection pressure: a kinetic study.

PubMed

Wan, Zhenmao; Goddard, Noel L

2012-10-01

Inter- and intraspecies horizontal gene transfer enabled by bacterial secretion systems is a powerful mechanism for bacterial genome plasticity. The type IV secretion system of Escherichia coli, encoded by the F plasmid, enables cell-to-cell contact and subsequent DNA transfer known as conjugation. Conjugation is compromised by phage infection that specifically targets the secretion machinery. Hence, the use of phages to regulate the spread of genes, such as acquired antibiotic resistance or as general biosanitation agents, has gained interest. To predict the potential efficacy, the competition kinetics must first be understood. Using quantitative PCR to enumerate genomic loci in a resource-limited batch culture, we quantify the infection kinetics of the nonlytic phage M13 and its impact on conjugation in the absence of selection pressure (isogenic set). Modeling the resulting experimental data reveals the cellular growth rate to be reduced to 60% upon phage infection. We also find a maximum phage infection rate of 3×10(-11) mL phage(-1) min(-1) which is only 1 order of magnitude slower than the maximum conjugation rate (3×10(-10) mL cell(-1) min(-1)), suggesting phages must be in significant abundance to be effective antagonists to horizontal gene transfer. In the regime where the number of susceptible cells (F(+)) and phages are equal upon initial infection, we observe the spread of the conjugative plasmid throughout the cell population despite phage infection, but only at 10% of the uninfected rate. This has interesting evolutionary implications, as even in the absence of selection pressure, cells maintain the ability to conjugate despite phage vulnerability and the associated growth consequences.

Community Composition of Nitrous Oxide-Related Genes in Salt Marsh Sediments Exposed to Nitrogen Enrichment.

PubMed

Angell, John H; Peng, Xuefeng; Ji, Qixing; Craick, Ian; Jayakumar, Amal; Kearns, Patrick J; Ward, Bess B; Bowen, Jennifer L

2018-01-01

Salt marshes provide many key ecosystem services that have tremendous ecological and economic value. One critical service is the removal of fixed nitrogen from coastal waters, which limits the negative effects of eutrophication resulting from increased nutrient supply. Nutrient enrichment of salt marsh sediments results in higher rates of nitrogen cycling and, commonly, a concurrent increase in the flux of nitrous oxide, an important greenhouse gas. Little is known, however, regarding controls on the microbial communities that contribute to nitrous oxide fluxes in marsh sediments. To address this disconnect, we generated profiles of microbial communities and communities of micro-organisms containing specific nitrogen cycling genes that encode several enzymes ( amoA, norB, nosZ) related to nitrous oxide flux from salt marsh sediments. We hypothesized that communities of microbes responsible for nitrogen transformations will be structured by nitrogen availability. Taxa that respond positively to high nitrogen inputs may be responsible for the elevated rates of nitrogen cycling processes measured in fertilized sediments. Our data show that, with the exception of ammonia-oxidizing archaea, the community composition of organisms involved in the production and consumption of nitrous oxide was altered under nutrient enrichment. These results suggest that previously measured rates of nitrous oxide production and consumption are likely the result of changes in community structure, not simply changes in microbial activity.

Analysis of Gene Expression in Escherichia coli in Response to Changes of Growth-Limiting Nutrient in Chemostat Cultures

PubMed Central

Hua, Qiang; Yang, Chen; Oshima, Taku; Mori, Hirotada; Shimizu, Kazuyuki

2004-01-01

Studies of steady-state metabolic fluxes in Escherichia coli grown in nutrient-limited chemostat cultures suggest remarkable flux alterations in response to changes of growth-limiting nutrient in the medium (Hua et al., J. Bacteriol. 185:7053-7067, 2003). To elucidate the physiological adaptation of cells to the nutrient condition through the flux change and understand the molecular mechanisms underlying the change in the flux, information on gene expression is of great importance. DNA microarray analysis was performed to investigate the global transcriptional responses of steady-state cells grown in chemostat cultures with limited glucose or ammonia while other environmental conditions and the growth rate were kept constant. In slow-growing cells (specific growth rate of 0.10 h−1), 9.8% of a total of 4,071 genes investigated, especially those involved in amino acid metabolism, central carbon and energy metabolism, transport system and cell envelope, were observed to be differentially expressed between the two nutrient-limited cultures. One important characteristic of E. coli grown under nutrient limitation was its capacity to scavenge carbon or nitrogen from the medium through elevating the expression of the corresponding transport and assimilation genes. The number of differentially expressed genes in faster-growing cells (specific growth rate of 0.55 h−1), however, decreased to below half of that in slow-growing cells, which could be explained by diverse transcriptional responses to the growth rate under different nutrient limitations. Independent of the growth rate, 92 genes were identified as being differentially expressed. Genes tightly related to the culture conditions were highlighted, some of which may be used to characterize nutrient-limited growth. PMID:15066832

Acetylcholinesterase genes within the Diptera: takeover and loss in true flies

PubMed Central

Huchard, Elise; Martinez, Michel; Alout, Haoues; Douzery, Emmanuel J.P; Lutfalla, Georges; Berthomieu, Arnaud; Berticat, Claire; Raymond, Michel; Weill, Mylène

2006-01-01

It has recently been reported that the synaptic acetylcholinesterase (AChE) in mosquitoes is encoded by the ace-1 gene, distinct and divergent from the ace-2 gene, which performs this function in Drosophila. This is an unprecedented situation within the Diptera order because both ace genes derive from an old duplication and are present in most insects and arthropods. Nevertheless, Drosophila possesses only the ace-2 gene. Thus, a secondary loss occurred during the evolution of Diptera, implying a vital function switch from one gene (ace-1) to the other (ace-2). We sampled 78 species, representing 50 families (27% of the Dipteran families) spread over all major subdivisions of the Diptera, and looked for ace-1 and ace-2 by systematic PCR screening to determine which taxonomic groups within the Diptera have this gene change. We show that this loss probably extends to all true flies (or Cyclorrhapha), a large monophyletic group of the Diptera. We also show that ace-2 plays a non-detectable role in the synaptic AChE in a lower Diptera species, suggesting that it has non-synaptic functions. A relative molecular evolution rate test showed that the intensity of purifying selection on ace-2 sequences is constant across the Diptera, irrespective of the presence or absence of ace-1, confirming the evolutionary importance of non-synaptic functions for this gene. We discuss the evolutionary scenarios for the takeover of ace-2 and the loss of ace-1, taking into account our limited knowledge of non-synaptic functions of ace genes and some specific adaptations of true flies. PMID:17002944

'Overgrowth' mutants in barley and wheat: new alleles and phenotypes of the 'Green Revolution' DELLA gene.

PubMed

Chandler, Peter Michael; Harding, Carol Anne

2013-04-01

A suppressor screen using dwarf mutants of barley (Hordeum vulgare L.) led to the isolation of 'overgrowth' derivatives, which retained the original dwarfing gene but grew at a faster rate because of a new mutation. The new mutations were in the Slender1 (Sln1) gene (11/13 cases), which encodes the DELLA protein central to gibberellin (GA) signalling, showed 100% genetic linkage to Sln1 (1/13), or were in the Spindly1 (Spy1) gene (1/13), which encodes another protein involved in GA signalling. The overgrowth mutants were characterized by increased GA signalling, although the extent still depended on the background GA biosynthesis capacity, GA receptor function, and DELLA activity. A comparison between two GA responses, α-amylase production and leaf growth rate, revealed degrees of specificity for both the overgrowth allele and the GA response under consideration. Many overgrowth mutants were also isolated in a dwarf line of bread wheat (Triticum aestivum L.) and 19 new alleles were identified in the Rht-B1 gene, one of the 'Green Revolution' semi-dwarfing genes and the orthologue of Sln1. The sites of amino acid substitutions in the DELLA proteins of both species provide insight into DELLA function, and included examples where identical but independent substitutions were observed. In both species, the starting lines were too dwarfed to be directly useful in breeding programmes, but new overgrowth derivatives with semidwarf heights have now been characterized. The variation they exhibit in GA-influenced traits identifies novel alleles with perfect markers that are of potential use in breeding.

In vitro characterization of six STUB1 variants in spinocerebellar ataxia 16 reveals altered structural properties for the encoded CHIP proteins

PubMed Central

Pakdaman, Yasaman; Sanchez-Guixé, Monica; Kleppe, Rune; Erdal, Sigrid; Bustad, Helene J.; Bjørkhaug, Lise; Haugarvoll, Kristoffer; Tzoulis, Charalampos; Heimdal, Ketil; Knappskog, Per M.; Johansson, Stefan

2017-01-01

Spinocerebellar ataxia, autosomal recessive 16 (SCAR16) is caused by biallelic mutations in the STIP1 homology and U-box containing protein 1 (STUB1) gene encoding the ubiquitin E3 ligase and dimeric co-chaperone C-terminus of Hsc70-interacting protein (CHIP). It has been proposed that the disease mechanism is related to CHIP’s impaired E3 ubiquitin ligase properties and/or interaction with its chaperones. However, there is limited knowledge on how these mutations affect the stability, folding, and protein structure of CHIP itself. To gain further insight, six previously reported pathogenic STUB1 variants (E28K, N65S, K145Q, M211I, S236T, and T246M) were expressed as recombinant proteins and studied using limited proteolysis, size-exclusion chromatography (SEC), and circular dichroism (CD). Our results reveal that N65S shows increased CHIP dimerization, higher levels of α-helical content, and decreased degradation rate compared with wild-type (WT) CHIP. By contrast, T246M demonstrates a strong tendency for aggregation, a more flexible protein structure, decreased levels of α-helical structures, and increased degradation rate compared with WT CHIP. E28K, K145Q, M211I, and S236T also show defects on structural properties compared with WT CHIP, although less profound than what observed for N65S and T246M. In conclusion, our results illustrate that some STUB1 mutations known to cause recessive SCAR16 have a profound impact on the protein structure, stability, and ability of CHIP to dimerize in vitro. These results add to the growing understanding on the mechanisms behind the disorder. PMID:28396517

In vitro characterization of six STUB1 variants in spinocerebellar ataxia 16 reveals altered structural properties for the encoded CHIP proteins.

PubMed

Pakdaman, Yasaman; Sanchez-Guixé, Monica; Kleppe, Rune; Erdal, Sigrid; Bustad, Helene J; Bjørkhaug, Lise; Haugarvoll, Kristoffer; Tzoulis, Charalampos; Heimdal, Ketil; Knappskog, Per M; Johansson, Stefan; Aukrust, Ingvild

2017-04-30

Spinocerebellar ataxia, autosomal recessive 16 (SCAR16) is caused by biallelic mutations in the STIP1 homology and U-box containing protein 1 ( STUB1 ) gene encoding the ubiquitin E3 ligase and dimeric co-chaperone C-terminus of Hsc70-interacting protein (CHIP). It has been proposed that the disease mechanism is related to CHIP's impaired E3 ubiquitin ligase properties and/or interaction with its chaperones. However, there is limited knowledge on how these mutations affect the stability, folding, and protein structure of CHIP itself. To gain further insight, six previously reported pathogenic STUB1 variants (E28K, N65S, K145Q, M211I, S236T, and T246M) were expressed as recombinant proteins and studied using limited proteolysis, size-exclusion chromatography (SEC), and circular dichroism (CD). Our results reveal that N65S shows increased CHIP dimerization, higher levels of α-helical content, and decreased degradation rate compared with wild-type (WT) CHIP. By contrast, T246M demonstrates a strong tendency for aggregation, a more flexible protein structure, decreased levels of α-helical structures, and increased degradation rate compared with WT CHIP. E28K, K145Q, M211I, and S236T also show defects on structural properties compared with WT CHIP, although less profound than what observed for N65S and T246M. In conclusion, our results illustrate that some STUB1 mutations known to cause recessive SCAR16 have a profound impact on the protein structure, stability, and ability of CHIP to dimerize in vitro. These results add to the growing understanding on the mechanisms behind the disorder. © 2017 The Author(s).

An edge preserving differential image coding scheme

NASA Technical Reports Server (NTRS)

Rost, Martin C.; Sayood, Khalid

1992-01-01

Differential encoding techniques are fast and easy to implement. However, a major problem with the use of differential encoding for images is the rapid edge degradation encountered when using such systems. This makes differential encoding techniques of limited utility, especially when coding medical or scientific images, where edge preservation is of utmost importance. A simple, easy to implement differential image coding system with excellent edge preservation properties is presented. The coding system can be used over variable rate channels, which makes it especially attractive for use in the packet network environment.

Transferred interbacterial antagonism genes augment eukaryotic innate immune function.

PubMed

Chou, Seemay; Daugherty, Matthew D; Peterson, S Brook; Biboy, Jacob; Yang, Youyun; Jutras, Brandon L; Fritz-Laylin, Lillian K; Ferrin, Michael A; Harding, Brittany N; Jacobs-Wagner, Christine; Yang, X Frank; Vollmer, Waldemar; Malik, Harmit S; Mougous, Joseph D

2015-02-05

Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.

CYTOMEGALOVIRUS VECTORS VIOLATE CD8+ T CELL EPITOPE RECOGNITION PARADIGMS

PubMed Central

Hansen, Scott G.; Sacha, Jonah B.; Hughes, Colette M.; Ford, Julia C.; Burwitz, Benjamin J.; Scholz, Isabel; Gilbride, Roxanne M.; Lewis, Matthew S.; Gilliam, Awbrey N.; Ventura, Abigail B.; Malouli, Daniel; Xu, Guangwu; Richards, Rebecca; Whizin, Nathan; Reed, Jason S.; Hammond, Katherine B.; Fischer, Miranda; Turner, John M.; Legasse, Alfred W.; Axthelm, Michael K.; Edlefsen, Paul T.; Nelson, Jay A.; Lifson, Jeffrey D.; Früh, Klaus; Picker, Louis J.

2013-01-01

CD8+ T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of anti-pathogen immunity. We found that simian immunodeficiency virus (SIV) protein-expressing Rhesus Cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8+ T cells that recognize unusual, diverse and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope-specific CD8+ T cell responses is suppressed by the RhCMV-encoded Rh189 (US11) gene, and the promiscuous MHC class I- and class II-restricted CD8+ T cell responses only occur in the absence of the Rh157.4-.6 (UL128-131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8+ T cell epitope recognition. PMID:23704576

Isolation and Characterization of a myo-inositol-1-phosphate Synthase Gene from Yellow Passion Fruit (Passiflora edulis f. flavicarpa) Expressed During Seed Development and Environmental Stress

PubMed Central

Abreu, Emanuel F. M.; Aragão, Francisco J. L.

2007-01-01

Background and Aims Myo-inositol-1l-phosphate synthase (MIPS) catalyses the conversion of d-glucose 6-phosphate to 1-l-myo-inositol-1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. Inositol phospholipids play a vital role in membrane trafficking and signalling pathways, auxin storage and transport, phytic acid biosynthesis, cell wall biosynthesis and production of stress-related molecules. In the present study, an MIPS cDNA from developing Passiflora edulis f. flavicarpa seeds was characterized and an investigation made into its spatial and differential expression, as well as changes in its transcription during exposure of growing plants to cold and heat stresses. Methods The MIPS-encoding gene was isolated by polymerase chain reaction (PCR) methods, and transcript levels were examined using semi-quantitative reverse transcription–PCR (RT–PCR) during seed development and in response to heat and cold stress. In addition, the copy number of the cloned PeMIPS1 gene in the genome of Passiflora edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea was determined by Southern blot analyses. Key Results A full-length cDNA clone of the PeMIPS1 from P. edulis was isolated and characterized. Southern blot analyses indicated that the genomic DNA might have diverse sequences of MIPS-encoding genes and one copy of the cloned PeMIPS1 gene in the genomes of P. edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea. RT–PCR expression analyses revealed the presence of PeMIPS1 transcripts in ovules, pollen grains and leaves, and during the seed developmental stages, where it peaked at 9 d after pollination. The PeMIPS1 gene is differentially regulated under cold and heat stress, presenting a light-responsive transcription. Conclusions Experimental data suggest that PeMIPS1 transcription plays an important role in the establishment of developmental programmes and during the response of plants to environmental changes. The PeMIPS1 is differentially transcribed during cold and heat stress, presenting a light response pattern, suggesting that it is important for environmental stress response. PMID:17138579

Genome complexity in the coelacanth is reflected in its adaptive immune system

USGS Publications Warehouse

Saha, Nil Ratan; Ota, Tatsuya; Litman, Gary W.; Hansen, John; Parra, Zuly; Hsu, Ellen; Buonocore, Francesco; Canapa, Adriana; Cheng, Jan-Fang; Amemiya, Chris T.

2014-01-01

We have analyzed the available genome and transcriptome resources from the coelacanth in order to characterize genes involved in adaptive immunity. Two highly distinctive IgW-encoding loci have been identified that exhibit a unique genomic organization, including a multiplicity of tandemly repeated constant region exons. The overall organization of the IgW loci precludes typical heavy chain class switching. A locus encoding IgM could not be identified either computationally or by using several different experimental strategies. Four distinct sets of genes encoding Ig light chains were identified. This includes a variant sigma-type Ig light chain previously identified only in cartilaginous fishes and which is now provisionally denoted sigma-2. Genes encoding α/β and γ/δ T-cell receptors, and CD3, CD4, and CD8 co-receptors also were characterized. Ig heavy chain variable region genes and TCR components are interspersed within the TCR α/δ locus; this organization previously was reported only in tetrapods and raises questions regarding evolution and functional cooption of genes encoding variable regions. The composition, organization and syntenic conservation of the major histocompatibility complex locus have been characterized. We also identified large numbers of genes encoding cytokines and their receptors, and other genes associated with adaptive immunity. In terms of sequence identity and organization, the adaptive immune genes of the coelacanth more closely resemble orthologous genes in tetrapods than those in teleost fishes, consistent with current phylogenomic interpretations. Overall, the work reported described herein highlights the complexity inherent in the coelacanth genome and provides a rich catalog of immune genes for future investigations.

DNA/RNA Helicase Gene Mutations in a Form of Juvenile Amyotrophic Lateral Sclerosis (ALS4)

PubMed Central

Chen, Ying-Zhang; Bennett, Craig L.; Huynh, Huy M.; Blair, Ian P.; Puls, Imke; Irobi, Joy; Dierick, Ines; Abel, Annette; Kennerson, Marina L.; Rabin, Bruce A.; Nicholson, Garth A.; Auer-Grumbach, Michaela; Wagner, Klaus; De Jonghe, Peter; Griffin, John W.; Fischbeck, Kenneth H.; Timmerman, Vincent; Cornblath, David R.; Chance, Phillip F.

2004-01-01

Juvenile amyotrophic lateral sclerosis (ALS4) is a rare autosomal dominant form of juvenile amyotrophic lateral sclerosis (ALS) characterized by distal muscle weakness and atrophy, normal sensation, and pyramidal signs. Individuals affected with ALS4 usually have an onset of symptoms at age <25 years, a slow rate of progression, and a normal life span. The ALS4 locus maps to a 1.7-Mb interval on chromosome 9q34 flanked by D9S64 and D9S1198. To identify the molecular basis of ALS4, we tested 19 genes within the ALS4 interval and detected missense mutations (T3I, L389S, and R2136H) in the Senataxin gene (SETX). The SETX gene encodes a novel 302.8-kD protein. Although its function remains unknown, SETX contains a DNA/RNA helicase domain with strong homology to human RENT1 and IGHMBP2, two genes encoding proteins known to have roles in RNA processing. These observations of ALS4 suggest that mutations in SETX may cause neuronal degeneration through dysfunction of the helicase activity or other steps in RNA processing. PMID:15106121

The Bacillus subtilis ywjI (glpX) gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the class III Fbp enzyme.

PubMed

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-05-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.

The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme▿

PubMed Central

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-01-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions. PMID:19270101

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

PubMed

Hurst, Laurence D; Ghanbarian, Avazeh T; Forrest, Alistair R R; Huminiecki, Lukasz

2015-12-01

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution.

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome

PubMed Central

Hurst, Laurence D.; Ghanbarian, Avazeh T.; Forrest, Alistair R. R.; Huminiecki, Lukasz

2015-01-01

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X’s gene content, gene expression, and evolution. PMID:26685068

Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

PubMed Central

Vasala, A; Dupont, L; Baumann, M; Ritzenthaler, P; Alatossava, T

1993-01-01

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module. Images PMID:8497043

Coherent-state constellations and polar codes for thermal Gaussian channels

NASA Astrophysics Data System (ADS)

Lacerda, Felipe; Renes, Joseph M.; Scholz, Volkher B.

2017-06-01

Optical communication channels are ultimately quantum mechanical in nature, and we must therefore look beyond classical information theory to determine their communication capacity as well as to find efficient encoding and decoding schemes of the highest rates. Thermal channels, which arise from linear coupling of the field to a thermal environment, are of particular practical relevance; their classical capacity has been recently established, but their quantum capacity remains unknown. While the capacity sets the ultimate limit on reliable communication rates, it does not promise that such rates are achievable by practical means. Here we construct efficiently encodable codes for thermal channels which achieve the classical capacity and the so-called Gaussian coherent information for transmission of classical and quantum information, respectively. Our codes are based on combining polar codes with a discretization of the channel input into a finite "constellation" of coherent states. Encoding of classical information can be done using linear optics.

Carbapenemase-producing Enterobacteriaceae: Risk factors for infection and impact of resistance on outcomes

PubMed Central

Mariappan, Shanthi; Sekar, Uma; Kamalanathan, Arunagiri

2017-01-01

Background: Carbapenemase-producing Enterobacteriaceae (CPE) have increased in recent years leading to limitations of treatment options. The present study was undertaken to detect CPE, risk factors for acquiring them and their impact on clinical outcomes. Methods: This retrospective observational study included 111 clinically significant Enterobacteriaceae resistant to cephalosporins subclass III and exhibiting a positive modified Hodge test. Screening for carbapenemase production was done by phenotypic methods, and polymerase chain reaction was performed to detect genes encoding them. Retrospectively, the medical records of the patients were perused to assess risk factors for infections with CPE and their impact. The data collected were duration of hospital stay, Intensive Care Unit (ICU) stay, use of invasive devices, mechanical ventilation, the presence of comorbidities, and antimicrobial therapy. The outcome was followed up. Univariate and multivariate analysis of the data were performed using SPSS software. Results: Carbapenemase-encoding genes were detected in 67 isolates. The genes detected were New Delhi metallo-β-lactamase, Verona integron-encoded metallo-β-lactamase, and oxacillinase-181.Although univariate analysis identified risk factors associated with acquiring CPE infections as ICU stay (P = 0.021), mechanical ventilation (P = 0.013), indwelling device (P = 0.011), diabetes mellitus (P = 0.036), usage of multiple antimicrobial agents (P = 0.007), administration of carbapenems (P = 0.042), presence of focal infection or sepsis (P = 0.013), and surgical interventions (P = 0.016), multivariate analysis revealed that all these factors were insignificant. Mortality rate was 56.7% in patients with CPE infections. By both univariate and multivariate analysis of impact of the variables on mortality in these patients, the significant factors were mechanical ventilation (odds ratio [OR]: 0.141, 95% confidence interval [CI]: 0.024–0.812) and presence of indwelling invasive device (OR: 8.034; 95% CI: 2.060–31.335). Conclusion: In this study, no specific factor was identified as an independent risk for acquisition of CPE infection. However, as it is evident by multivariate analysis, there is an increased risk of mortality in patients with CPE infections when they are ventilated and are supported by indwelling devices. PMID:28251105

Comprehensive identification of mutations responsible for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA)-to-VISA conversion in laboratory-generated VISA strains derived from hVISA clinical strain Mu3.

PubMed

Matsuo, Miki; Cui, Longzhu; Kim, Jeeyoung; Hiramatsu, Keiichi

2013-12-01

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10(-6) or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.

Dimethylsulfoniopropionate biosynthesis in a diatom Thalassiosira pseudonana: Identification of a gene encoding MTHB-methyltransferase.

PubMed

Kageyama, Hakuto; Tanaka, Yoshito; Shibata, Ayumi; Waditee-Sirisattha, Rungaroon; Takabe, Teruhiro

2018-05-01

Dimethylsulfoniopropionate (DMSP) is one of the most abundant molecules on earth and plays a pivotal role in the marine sulfur cycle. DMSP is believed to be synthesized from methionine by a four-step reaction pathway in marine algae. The genes responsible for biosynthesis of DMSP remain unidentified. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems and contributes greatly to the world's primary production. In this study, through genome search, in vivo activity and functional studies of cDNA products, a gene encoding Thalassiosira methyltransferase (TpMMT) which catalyzes the key step of DMSP synthesis formation of 4-methylthio-2-hydroxybutyrate (DMSHB) from 4-methylthio-2-oxobutyrate (MTHB), was identified. The amino acid sequence of TpMMT was homologous to the methyltransferase from Phaeodactylum tricornutum CCAP 1055/1, but not the recently identified bacterium gene. High salinity and nitrogen limitation stresses caused the increase of DMSP content and TpMMT protein in Thalassiosira. In addition to TpMMT, the enzyme activities for the first three steps could be detected and enhanced under high salinity, suggesting the importance of four-step DMSP synthetic pathway in Thalassiosira. Copyright © 2018 Elsevier Inc. All rights reserved.

Evidence for a major role of antisense RNAs in cyanobacterial gene regulation

PubMed Central

Georg, Jens; Voß, Björn; Scholz, Ingeborg; Mitschke, Jan; Wilde, Annegret; Hess, Wolfgang R

2009-01-01

Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon-limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light-dependent manner and may be required for processing the L11 r-operon and the SyR7 noncoding RNA, which is antisense to the murF 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ∼10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks. PMID:19756044

Evidence for a major role of antisense RNAs in cyanobacterial gene regulation.

PubMed

Georg, Jens; Voss, Björn; Scholz, Ingeborg; Mitschke, Jan; Wilde, Annegret; Hess, Wolfgang R

2009-01-01

Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium Synechocystis sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon-limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to rpl1, which accumulates in a light-dependent manner and may be required for processing the L11 r-operon and the SyR7 noncoding RNA, which is antisense to the murF 5' UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, approximately 10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks.

Consistency of gene starts among Burkholderia genomes

PubMed Central

2011-01-01

Background Evolutionary divergence in the position of the translational start site among orthologous genes can have significant functional impacts. Divergence can alter the translation rate, degradation rate, subcellular location, and function of the encoded proteins. Results Existing Genbank gene maps for Burkholderia genomes suggest that extensive divergence has occurred--53% of ortholog sets based on Genbank gene maps had inconsistent gene start sites. However, most of these inconsistencies appear to be gene-calling errors. Evolutionary divergence was the most plausible explanation for only 17% of the ortholog sets. Correcting probable errors in the Genbank gene maps decreased the percentage of ortholog sets with inconsistent starts by 68%, increased the percentage of ortholog sets with extractable upstream intergenic regions by 32%, increased the sequence similarity of intergenic regions and predicted proteins, and increased the number of proteins with identifiable signal peptides. Conclusions Our findings highlight an emerging problem in comparative genomics: single-digit percent errors in gene predictions can lead to double-digit percentages of inconsistent ortholog sets. The work demonstrates a simple approach to evaluate and improve the quality of gene maps. PMID:21342528

Recombinant DNA encoding a desulfurization biocatalyst

DOEpatents

Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

1994-01-01

This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

Metagenomic evidence for metabolism of trace atmospheric gases by high-elevation desert Actinobacteria

PubMed Central

Lynch, Ryan C.; Darcy, John L.; Kane, Nolan C.; Nemergut, Diana R.; Schmidt, Steve K.

2014-01-01

Previous surveys of very dry Atacama Desert mineral soils have consistently revealed sparse communities of non-photosynthetic microbes. The functional nature of these microorganisms remains debatable given the harshness of the environment and low levels of biomass and diversity. The aim of this study was to gain an understanding of the phylogenetic community structure and metabolic potential of a low-diversity mineral soil metagenome that was collected from a high-elevation Atacama Desert volcano debris field. We pooled DNA extractions from over 15 g of volcanic material, and using whole genome shotgun sequencing, observed only 75–78 total 16S rRNA gene OTUs3%. The phylogenetic structure of this community is significantly under dispersed, with actinobacterial lineages making up 97.9–98.6% of the 16S rRNA genes, suggesting a high degree of environmental selection. Due to this low diversity and uneven community composition, we assembled and analyzed the metabolic pathways of the most abundant genome, a Pseudonocardia sp. (56–72% of total 16S genes). Our assembly and binning efforts yielded almost 4.9 Mb of Pseudonocardia sp. contigs, which accounts for an estimated 99.3% of its non-repetitive genomic content. This genome contains a limited array of carbohydrate catabolic pathways, but encodes for CO2 fixation via the Calvin cycle. The genome also encodes complete pathways for the catabolism of various trace gases (H2, CO and several organic C1 compounds) and the assimilation of ammonia and nitrate. We compared genomic content among related Pseudonocardia spp. and estimated rates of non-synonymous and synonymous nucleic acid substitutions between protein coding homologs. Collectively, these comparative analyses suggest that the community structure and various functional genes have undergone strong selection in the nutrient poor desert mineral soils and high-elevation atmospheric conditions. PMID:25566214

A highly divergent gene cluster in honey bees encodes a novel silk family.

PubMed

Sutherland, Tara D; Campbell, Peter M; Weisman, Sarah; Trueman, Holly E; Sriskantha, Alagacone; Wanjura, Wolfgang J; Haritos, Victoria S

2006-11-01

The pupal cocoon of the domesticated silk moth Bombyx mori is the best known and most extensively studied insect silk. It is not widely known that Apis mellifera larvae also produce silk. We have used a combination of genomic and proteomic techniques to identify four honey bee fiber genes (AmelFibroin1-4) and two silk-associated genes (AmelSA1 and 2). The four fiber genes are small, comprise a single exon each, and are clustered on a short genomic region where the open reading frames are GC-rich amid low GC intergenic regions. The genes encode similar proteins that are highly helical and predicted to form unusually tight coiled coils. Despite the similarity in size, structure, and composition of the encoded proteins, the genes have low primary sequence identity. We propose that the four fiber genes have arisen from gene duplication events but have subsequently diverged significantly. The silk-associated genes encode proteins likely to act as a glue (AmelSA1) and involved in silk processing (AmelSA2). Although the silks of honey bees and silkmoths both originate in larval labial glands, the silk proteins are completely different in their primary, secondary, and tertiary structures as well as the genomic arrangement of the genes encoding them. This implies independent evolutionary origins for these functionally related proteins.

Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification.

PubMed

Paris, Daniel H; Blacksell, Stuart D; Newton, Paul N; Day, Nicholas P J

2008-12-01

We present a loop-mediated isothermal PCR assay (LAMP) targeting the groEL gene, which encodes the 60kDa heat shock protein of Orientia tsutsugamushi. Evaluation included testing of 63 samples of contemporary in vitro isolates, buffy coats and whole blood samples from patients with fever. Detection limits for LAMP were assessed by serial dilutions and quantitation by real-time PCR assay based on the same target gene: three copies/microl for linearized plasmids, 26 copies/microl for VERO cell culture isolates, 14 copies/microl for full blood samples and 41 copies/microl for clinical buffy coats. Based on a limited sample number, the LAMP assay is comparable in sensitivity with conventional nested PCR (56kDa gene), with limits of detection well below the range of known admission bacterial loads of patients with scrub typhus. This inexpensive method requires no sophisticated equipment or sample preparation, and may prove useful as a diagnostic assay in financially poor settings; however, it requires further prospective validation in the field setting.

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

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

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

NASA Astrophysics Data System (ADS)

Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

2016-06-01

Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.

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

PubMed

Adewoye, L O; Worobec, E A

2000-08-08

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

Comparative transcriptome analysis of different chemotypes elucidates withanolide biosynthesis pathway from medicinal plant Withania somnifera

PubMed Central

Gupta, Parul; Goel, Ridhi; Agarwal, Aditya Vikram; Asif, Mehar Hasan; Sangwan, Neelam Singh; Sangwan, Rajender Singh; Trivedi, Prabodh Kumar

2015-01-01

Withania somnifera is one of the most valuable medicinal plants synthesizing secondary metabolites known as withanolides. Despite pharmaceutical importance, limited information is available about the biosynthesis of withanolides. Chemo-profiling of leaf and root tissues of Withania suggest differences in the content and/or nature of withanolides in different chemotypes. To identify genes involved in chemotype and/or tissue-specific withanolide biosynthesis, we established transcriptomes of leaf and root tissues of distinct chemotypes. Genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis with their alternatively spliced forms and paralogous have been identified. Analysis suggests differential expression of large number genes among leaf and root tissues of different chemotypes. Study also identified differentially expressing transcripts encoding cytochrome P450s, glycosyltransferases, methyltransferases and transcription factors which might be involved in chemodiversity in Withania. Virus induced gene silencing of the sterol ∆7-reductase (WsDWF5) involved in the synthesis of 24-methylene cholesterol, withanolide backbone, suggests role of this enzyme in biosynthesis of withanolides. Information generated, in this study, provides a rich resource for functional analysis of withanolide-specific genes to elucidate chemotype- as well as tissue-specific withanolide biosynthesis. This genomic resource will also help in development of new tools for functional genomics and breeding in Withania. PMID:26688389

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

A Novel Phytophthora sojae Resistance Rps12 Gene Mapped to a Genomic Region That Contains Several Rps Genes.

PubMed

Sahoo, Dipak K; Abeysekara, Nilwala S; Cianzio, Silvia R; Robertson, Alison E; Bhattacharyya, Madan K

2017-01-01

Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance.

Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns.

PubMed

Berthelet, M; Whyte, L G; Greer, C W

1996-04-15

Polyvinylpolypyrrolidone spin columns were used to rapidly purify crude soil DNA extracts from humic materials for polymerase chain reaction (PCR) analysis. The PCR detection limit for the tfdC gene, encoding chlorocatechol dioxygenase from the 2,4-dichlorophenoxyacetic acid degradation pathway, was 10(1)-10(2) cells/g soil in inoculated soils. The procedure could be applied to the amplification of biodegradative genes from indigenous microbial populations from a wide variety of soil types, and the entire analysis could be performed within 8 h.

Extended biofilm susceptibility assay for Staphylococcus aureus bovine mastitis isolates: evidence for association between genetic makeup and biofilm susceptibility.

PubMed

Melchior, M B; van Osch, M H J; Lam, T J G M; Vernooij, J C M; Gaastra, W; Fink-Gremmels, J

2011-12-01

Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to Clinical and Laboratory Standards Institute standards. However, various authors have shown a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that in vivo biofilm formation by Staph. aureus, which is not assessed in the antimicrobial susceptibility tests, is associated with this problem, resulting in disappointing cure rates, especially for infections of longer duration. Previous data obtained with a limited number of strains showed that the extended biofilm antimicrobial susceptibility (EBS) assay reveals differences between strains, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test. The objective of this study was to test a collection of Staph. aureus bovine mastitis strains in the EBS assay and to model the effect of antimicrobial exposure, duration of antimicrobial exposure, and genotype profile of the strains on antimicrobial susceptibility. With the results from a previous study with the same collection of strains, the effect of genotype represented by accessory gene regulator gene (agr-type), the presence of insertional sequence 257 (IS257), intercellular adhesion (ica), and the β-lactamase (blaZ) gene were entered as explanatory factors in a logistic regression model. The agr locus of Staph. aureus controls the expression of most of the virulence factors, represses the transcription of several cell wall-associated proteins, and activates several exoproteins during the post-exponential phase. The IS257 gene has been related to biofilm formation in vitro and was found earlier in 50% of the agr-type 2 strains. The ica gene cluster encodes for the production of an extracellular polysaccharide adhesin, termed polysaccharide intercellular adhesin, which appears to have an important role in pathogenic Staph. aureus infections. The blaZ gene encodes the presence of the penicillin resistance in the strain. The EBS assay together with the logistic regression model revealed that the duration of therapy is the most important factor of therapy outcome in this in vitro model. Furthermore, the effect of genotypic differences seems to be more important for therapy outcome than the antimicrobial used in this model. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Natural mismatch repair mutations mediate phenotypic diversity and drug resistance in Cryptococcus deuterogattii.

PubMed

Billmyre, R Blake; Clancey, Shelly Applen; Heitman, Joseph

2017-09-26

Pathogenic microbes confront an evolutionary conflict between the pressure to maintain genome stability and the need to adapt to mounting external stresses. Bacteria often respond with elevated mutation rates, but little evidence exists of stable eukaryotic hypermutators in nature. Whole genome resequencing of the human fungal pathogen Cryptococcus deuterogattii identified an outbreak lineage characterized by a nonsense mutation in the mismatch repair component MSH2. This defect results in a moderate mutation rate increase in typical genes, and a larger increase in genes containing homopolymer runs. This allows facile inactivation of genes with coding homopolymer runs including FRR1 , which encodes the target of the immunosuppresive antifungal drugs FK506 and rapamycin. Our study identifies a eukaryotic hypermutator lineage spread over two continents and suggests that pathogenic eukaryotic microbes may experience similar selection pressures on mutation rate as bacterial pathogens, particularly during long periods of clonal growth or while expanding into new environments.

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

PubMed

Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

2017-09-06

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

Recombinant DNA encoding a desulfurization biocatalyst

DOEpatents

Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

1994-10-18

This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

Structure, Function, Interaction, Co-evolution of Rice Blast Resistance Genes

USDA-ARS?s Scientific Manuscript database

Rice blast disease caused by the fungal pathogen Magnaporthe oryzae is one of the most destructive rice diseases worldwide. Resistance (R) genes to blast encode proteins that detect pathogen signaling molecules encoded by M. oryzae avirulence (AVR) genes. R genes can be a single or a member of clu...

A DEMETER-like DNA demethylase governs tomato fruit ripening

PubMed Central

Liu, Ruie; How-Kit, Alexandre; Stammitti, Linda; Teyssier, Emeline; Rolin, Dominique; Mortain-Bertrand, Anne; Halle, Stefanie; Liu, Mingchun; Kong, Junhua; Wu, Chaoqun; Degraeve-Guibault, Charlotte; Chapman, Natalie H.; Maucourt, Mickael; Hodgman, T. Charlie; Tost, Jörg; Bouzayen, Mondher; Hong, Yiguo; Seymour, Graham B.; Giovannoni, James J.; Gallusci, Philippe

2015-01-01

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening— an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato. PMID:26261318

Molecular genetics of Erwinia amylovora involved in the development of fire blight.

PubMed

Oh, Chang-Sik; Beer, Steven V

2005-12-15

The bacterial plant pathogen, Erwinia amylovora, causes the devastating disease known as fire blight in some Rosaceous plants like apple, pear, quince, raspberry and several ornamentals. Knowledge of the factors affecting the development of fire blight has mushroomed in the last quarter century. On the molecular level, genes encoding a Hrp type III secretion system, genes encoding enzymes involved in synthesis of extracellular polysaccharides and genes facilitating the growth of E. amylovora in its host plants have been characterized. The Hrp pathogenicity island, delimited by genes suggesting horizontal gene transfer, is composed of four distinct regions, the hrp/hrc region, the HEE (Hrp effectors and elicitors) region, the HAE (Hrp-associated enzymes) region, and the IT (Island transfer) region. The Hrp pathogenicity island encodes a Hrp type III secretion system (TTSS), which delivers several proteins from bacteria to plant apoplasts or cytoplasm. E. amylovora produces two exopolysaccharides, amylovoran and levan, which cause the characteristic fire blight wilting symptom in host plants. In addition, other genes, and their encoded proteins, have been characterized as virulence factors of E. amylovora that encode enzymes facilitating sorbitol metabolism, proteolytic activity and iron harvesting. This review summarizes our understanding of the genes and gene products of E. amylovora that are involved in the development of the fire blight disease.

The Drosophila pigmentation gene pink (p) encodes a homologue of human Hermansky-Pudlak syndrome 5 (HPS5).

PubMed

Falcón-Pérez, Juan M; Romero-Calderón, Rafael; Brooks, Elizabeth S; Krantz, David E; Dell'Angelica, Esteban C

2007-02-01

Lysosome-related organelles comprise a group of specialized intracellular compartments that include melanosomes and platelet dense granules (in mammals) and eye pigment granules (in insects). In humans, the biogenesis of these organelles is defective in genetic disorders collectively known as Hermansky-Pudlak syndrome (HPS). Patients with HPS-2, and two murine HPS models, carry mutations in genes encoding subunits of adaptor protein (AP)-3. Other genes mutated in rodent models include those encoding VPS33A and Rab38. Orthologs of all of these genes in Drosophila melanogaster belong to the 'granule group' of eye pigmentation genes. Other genes associated with HPS encode subunits of three complexes of unknown function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3, for which the Drosophila counterparts had not been characterized. Here, we report that the gene encoding the Drosophila ortholog of the HPS5 subunit of BLOC-2 is identical to the granule group gene pink (p), which was first studied in 1910 but had not been identified at the molecular level. The phenotype of pink mutants was exacerbated by mutations in AP-3 subunits or in the orthologs of VPS33A and Rab38. These results validate D. melanogaster as a genetic model to study the function of the BLOCs.

An integrated metagenome and -proteome analysis of the microbial community residing in a biogas production plant.

PubMed

Ortseifen, Vera; Stolze, Yvonne; Maus, Irena; Sczyrba, Alexander; Bremges, Andreas; Albaum, Stefan P; Jaenicke, Sebastian; Fracowiak, Jochen; Pühler, Alfred; Schlüter, Andreas

2016-08-10

To study the metaproteome of a biogas-producing microbial community, fermentation samples were taken from an agricultural biogas plant for microbial cell and protein extraction and corresponding metagenome analyses. Based on metagenome sequence data, taxonomic community profiling was performed to elucidate the composition of bacterial and archaeal sub-communities. The community's cytosolic metaproteome was represented in a 2D-PAGE approach. Metaproteome databases for protein identification were compiled based on the assembled metagenome sequence dataset for the biogas plant analyzed and non-corresponding biogas metagenomes. Protein identification results revealed that the corresponding biogas protein database facilitated the highest identification rate followed by other biogas-specific databases, whereas common public databases yielded insufficient identification rates. Proteins of the biogas microbiome identified as highly abundant were assigned to the pathways involved in methanogenesis, transport and carbon metabolism. Moreover, the integrated metagenome/-proteome approach enabled the examination of genetic-context information for genes encoding identified proteins by studying neighboring genes on the corresponding contig. Exemplarily, this approach led to the identification of a Methanoculleus sp. contig encoding 16 methanogenesis-related gene products, three of which were also detected as abundant proteins within the community's metaproteome. Thus, metagenome contigs provide additional information on the genetic environment of identified abundant proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

SHV-7, a novel cefotaxime-hydrolyzing beta-lactamase, identified in Escherichia coli isolates from hospitalized nursing home patients.

PubMed Central

Bradford, P A; Urban, C; Jaiswal, A; Mariano, N; Rasmussen, B A; Projan, S J; Rahal, J J; Bush, K

1995-01-01

Four ceftazidime-resistant Escherichia coli strains were isolated from elderly nursing home patients in a New York hospital during 1993. Strains MCQ-2, MCQ-3, and MCQ-4 were determined to be identical by pulsed-field gel electrophoresis and plasmid profiles, whereas strain MCQ-1 was unique. Strain MCQ-1 was determined to produce a TEM-10 beta-lactamase. Strains MCQ-2, MCQ-3, and MCQ-4 were also noted to be resistant to cefotaxime. These three strains produced two beta-lactamases with pIs of 5.4 (TEM-1) and 7.6. beta-Lactamase assays revealed that the pI 7.6 enzyme hydrolyzed cefotaxime faster (at a relative hydrolysis rate of 30% compared with that of benzylpenicillin) than either ceftazidime or aztreonam (relative hydrolysis rates of 13 and 3.3%, respectively). Nucleotide sequencing of the gene encoding the pI 7.6 beta-lactamase from strain MCQ-3 revealed a blaSHV-type gene differing from the gene encoding SHV-1 at four nucleotides which resulted in amino acid substitutions: phenylalanine for isoleucine at position 8, serine for arginine at position 43, serine for glycine at position 238, and lysine for glutamate at position 240. This novel SHV-type extended-spectrum beta-lactamase is designated SHV-7. PMID:7785992

Molecular Determinants of Antiestrogen and Drug Sensitivity in Breast Carcinoma Cells

DTIC Science & Technology

1996-08-01

00 ~cd -olC CC) 00, COq -6 0 00d C5 kr0) C~U, 23l Effects of infection rate and selection pressure on gene expression from an internal promoter of a...Hybridization probes were prepared by restriction enzyme digestion of the LNCIuc plasmid, followed by the isolation of the desired fragments by...sensitivity to this drug. The bacterial neo gene encodes neomycin phosphotransferase, an enzyme that metabolically inactivates G418, with the extent of

Experimental study on discretely modulated continuous-variable quantum key distribution

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

Shen Yong; Zou Hongxin; Chen Pingxing

2010-08-15

We present a discretely modulated continuous-variable quantum key distribution system in free space by using strong coherent states. The amplitude noise in the laser source is suppressed to the shot-noise limit by using a mode cleaner combined with a frequency shift technique. Also, it is proven that the phase noise in the source has no impact on the final secret key rate. In order to increase the encoding rate, we use broadband homodyne detectors and the no-switching protocol. In a realistic model, we establish a secret key rate of 46.8 kbits/s against collective attacks at an encoding rate of 10more » MHz for a 90% channel loss when the modulation variance is optimal.« less

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

Mutation in PLK4, encoding a master regulator of centriole formation, defines a novel locus for primordial dwarfism.

PubMed

Shaheen, Ranad; Al Tala, Saeed; Almoisheer, Agaadir; Alkuraya, Fowzan S

2014-12-01

Primordial dwarfism (PD) is a heterogeneous clinical entity characterised by severe prenatal and postnatal growth deficiency. Despite the recent wave of disease gene discovery, the causal mutations in many PD patients remain unknown. To describe a PD family that maps to a novel locus. Clinical, imaging and laboratory phenotyping of a new family with PD followed by autozygosity mapping, linkage analysis and candidate gene sequencing. We describe a multiplex consanguineous Saudi family in which two full siblings and one half-sibling presented with classical features of Seckel syndrome in addition to optic nerve hypoplasia. We were able to map the phenotype to a single novel locus on 4q25-q28.2, in which we identified a five base-pair deletion in PLK4, which encodes a master regulator of centriole duplication. Our discovery further confirms the role of genes involved in centriole biology in the pathogenesis of PD. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Identification and Characterization of a Cis-Encoded Antisense RNA Associated with the Replication Process of Salmonella enterica Serovar Typhi

PubMed Central

Dadzie, Isaac; Xu, Shungao; Ni, Bin; Zhang, Xiaolei; Zhang, Haifang; Sheng, Xiumei; Xu, Huaxi; Huang, Xinxiang

2013-01-01

Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise whiles others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cell's transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned. PMID:23637809

Genome Content and Phylogenomics Reveal both Ancestral and Lateral Evolutionary Pathways in Plant-Pathogenic Streptomyces Species

PubMed Central

Huguet-Tapia, Jose C.; Lefebure, Tristan; Badger, Jonathan H.; Guan, Dongli; Stanhope, Michael J.

2016-01-01

Streptomyces spp. are highly differentiated actinomycetes with large, linear chromosomes that encode an arsenal of biologically active molecules and catabolic enzymes. Members of this genus are well equipped for life in nutrient-limited environments and are common soil saprophytes. Out of the hundreds of species in the genus Streptomyces, a small group has evolved the ability to infect plants. The recent availability of Streptomyces genome sequences, including four genomes of pathogenic species, provided an opportunity to characterize the gene content specific to these pathogens and to study phylogenetic relationships among them. Genome sequencing, comparative genomics, and phylogenetic analysis enabled us to discriminate pathogenic from saprophytic Streptomyces strains; moreover, we calculated that the pathogen-specific genome contains 4,662 orthologs. Phylogenetic reconstruction suggested that Streptomyces scabies and S. ipomoeae share an ancestor but that their biosynthetic clusters encoding the required virulence factor thaxtomin have diverged. In contrast, S. turgidiscabies and S. acidiscabies, two relatively unrelated pathogens, possess highly similar thaxtomin biosynthesis clusters, which suggests that the acquisition of these genes was through lateral gene transfer. PMID:26826232

A Comprehensive Analysis of Nuclear-Encoded Mitochondrial Genes in Schizophrenia.

PubMed

Gonçalves, Vanessa F; Cappi, Carolina; Hagen, Christian M; Sequeira, Adolfo; Vawter, Marquis P; Derkach, Andriy; Zai, Clement C; Hedley, Paula L; Bybjerg-Grauholm, Jonas; Pouget, Jennie G; Cuperfain, Ari B; Sullivan, Patrick F; Christiansen, Michael; Kennedy, James L; Sun, Lei

2018-05-01

The genetic risk factors of schizophrenia (SCZ), a severe psychiatric disorder, are not yet fully understood. Multiple lines of evidence suggest that mitochondrial dysfunction may play a role in SCZ, but comprehensive association studies are lacking. We hypothesized that variants in nuclear-encoded mitochondrial genes influence susceptibility to SCZ. We conducted gene-based and gene-set analyses using summary association results from the Psychiatric Genomics Consortium Schizophrenia Phase 2 (PGC-SCZ2) genome-wide association study comprising 35,476 cases and 46,839 control subjects. We applied the MAGMA method to three sets of nuclear-encoded mitochondrial genes: oxidative phosphorylation genes, other nuclear-encoded mitochondrial genes, and genes involved in nucleus-mitochondria crosstalk. Furthermore, we conducted a replication study using the iPSYCH SCZ sample of 2290 cases and 21,621 control subjects. In the PGC-SCZ2 sample, 1186 mitochondrial genes were analyzed, among which 159 had p values < .05 and 19 remained significant after multiple testing correction. A meta-analysis of 818 genes combining the PGC-SCZ2 and iPSYCH samples resulted in 104 nominally significant and nine significant genes, suggesting a polygenic model for the nuclear-encoded mitochondrial genes. Gene-set analysis, however, did not show significant results. In an in silico protein-protein interaction network analysis, 14 mitochondrial genes interacted directly with 158 SCZ risk genes identified in PGC-SCZ2 (permutation p = .02), and aldosterone signaling in epithelial cells and mitochondrial dysfunction pathways appeared to be overrepresented in this network of mitochondrial and SCZ risk genes. This study provides evidence that specific aspects of mitochondrial function may play a role in SCZ, but we did not observe its broad involvement even using a large sample. Copyright © 2018 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Multidrug resistance in fungi: regulation of transporter-encoding gene expression

PubMed Central

Paul, Sanjoy; Moye-Rowley, W. Scott

2014-01-01

A critical risk to the continued success of antifungal chemotherapy is the acquisition of resistance; a risk exacerbated by the few classes of effective antifungal drugs. Predictably, as the use of these drugs increases in the clinic, more resistant organisms can be isolated from patients. A particularly problematic form of drug resistance that routinely emerges in the major fungal pathogens is known as multidrug resistance. Multidrug resistance refers to the simultaneous acquisition of tolerance to a range of drugs via a limited or even single genetic change. This review will focus on recent progress in understanding pathways of multidrug resistance in fungi including those of most medical relevance. Analyses of multidrug resistance in Saccharomyces cerevisiae have provided the most detailed outline of multidrug resistance in a eukaryotic microorganism. Multidrug resistant isolates of S. cerevisiae typically result from changes in the activity of a pair of related transcription factors that in turn elicit overproduction of several target genes. Chief among these is the ATP-binding cassette (ABC)-encoding gene PDR5. Interestingly, in the medically important Candida species, very similar pathways are involved in acquisition of multidrug resistance. In both C. albicans and C. glabrata, changes in the activity of transcriptional activator proteins elicits overproduction of a protein closely related to S. cerevisiae Pdr5 called Cdr1. The major filamentous fungal pathogen, Aspergillus fumigatus, was previously thought to acquire resistance to azole compounds (the principal antifungal drug class) via alterations in the azole drug target-encoding gene cyp51A. More recent data indicate that pathways in addition to changes in the cyp51A gene are important determinants in A. fumigatus azole resistance. We will discuss findings that suggest azole resistance in A. fumigatus and Candida species may share more mechanistic similarities than previously thought. PMID:24795641

The Rodin-Ohno hypothesis that two enzyme superfamilies descended from one ancestral gene: an unlikely scenario for the origins of translation that will not be dismissed

PubMed Central

2014-01-01

Background Because amino acid activation is rate-limiting for uncatalyzed protein synthesis, it is a key puzzle in understanding the origin of the genetic code. Two unrelated classes (I and II) of contemporary aminoacyl-tRNA synthetases (aaRS) now translate the code. Observing that codons for the most highly conserved, Class I catalytic peptides, when read in the reverse direction, are very nearly anticodons for Class II defining catalytic peptides, Rodin and Ohno proposed that the two superfamilies descended from opposite strands of the same ancestral gene. This unusual hypothesis languished for a decade, perhaps because it appeared to be unfalsifiable. Results The proposed sense/antisense alignment makes important predictions. Fragments that align in antiparallel orientations, and contain the respective active sites, should catalyze the same two reactions catalyzed by contemporary synthetases. Recent experiments confirmed that prediction. Invariant cores from both classes, called Urzymes after Ur = primitive, authentic, plus enzyme and representing ~20% of the contemporary structures, can be expressed and exhibit high, proportionate rate accelerations for both amino-acid activation and tRNA acylation. A major fraction (60%) of the catalytic rate acceleration by contemporary synthetases resides in segments that align sense/antisense. Bioinformatic evidence for sense/antisense ancestry extends to codons specifying the invariant secondary and tertiary structures outside the active sites of the two synthetase classes. Peptides from a designed, 46-residue gene constrained by Rosetta to encode Class I and II ATP binding sites with fully complementary sequences both accelerate amino acid activation by ATP ~400 fold. Conclusions Biochemical and bioinformatic results substantially enhance the posterior probability that ancestors of the two synthetase classes arose from opposite strands of the same ancestral gene. The remarkable acceleration by short peptides of the rate-limiting step in uncatalyzed protein synthesis, together with the synergy of synthetase Urzymes and their cognate tRNAs, introduce a new paradigm for the origin of protein catalysts, emphasize the potential relevance of an operational RNA code embedded in the tRNA acceptor stems, and challenge the RNA-World hypothesis. Reviewers This article was reviewed by Dr. Paul Schimmel (nominated by Laura Landweber), Dr. Eugene Koonin and Professor David Ardell. PMID:24927791

The role of the F-box gene TaFBA1 from wheat (Triticum aestivum L.) in drought tolerance.

PubMed

Zhou, Shumei; Sun, Xiudong; Yin, Suhong; Kong, Xiangzhu; Zhou, Shan; Xu, Ying; Luo, Yin; Wang, Wei

2014-11-01

Drought is one of the most important factors limiting plant growth and development. We identified a gene in wheat (Triticum aestivum L.) under drought stress named TaFBA1. TaFBA1 encodes a putative 325-amino-acid F-box protein with a conserved N-terminal F-box domain and a C-terminal AMN1 domain. Real-time RT-PCR analysis revealed that TaFBA1 transcript accumulation was upregulated by high-salinity, water stress, and abscisic acid (ABA) treatment. To evaluate the functions of TaFBA1 in the regulation of drought stress responses, we produced transgenic tobacco lines overexpressing TaFBA1. Under water stress conditions, the transgenic tobacco plants had a higher germination rate, higher relative water content, net photosynthesis rate (Pn), less chlorophyll loss, and less growth inhibition than WT. These results demonstrate the high tolerance of the transgenic plants to drought stress compared to the WT. The enhanced oxidative stress tolerance of these plants, which may be involved in their drought tolerance, was indicated by their lower levels of reactive oxygen species (ROS) accumulation, MDA content, and cell membrane damage under drought stress compared to WT. The antioxidant enzyme activities were higher in the transgenic plants than in WT, which may be related to the upregulated expression of some antioxidant genes via overexpression of TaFBA1. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Constitutive Transcription and Stable RNA Accumulation in Plastids during the Conversion of Chloroplasts to Chromoplasts in Ripening Tomato Fruits 1

PubMed Central

Marano, María Rosa; Carrillo, Néstor

1992-01-01

The size distribution of plastid transcripts during chromoplast differentiation in ripening tomato (Lycopersicon esculentum L.) fruits was determined using northern blot analysis. Hybridization of total cellular RNA from leaves and fruits with several tobacco chloroplast DNA probes showed distinct transcript patterns in chloroplasts and chromoplasts. We also compared transcriptional rates by probing immobilized DNA fragments of small size (representing about 85% of the plastid genome) with run-on transcripts from tomato plastids. The relative rates of transcription of the various DNA regions were very similar in chloro- and chromoplasts. Parallel determination of the steady-state levels of plastid RNA showed no strict correlation between synthesis rate and RNA accumulation. Differences in the relative abundance of transcripts between chloro- and chromoplasts were not very pronounced and were limited to a small number of genes. The results indicate that the conversion of chloroplasts to chromoplasts at the onset of tomato fruit ripening proceeds with no important variations in the relative transcription rates and with only moderate changes in the relative stability of plastid-encoded transcripts. Images Figure 1 Figure 4 PMID:16653091

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

PubMed Central

Chambouvet, Aurélie; Milner, David S.; Attah, Victoria; Terrado, Ramón; Lovejoy, Connie; Moreau, Hervé; Derelle, Évelyne; Richards, Thomas A.

2017-01-01

Phytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer. PMID:28827361

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

[High gene conversion frequency between genes encoding 2-deoxyglucose-6-phosphate phosphatase in 3 Saccharomyces species].

PubMed

Piscopo, Sara-Pier; Drouin, Guy

2014-05-01

Gene conversions are nonreciprocal sequence exchanges between genes. They are relatively common in Saccharomyces cerevisiae, but few studies have investigated the evolutionary fate of gene conversions or their functional impacts. Here, we analyze the evolution and impact of gene conversions between the two genes encoding 2-deoxyglucose-6-phosphate phosphatase in S. cerevisiae, Saccharomyces paradoxus and Saccharomyces mikatae. Our results demonstrate that the last half of these genes are subject to gene conversions among these three species. The greater similarity and the greater percentage of GC nucleotides in the converted regions, as well as the absence of long regions of adjacent common converted sites, suggest that these gene conversions are frequent and occur independently in all three species. The high frequency of these conversions probably result from the fact that they have little impact on the protein sequences encoded by these genes.

Comparative genome analysis reveals genetic adaptation to versatile environmental conditions and importance of biofilm lifestyle in Comamonas testosteroni.

PubMed

Wu, Yichao; Arumugam, Krithika; Tay, Martin Qi Xiang; Seshan, Hari; Mohanty, Anee; Cao, Bin

2015-04-01

Comamonas testosteroni is an important environmental bacterium capable of degrading a variety of toxic aromatic pollutants and has been demonstrated to be a promising biocatalyst for environmental decontamination. This organism is often found to be among the primary surface colonizers in various natural and engineered ecosystems, suggesting an extraordinary capability of this organism in environmental adaptation and biofilm formation. The goal of this study was to gain genetic insights into the adaption of C. testosteroni to versatile environments and the importance of a biofilm lifestyle. Specifically, a draft genome of C. testosteroni I2 was obtained. The draft genome is 5,778,710 bp in length and comprises 110 contigs. The average G+C content was 61.88 %. A total of 5365 genes with 5263 protein-coding genes were predicted, whereas 4324 (80.60 % of total genes) protein-encoding genes were associated with predicted functions. The catabolic genes responsible for biodegradation of steroid and other aromatic compounds on draft genome were identified. Plasmid pI2 was found to encode a complete pathway for aniline degradation and a partial catabolic pathway for chloroaniline. This organism was found to be equipped with a sophisticated signaling system which helps it find ideal niches and switch between planktonic and biofilm lifestyles. A large number of putative multi-drug-resistant genes coding for abundant outer membrane transporters, chaperones, and heat shock proteins for the protection of cellular function were identified in the genome of strain I2. In addition, the genome of strain I2 was predicted to encode several proteins involved in producing, secreting, and uptaking siderophores under iron-limiting conditions. The genome of strain I2 contains a number of genes responsible for the synthesis and secretion of exopolysaccharides, an extracellular component essential for biofilm formation. Overall, our results reveal the genomic features underlying the adaption of C. testosteroni to versatile environments and highlighting the importance of its biofilm lifestyle.

Lentiviral hematopoietic cell gene therapy for X-linked adrenoleukodystrophy.

PubMed

Cartier, Nathalie; Hacein-Bey-Abina, Salima; Bartholomae, Cynthia C; Bougnères, Pierre; Schmidt, Manfred; Kalle, Christof Von; Fischer, Alain; Cavazzana-Calvo, Marina; Aubourg, Patrick

2012-01-01

X-linked adrenoleukodystrophy (X-ALD) is a severe genetic demyelinating disease caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. When performed at an early stage of the disease, allogeneic hematopoietic stem cell transplantation (HCT) can arrest the progression of cerebral demyelinating lesions. To overcome the limitations of allogeneic HCT, hematopoietic stem cell (HSC) gene therapy strategy aiming to perform autologous transplantation of lentivirally corrected cells was developed. We demonstrated the preclinical feasibility of HSC gene therapy for ALD based on the correction of CD34+ cells from X-ALD patients using an HIV1-derived lentiviral vector. These results prompted us to initiate an HSC gene therapy trial in two X-ALD patients who had developed progressive cerebral demyelination, were candidates for allogeneic HCT, but had no HLA-matched donors or cord blood. Autologous CD34+ cells were purified from the peripheral blood after G-CSF stimulation, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1 cDNA, and then reinfused into the patients after they had received full myeloablative conditioning. Over 3 years of follow-up, the hematopoiesis remained polyclonal in the two patients treated with 7-14% of granulocytes, monocytes, and T and B lymphocytes expressing the lentivirally encoded ALD protein. There was no evidence of clonal dominance or skewing based on the retrieval of lentiviral insertion repertoire in different hematopoietic lineages by deep sequencing. Cerebral demyelination was arrested 14 and 16months, respectively, in the two treated patients, without further progression up to the last follow-up, a clinical outcome that is comparable to that observed after allogeneic HCT. Longer follow-up of these two treated patients and HSC gene therapy performed in additional ALD patients are however needed to evaluate the safety and efficacy of lentiviral HSC gene therapy in cerebral forms of X-ALD. Copyright © 2012 Elsevier Inc. All rights reserved.

The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS.

PubMed

Dandoy, Damien; Fremaux, Christophe; de Frahan, Marie Henry; Horvath, Philippe; Boyaval, Patrick; Hols, Pascal; Fontaine, Laetitia

2011-08-30

In industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products. In the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transferred by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus. We have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.

Comparative genomics of the lactic acid bacteria

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

Makarova, K.; Slesarev, A.; Wolf, Y.

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive genemore » loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.« less

Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

Treesearch

Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

2003-01-01

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

The transcriptomic response of mixed neuron-glial cell cultures to 1,25-dihydroxyvitamin d3 includes genes limiting the progression of neurodegenerative diseases.

PubMed

Nissou, Marie-France; Brocard, Jacques; El Atifi, Michèle; Guttin, Audrey; Andrieux, Annie; Berger, François; Issartel, Jean-Paul; Wion, Didier

2013-01-01

Seasonal or chronic vitamin D deficiency and/or insufficiency is highly prevalent in the human population. Receptors for 1,25-dihydroxyvitamin D3, the hormonal metabolite of vitamin D, are found throughout the brain. To provide further information on the role of this hormone on brain function, we analyzed the transcriptomic profiles of mixed neuron-glial cell cultures in response to 1,25-dihydroxyvitamin D3. 1,25-dihydroxyvitamin D3 treatment increases the mRNA levels of 27 genes by at least 1.9 fold. Among them, 17 genes were related to neurodegenerative and psychiatric diseases, or brain morphogenesis. Notably, 10 of these genes encode proteins potentially limiting the progression of Alzheimer's disease. These data provide support for a role of 1,25-dihydroxyvitamin D3 in brain disease prevention. The possible consequences of circannual or chronic vitamin D insufficiencies on a tissue with a low regenerative potential such as the brain should be considered.

A deep auto-encoder model for gene expression prediction.

PubMed

Xie, Rui; Wen, Jia; Quitadamo, Andrew; Cheng, Jianlin; Shi, Xinghua

2017-11-17

Gene expression is a key intermediate level that genotypes lead to a particular trait. Gene expression is affected by various factors including genotypes of genetic variants. With an aim of delineating the genetic impact on gene expression, we build a deep auto-encoder model to assess how good genetic variants will contribute to gene expression changes. This new deep learning model is a regression-based predictive model based on the MultiLayer Perceptron and Stacked Denoising Auto-encoder (MLP-SAE). The model is trained using a stacked denoising auto-encoder for feature selection and a multilayer perceptron framework for backpropagation. We further improve the model by introducing dropout to prevent overfitting and improve performance. To demonstrate the usage of this model, we apply MLP-SAE to a real genomic datasets with genotypes and gene expression profiles measured in yeast. Our results show that the MLP-SAE model with dropout outperforms other models including Lasso, Random Forests and the MLP-SAE model without dropout. Using the MLP-SAE model with dropout, we show that gene expression quantifications predicted by the model solely based on genotypes, align well with true gene expression patterns. We provide a deep auto-encoder model for predicting gene expression from SNP genotypes. This study demonstrates that deep learning is appropriate for tackling another genomic problem, i.e., building predictive models to understand genotypes' contribution to gene expression. With the emerging availability of richer genomic data, we anticipate that deep learning models play a bigger role in modeling and interpreting genomics.

Different Alleles of a Gene Encoding Leucoanthocyanidin Reductase (PaLAR3) Influence Resistance against the Fungus Heterobasidion parviporum in Picea abies1

PubMed Central

Ihrmark, Katarina

2016-01-01

Despite the fact that fungal diseases are a growing menace for conifers in modern silviculture, only a very limited number of molecular markers for pathogen resistance have been validated in conifer species. A previous genetic study indicated that the resistance of Norway spruce (Picea abies) to Heterobasidion annosum s.l., a pathogenic basidiomycete species complex, is linked to a quantitative trait loci that associates with differences in fungal growth in sapwood (FGS) that includes a gene, PaLAR3, which encodes a leucoanthocyanidin reductase. In this study, gene sequences showed the presence of two PaLAR3 allelic lineages in P. abies. Higher resistance was associated with the novel allele, which was found in low frequency in the four P. abies populations that we studied. Norway spruce plants carrying at least one copy of the novel allele showed a significant reduction in FGS after inoculation with Heterobasidion parviporum compared to their half-siblings carrying no copies, indicating dominance of this allele. The amount of (+) catechin, the enzymatic product of PaLAR3, was significantly higher in bark of trees homozygous for the novel allele. Although we observed that the in vitro activities of the enzymes encoded by the two alleles were similar, we could show that allele-specific transcript levels were significantly higher for the novel allele, indicating that regulation of gene expression is responsible for the observed effects in resistance, possibly caused by differences in cis-acting elements that we observe in the promoter region of the two alleles. PMID:27317690

The VBP and a1/EBP leucine zipper factors bind overlapping subsets of avian retroviral long terminal repeat CCAAT/enhancer elements.

PubMed

Smith, C D; Baglia, L A; Curristin, S M; Ruddell, A

1994-10-01

Two long terminal repeat (LTR) enhancer-binding proteins which may regulate high rates of avian leukosis virus (ALV) LTR-enhanced c-myc transcription during bursal lymphomagenesis have been identified (A. Ruddell, M. Linial, and M. Groudine, Mol. Cell. Biol. 9:5660-5668, 1989). The genes encoding the a1/EBP and a3/EBP binding factors were cloned by expression screening of a lambda gt11 cDNA library from chicken bursal lymphoma cells. The a1/EBP cDNA encodes a novel leucine zipper transcription factor (W. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). The partial a3/EBP cDNA clone encodes amino acids 84 to 313 of vitellogenin gene-binding protein (VBP), a leucine zipper factor that binds the avian vitellogenin II gene promoter (S. Iyer, D. Davis, and J. Burch, Mol. Cell. Biol. 11:4863-4875, 1991). Multiple VBP mRNAs are expressed in B cells in a pattern identical to that previously observed for VBP in other cell types. The LTR-binding activities of VBP, a1/EBP, and B-cell nuclear extract protein were compared and mapped by gel shift, DNase I footprinting, and methylation interference assays. The purified VBP and a1/EBP bacterial fusion proteins bind overlapping but distinct subsets of CCAAT/enhancer elements in the closely related ALV and Rous sarcoma virus (RSV) LTR enhancers. Protein binding to these CCAAT/enhancer elements accounts for most of the labile LTR enhancer-binding activity observed in B-cell nuclear extracts. VBP and a1/EBP could mediate the high rates of ALV and RSV LTR-enhanced transcription in bursal lymphoma cells and many other cell types.

Loss of braking signals during inflammation: a factor affecting the development and disease course of multiple sclerosis.

PubMed

Gilli, Francesca; Navone, Nicole Désirée; Perga, Simona; Marnetto, Fabiana; Caldano, Marzia; Capobianco, Marco; Pulizzi, Annalisa; Malucchi, Simona; Bertolotto, Antonio

2011-07-01

In a recent genome-wide transcriptional analysis, we identified a gene signature for multiple sclerosis (MS), which reverted back to normal during pregnancy. Reversion was particularly evident for 7 genes: SOCS2, TNFAIP3, NR4A2, CXCR4, POLR2J, FAM49B, and STAG3L1, most of which encode negative regulators of inflammation. To corroborate dysregulation of genes, to evaluate the prognostic value of genes, and to study modulation of genes during different treatments. Comparison study. Italian referral center for MS. Quantitative polymerase chain reaction measurements were performed for 274 patients with MS and 60 healthy controls. Of the 274 patients with MS, 113 were treatment-naive patients in the initial stages of their disorder who were followed up in real-world clinical settings and categorized on the basis of disease course. The remaining 161 patients with MS received disease-modifying therapies (55 patients were treated with interferon beta, 52 with glatiramer acetate, and 54 with natalizumab) for a mean (SD) of 12 (2) months. Gene expression levels, relapse rate, and change in Expanded Disability Status Scale. We found a dysregulated gene pathway (P ≤ .006), with a downregulation of genes encoding negative regulators. The SOCS2, NR4A2, and TNFAIP3 genes were inversely correlated with both relapse rate (P ≤ .002) and change in Expanded Disability Status Scale (P ≤ .005). SOCS2 was modulated by both interferon beta and glatiramer acetate, TNFAIP3 was modulated by glatiramer acetate, and NR4A2 was not altered at all. No changes were induced by natalizumab. We demonstrate that there is a new molecular pathogenic mechanism that underlies the initiation and progression of MS. Defects in negative-feedback loops of inflammation lead to an overactivation of the immune system so as to predispose the brain to inflammation-sensitive MS.

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.

Is Mutation Random or Targeted?: No Evidence for Hypermutability in Snail Toxin Genes.

PubMed

Roy, Scott W

2016-10-01

Ever since Luria and Delbruck, the notion that mutation is random with respect to fitness has been foundational to modern biology. However, various studies have claimed striking exceptions to this rule. One influential case involves toxin-encoding genes in snails of the genus Conus, termed conotoxins, a large gene family that undergoes rapid diversification of their protein-coding sequences by positive selection. Previous reconstructions of the sequence evolution of conotoxin genes claimed striking patterns: (1) elevated synonymous change, interpreted as being due to targeted "hypermutation" in this region; (2) elevated transversion-to-transition ratios, interpreted as reflective of the particular mechanism of hypermutation; and (3) much lower rates of synonymous change in the codons encoding several highly conserved cysteine residues, interpreted as strong position-specific codon bias. This work has spawned a variety of studies on the potential mechanisms of hypermutation and on causes for cysteine codon bias, and has inspired hypermutation hypotheses for various other fast-evolving genes. Here, I show that all three findings are likely to be artifacts of statistical reconstruction. First, by simulating nonsynonymous change I show that high rates of dN can lead to overestimation of dS. Second, I show that there is no evidence for any of these three patterns in comparisons of closely related conotoxin sequences, suggesting that the reported findings are due to breakdown of statistical methods at high levels of sequence divergence. The current findings suggest that mutation and codon bias in conotoxin genes may not be atypical, and that random mutation and selection can explain the evolution of even these exceptional loci. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Molecular evolution of nitrogen assimilatory enzymes in marine prasinophytes.

PubMed

Ghoshroy, Sohini; Robertson, Deborah L

2015-01-01

Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex history is observed. Phylogenetic analyses suggest that genes encoding glutamine synthetase (GS) III in the prasinophytes evolved by horizontal gene transfer from a member of the heterokonts. In contrast, genes encoding GSIIE, a canonical vascular plant and green algal enzyme, were found in the Micromonas genomes but have been lost from Ostreococcus. Phylogenetic analyses placed the Micromonas GSIIs in a larger chlorophyte/vascular plant clade; a similar topology was observed for ferredoxin-dependent nitrite reductase (Fd-NiR), indicating the genes encoding GSII and Fd-NiR in these prasinophytes evolved via vertical transmission. Our results show that genes encoding the nitrogen-assimilating enzymes in Micromonas and Ostreococcus have been differentially lost and as well as recruited from different evolutionary lineages, suggesting that the regulation of nitrogen assimilation in prasinophytes will differ from other green algae.

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.

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones

PubMed Central

Glass, Jennifer B.; Kretz, Cecilia B.; Ganesh, Sangita; Ranjan, Piyush; Seston, Sherry L.; Buck, Kristen N.; Landing, William M.; Morton, Peter L.; Moffett, James W.; Giovannoni, Stephen J.; Vergin, Kevin L.; Stewart, Frank J.

2015-01-01

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO3−, NO2−, Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu. PMID:26441925

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones.

PubMed

Glass, Jennifer B; Kretz, Cecilia B; Ganesh, Sangita; Ranjan, Piyush; Seston, Sherry L; Buck, Kristen N; Landing, William M; Morton, Peter L; Moffett, James W; Giovannoni, Stephen J; Vergin, Kevin L; Stewart, Frank J

2015-01-01

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO[Formula: see text], NO[Formula: see text], Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu.

Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae).

PubMed

Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M; Zhang, Shuyi

2014-01-01

Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

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

RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance

PubMed Central

Gong, Zhizhong; Lee, Hojoung; Xiong, Liming; Jagendorf, André; Stevenson, Becky; Zhu, Jian-Kang

2002-01-01

Susceptibility to chilling injury prevents the cultivation of many important crops and limits the extended storage of horticultural commodities. Although freezing tolerance is acquired through cold-induced gene expression changes mediated in part by the CBF family of transcriptional activators, whether plant chilling resistance or sensitivity involves the CBF genes is not known. We report here that an Arabidopsis thaliana mutant impaired in the cold-regulated expression of CBF genes and their downstream target genes is sensitive to chilling stress. Expression of CBF3 under a strong constitutive promoter restores chilling resistance to the mutant plants. The mutated gene was cloned and found to encode a nuclear localized RNA helicase. Our results identify a regulator of CBF genes, and demonstrate the importance of gene regulation and the CBF transcriptional activators in plant chilling resistance. PMID:12165572

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

PubMed

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-08-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

PubMed Central

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G.; Corydon, Thomas J.; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-01-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo. PMID:26204415

Screening for ATM Mutations in an African-American Population to Identify a Predictor of Breast Cancer Susceptibility

DTIC Science & Technology

2006-07-01

ATM genetic variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for each mutation examined. 15. SUBJECT...women without breast cancer. An additional objective is to determine the functional impact upon the protein encoded by the ATM gene for each mutation ...each ATM variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for mutations identified. Body STATEMENT

Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

PubMed

Suter, T M; Viswanathan, V K; Cianciotto, N P

1997-06-01

A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae.

Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

PubMed Central

Suter, T M; Viswanathan, V K; Cianciotto, N P

1997-01-01

A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae. PMID:9174205

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.

Escherichia coli yjjPB genes encode a succinate transporter important for succinate production.

PubMed

Fukui, Keita; Nanatani, Kei; Hara, Yoshihiko; Yamakami, Suguru; Yahagi, Daiki; Chinen, Akito; Tokura, Mitsunori; Abe, Keietsu

2017-09-01

Under anaerobic conditions, Escherichia coli produces succinate from glucose via the reductive tricarboxylic acid cycle. To date, however, no genes encoding succinate exporters have been established in E. coli. Therefore, we attempted to identify genes encoding succinate exporters by screening an E. coli MG1655 genome library. We identified the yjjPB genes as candidates encoding a succinate transporter, which enhanced succinate production in Pantoea ananatis under aerobic conditions. A complementation assay conducted in Corynebacterium glutamicum strain AJ110655ΔsucE1 demonstrated that both YjjP and YjjB are required for the restoration of succinate production. Furthermore, deletion of yjjPB decreased succinate production in E. coli by 70% under anaerobic conditions. Taken together, these results suggest that YjjPB constitutes a succinate transporter in E. coli and that the products of both genes are required for succinate export.

RNAi-mediated resistance to rice black-streaked dwarf virus in transgenic rice.

PubMed

Ahmed, Mohamed M S; Bian, Shiquan; Wang, Muyue; Zhao, Jing; Zhang, Bingwei; Liu, Qiaoquan; Zhang, Changquan; Tang, Shuzhu; Gu, Minghong; Yu, Hengxiu

2017-04-01

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, causes significant economic losses in rice production in China and many other Asian countries. Development of resistant varieties by using conventional breeding methods is limited, as germplasm with high level of resistance to RBSDV have not yet been found. One of the most promising methods to confer resistance against RBSDV is the use of RNA interference (RNAi) technology. RBSDV non-structural protein P7-2, encoded by S7-2 gene, is a potential F-box protein and involved in the plant-virus interaction through the ubiquitination pathway. P8, encoded by S8 gene, is the minor core protein that possesses potent active transcriptional repression activity. In this study, we transformed rice calli using a mini-twin T-DNA vector harboring RNAi constructs of the RBSDV genes S7-2 or S8, and obtained plants harboring the target gene constructs and the selectable marker gene, hygromycin phosphotransferase (HPT). From the offspring of these transgenic plants, we obtained selectable marker (HPT gene)-free plants. Homozygous T 5 transgenic lines which harbored either S7-2-RNAi or S8-RNAi exhibited high level resistance against RBSDV under field infection pressure from indigenous viruliferous small brown planthoppers. Thus, our results showed that RNA interference with the expression of S7-2 or S8 genes seemed an effective way to induce high level resistance in rice against RBSD disease.

Origins of neurogenesis, a cnidarian view.

PubMed

Galliot, Brigitte; Quiquand, Manon; Ghila, Luiza; de Rosa, Renaud; Miljkovic-Licina, Marijana; Chera, Simona

2009-08-01

New perspectives on the origin of neurogenesis emerged with the identification of genes encoding post-synaptic proteins as well as many "neurogenic" regulators as the NK, Six, Pax, bHLH proteins in the Demosponge genome, a species that might differentiate sensory cells but no neurons. However, poriferans seem to miss some key regulators of the neurogenic circuitry as the Hox/paraHox and Otx-like gene families. Moreover as a general feature, many gene families encoding evolutionarily-conserved signaling proteins and transcription factors were submitted to a wave of gene duplication in the last common eumetazoan ancestor, after Porifera divergence. In contrast gene duplications in the last common bilaterian ancestor, Urbilateria, are limited, except for the bHLH Atonal-class. Hence Cnidaria share with Bilateria a large number of genetic tools. The expression and functional analyses currently available suggest a neurogenic function for numerous orthologs in developing or adult cnidarians where neurogenesis takes place continuously. As an example, in the Hydra polyp, the Clytia medusa and the Acropora coral, the Gsx/cnox2/Anthox-2 ParaHox gene likely supports neurogenesis. Also neurons and nematocytes (mechanosensory cells) share in hydrozoans a common stem cell and several regulatory genes indicating that they can be considered as sister cells. Performed in anthozoan and medusozoan species, these studies should tell us more about the way(s) evolution hazards achieved the transition from epithelial to neuronal cell fate, and about the robustness of the genetic circuitry that allowed neuromuscular transmission to arise and be maintained across evolution.

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

Experimental verification of a predicted novel microRNA located in human PIK3CA gene with a potential oncogenic function in colorectal cancer.

PubMed

Saleh, Ali Jason; Soltani, Bahram M; Dokanehiifard, Sadat; Medlej, Abdallah; Tavalaei, Mahmoud; Mowla, Seyed Javad

2016-10-01

PI3K/AKT signaling is involved in cell survival, proliferation, and migration. In this pathway, PI3Kα enzyme is composed of a regulatory protein encoded by p85 gene and a catalytic protein encoded by PIK3CA gene. Human PIK3CA locus is amplified in several cancers including lung and colorectal cancer (CRC). Therefore, microRNAs (miRNAs) that are encoded within the PIK3CA gene might have a role in cancer development. Here, we report a novel microRNA named PIK3CA-miR1 (EBI accession no. LN626315), which is located within PIK3CA gene. A DNA segment corresponding to PIK3CA-premir1 sequence was transfected in human cell lines that resulted in generation of mature exogenous PIK3CA-miR1. Following the overexpression of PIK3CA-miR1, its predicted target genes (APPL1 and TrkC) were significantly downregulated in the CRC-originated HCT116 and SW480 cell lines, detected by qRT-PCR. Then, dual luciferase assay supported the interaction of PIK3CA-miR1 with APPL1 and TrkC transcripts. Endogenous PIK3CA-miR1 expression was also detected in several cell lines (highly in HCT116 and SW480) and highly in CRC specimens. Consistently, overexpression of PIK3CA-premir1 in HCT116 and SW480 cells resulted in significant reduction of the sub-G1 cell distribution and apoptotic cell rate, as detected by flowcytometry, and resulted in increased cell proliferation, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PIK3CA-miR1 overexpression also resulted in Wnt signaling upregulation detected by Top/Fop assay. Overall, accumulative evidences indicated the presence of a bona fide novel onco-miRNA encoded within the PIK3CA oncogene, which is highly expressed in colorectal cancer and has a survival effect in CRC-originated cells.

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

PubMed

Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

1992-01-01

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

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.

Computational screening of disease-associated mutations in OCA2 gene.

PubMed

Kamaraj, Balu; Purohit, Rituraj

2014-01-01

Oculocutaneous albinism type 2 (OCA2), caused by mutations of OCA2 gene, is an autosomal recessive disorder characterized by reduced biosynthesis of melanin pigment in the skin, hair, and eyes. The OCA2 gene encodes instructions for making a protein called the P protein. This protein plays a crucial role in melanosome biogenesis, and controls the eumelanin content in melanocytes in part via the processing and trafficking of tyrosinase which is the rate-limiting enzyme in melanin synthesis. In this study we analyzed the pathogenic effect of 95 non-synonymous single nucleotide polymorphisms reported in OCA2 gene using computational methods. We found R305W mutation as most deleterious and disease associated using SIFT, PolyPhen, PANTHER, PhD-SNP, Pmut, and MutPred tools. To understand the atomic arrangement in 3D space, the native and mutant (R305W) structures were modeled. Molecular dynamics simulation was conducted to observe the structural significance of computationally prioritized disease-associated mutation (R305W). Root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessibility surface area, hydrogen bond (NH bond), trace of covariance matrix, eigenvector projection analysis, and density analysis results showed prominent loss of stability and rise in mutant flexibility values in 3D space. This study presents a well designed computational methodology to examine the albinism-associated SNPs.

Differential response of ammonia-oxidizing archaea and bacteria to the wetting of salty arid soil.

PubMed

Sher, Yonatan; Ronen, Zeev; Nejidat, Ali

2016-08-01

Ammonia-oxidizing archaea and bacteria (AOA, AOB) catalyze the first and rate-limiting step of nitrification. To examine their differential responses to the wetting of dry and salty arid soil, AOA and AOB amoA genes (encoding subunit A of the ammonia monooxygenase) and transcripts were enumerated in dry (summer) and wet (after the first rainfall) soil under the canopy of halophytic shrubs and between the shrubs. AOA and AOB were more abundant under shrub canopies than between shrubs in both the dry and wetted soil. Soil wetting caused a significant decrease in AOB abundance under the canopy and an increase of AOA between the shrubs. The abundance of the archaeal amoA gene transcript was similar for both the wet and dry soil, and the transcript-to-gene ratios were < 1 independent of niche or water content. In contrast, the bacterial amoA transcript-to-gene ratios were between 78 and 514. The lowest ratio was in dry soil under the canopy and the highest in the soil between the shrubs. The results suggest that the AOA are more resilient to stress conditions and maintain a basic activity in arid ecosystems, while the AOB are more responsive to changes in the biotic and abiotic conditions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Arxula adeninivorans (Blastobotrys adeninivorans) — A Dimorphic Yeast of Great Biotechnological Potential

NASA Astrophysics Data System (ADS)

Böer, Erik; Steinborn, Gerhard; Florschütz, Kristina; Körner, Martina; Gellissen, Gerd; Kunze, Gotthard

The dimorphic ascomycetous yeast Arxula adeninivorans exhibits some unusual properties. Being a thermo- and halotolerant species it is able to assimilate and ferment many compounds as sole carbon and/or nitrogen source. It utilises n-alkanes and is capable of degrading starch. Due to these unusual biochemical properties A. adeninivorans can be exploited as a gene donor for the production of enzymes with attractive biotechnological characteristics. Examples of A. adeninivorans-derived genes that are overexpressed include the ALIP1 gene encoding a secretory lipase, the AINV encoding invertase, the AXDH encoding xylitol dehydrogenase and the APHY encoding a secretory phosphatase with phytase activity.

Size-fraction partitioning of community gene transcription and nitrogen metabolism in a marine oxygen minimum zone.

PubMed

Ganesh, Sangita; Bristow, Laura A; Larsen, Morten; Sarode, Neha; Thamdrup, Bo; Stewart, Frank J

2015-12-01

The genetic composition of marine microbial communities varies at the microscale between particle-associated (PA; >1.6 μm) and free-living (FL; 0.2-1.6 μm) niches. It remains unclear, however, how metabolic activities differ between PA and FL fractions. We combined rate measurements with metatranscriptomics to quantify PA and FL microbial activity in the oxygen minimum zone (OMZ) of the Eastern Tropical North Pacific, focusing on dissimilatory processes of the nitrogen (N) cycle. Bacterial gene counts were 8- to 15-fold higher in the FL compared with the PA fraction. However, rates of all measured N cycle processes, excluding ammonia oxidation, declined significantly following particle (>1.6 μm) removal. Without particles, rates of nitrate reduction to nitrite (1.5-9.4nMNd(-1)) fell to zero and N2 production by denitrification (0.5-1.7nMNd(-1)) and anammox (0.3-1.9nMNd(-1)) declined by 53-85%. The proportional representation of major microbial taxa and N cycle gene transcripts in metatranscriptomes followed fraction-specific trends. Transcripts encoding nitrate reductase were uniform among PA and FL fractions, whereas anammox-associated transcripts were proportionately enriched up to 15-fold in the FL fraction. In contrast, transcripts encoding enzymes for N2O and N2 production by denitrification were enriched up to 28-fold in PA samples. These patterns suggest that the majority of N cycle activity, excluding N2O and N2 production by denitrification, is confined to a FL majority that is critically dependent on access to particles, likely as a source of organic carbon and inorganic N. Variable particle distributions may drive heterogeneity in N cycle activity and gene expression in OMZs.

Size-fraction partitioning of community gene transcription and nitrogen metabolism in a marine oxygen minimum zone

PubMed Central

Ganesh, Sangita; Bristow, Laura A; Larsen, Morten; Sarode, Neha; Thamdrup, Bo; Stewart, Frank J

2015-01-01

The genetic composition of marine microbial communities varies at the microscale between particle-associated (PA; >1.6 μm) and free-living (FL; 0.2–1.6 μm) niches. It remains unclear, however, how metabolic activities differ between PA and FL fractions. We combined rate measurements with metatranscriptomics to quantify PA and FL microbial activity in the oxygen minimum zone (OMZ) of the Eastern Tropical North Pacific, focusing on dissimilatory processes of the nitrogen (N) cycle. Bacterial gene counts were 8- to 15-fold higher in the FL compared with the PA fraction. However, rates of all measured N cycle processes, excluding ammonia oxidation, declined significantly following particle (>1.6 μm) removal. Without particles, rates of nitrate reduction to nitrite (1.5–9.4nMNd−1) fell to zero and N2 production by denitrification (0.5–1.7nMNd−1) and anammox (0.3–1.9nMNd−1) declined by 53–85%. The proportional representation of major microbial taxa and N cycle gene transcripts in metatranscriptomes followed fraction-specific trends. Transcripts encoding nitrate reductase were uniform among PA and FL fractions, whereas anammox-associated transcripts were proportionately enriched up to 15-fold in the FL fraction. In contrast, transcripts encoding enzymes for N2O and N2 production by denitrification were enriched up to 28-fold in PA samples. These patterns suggest that the majority of N cycle activity, excluding N2O and N2 production by denitrification, is confined to a FL majority that is critically dependent on access to particles, likely as a source of organic carbon and inorganic N. Variable particle distributions may drive heterogeneity in N cycle activity and gene expression in OMZs. PMID:25848875

Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose.

PubMed

Chin, Young-Wook; Park, Jin-Byung; Park, Yong-Cheol; Kim, Kyoung Heon; Seo, Jin-Ho

2013-06-01

Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (GDP)-L-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-L-fucose from GDP-D-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-L-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-D-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-L-fucose at the specific rate of 0.11 mg g cell(-1) h(-1). The specific GDP-L-fucose content reached 5.5 mg g cell(-1), which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.

Dissecting Molecular Evolution in the Highly Diverse Plant Clade Caryophyllales Using Transcriptome Sequencing

PubMed Central

Yang, Ya; Moore, Michael J.; Brockington, Samuel F.; Soltis, Douglas E.; Wong, Gane Ka-Shu; Carpenter, Eric J.; Zhang, Yong; Chen, Li; Yan, Zhixiang; Xie, Yinlong; Sage, Rowan F.; Covshoff, Sarah; Hibberd, Julian M.; Nelson, Matthew N.; Smith, Stephen A.

2015-01-01

Many phylogenomic studies based on transcriptomes have been limited to “single-copy” genes due to methodological challenges in homology and orthology inferences. Only a relatively small number of studies have explored analyses beyond reconstructing species relationships. We sampled 69 transcriptomes in the hyperdiverse plant clade Caryophyllales and 27 outgroups from annotated genomes across eudicots. Using a combined similarity- and phylogenetic tree-based approach, we recovered 10,960 homolog groups, where each was represented by at least eight ingroup taxa. By decomposing these homolog trees, and taking gene duplications into account, we obtained 17,273 ortholog groups, where each was represented by at least ten ingroup taxa. We reconstructed the species phylogeny using a 1,122-gene data set with a gene occupancy of 92.1%. From the homolog trees, we found that both synonymous and nonsynonymous substitution rates in herbaceous lineages are up to three times as fast as in their woody relatives. This is the first time such a pattern has been shown across thousands of nuclear genes with dense taxon sampling. We also pinpointed regions of the Caryophyllales tree that were characterized by relatively high frequencies of gene duplication, including three previously unrecognized whole-genome duplications. By further combining information from homolog tree topology and synonymous distance between paralog pairs, phylogenetic locations for 13 putative genome duplication events were identified. Genes that experienced the greatest gene family expansion were concentrated among those involved in signal transduction and oxidoreduction, including a cytochrome P450 gene that encodes a key enzyme in the betalain synthesis pathway. Our approach demonstrates a new approach for functional phylogenomic analysis in nonmodel species that is based on homolog groups in addition to inferred ortholog groups. PMID:25837578

The CIDEA gene V115F polymorphism is associated with obesity in Swedish subjects.

PubMed

Dahlman, Ingrid; Kaaman, Maria; Jiao, Hong; Kere, Juha; Laakso, Markku; Arner, Peter

2005-10-01

The cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) gene is implicated as an important regulator of body weight in mice and humans and is therefore a candidate gene for human obesity. Here, we characterize common CIDEA gene polymorphisms and investigate them for association with obesity in two independent Swedish samples; the first comprised 981 women and the second 582 men. Both samples display a large variation in BMI. The only detected coding polymorphism encodes an exon 4 V115F amino acid substitution, which is associated with BMI in both sexes (P = 0.021 for women, P = 0.023 for men, and P = 0.0015 for joint analysis). These results support a role for CIDEA alleles in human obesity. CIDEA-deficient mice display higher metabolic rate, and the gene cross-talks with tumor necrosis factor-alpha (TNF-alpha) in fat cells. We hypothesize that CIDEA alleles regulate human obesity through impact on basal metabolic rate and adipocyte TNF-alpha signaling.

Nutrient control of eukaryote cell growth: a systems biology study in yeast.

PubMed

Gutteridge, Alex; Pir, Pinar; Castrillo, Juan I; Charles, Philip D; Lilley, Kathryn S; Oliver, Stephen G

2010-05-24

To elucidate the biological processes affected by changes in growth rate and nutrient availability, we have performed a comprehensive analysis of the transcriptome, proteome and metabolome responses of chemostat cultures of the yeast, Saccharomyces cerevisiae, growing at a range of growth rates and in four different nutrient-limiting conditions. We find significant changes in expression for many genes in each of the four nutrient-limited conditions tested. We also observe several processes that respond differently to changes in growth rate and are specific to each nutrient-limiting condition. These include carbohydrate storage, mitochondrial function, ribosome synthesis, and phosphate transport. Integrating transcriptome data with proteome measurements allows us to identify previously unrecognized examples of post-transcriptional regulation in response to both nutrient and growth-rate signals. Our results emphasize the unique properties of carbon metabolism and the carbon substrate, the limitation of which induces significant changes in gene regulation at the transcriptional and post-transcriptional level, as well as altering how many genes respond to growth rate. By comparison, the responses to growth limitation by other nutrients involve a smaller set of genes that participate in specific pathways. See associated commentary http://www.biomedcentral.com/1741-7007/8/62.

Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes That Cause Thoracic Aortic Aneurysms and Dissections Disrupt This Structure.

PubMed

Karimi, Ashkan; Milewicz, Dianna M

2016-01-01

The medial layer of the aorta confers elasticity and strength to the aortic wall and is composed of alternating layers of smooth muscle cells (SMCs) and elastic fibres. The SMC elastin-contractile unit is a structural unit that links the elastin fibres to the SMCs and is characterized by the following: (1) layers of elastin fibres that are surrounded by microfibrils; (2) microfibrils that bind to the integrin receptors in focal adhesions on the cell surface of the SMCs; and (3) SMC contractile filaments that are linked to the focal adhesions on the inner side of the membrane. The genes that are altered to cause thoracic aortic aneurysms and aortic dissections encode proteins involved in the structure or function of the SMC elastin-contractile unit. Included in this gene list are the genes encoding protein that are structural components of elastin fibres and microfibrils, FBN1, MFAP5, ELN, and FBLN4. Also included are genes that encode structural proteins in the SMC contractile unit, including ACTA2, which encodes SMC-specific α-actin and MYH11, which encodes SMC-specific myosin heavy chain, along with MYLK and PRKG1, which encode kinases that control SMC contraction. Finally, mutations in the gene encoding the protein linking integrin receptors to the contractile filaments, FLNA, also predispose to thoracic aortic disease. Thus, these data suggest that functional SMC elastin-contractile units are important for maintaining the structural integrity of the aorta. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

A Carbon Dioxide Limitation-Inducible Protein, ColA, Supports the Growth of Synechococcus sp. PCC 7002

PubMed Central

Shimakawa, Ginga; Watanabe, Satoru; Miyake, Chikahiro

2017-01-01

A limitation in carbon dioxide (CO2), which occurs as a result of natural environmental variation, suppresses photosynthesis and has the potential to cause photo-oxidative damage to photosynthetic cells. Oxygenic phototrophs have strategies to alleviate photo-oxidative damage to allow life in present atmospheric CO2 conditions. However, the mechanisms for CO2 limitation acclimation are diverse among the various oxygenic phototrophs, and many mechanisms remain to be discovered. In this study, we found that the gene encoding a CO2 limitation-inducible protein, ColA, is required for the cyanobacterium Synechococcus sp. PCC 7002 (S. 7002) to acclimate to limited CO2 conditions. An S. 7002 mutant deficient in ColA (ΔcolA) showed lower chlorophyll content, based on the amount of nitrogen, than that in S. 7002 wild-type (WT) under ambient air but not high CO2 conditions. Both thermoluminescence and protein carbonylation detected in the ambient air grown cells indicated that the lack of ColA promotes oxidative stress in S. 7002. Alterations in the photosynthetic O2 evolution rate and relative electron transport rate in the short-term response, within an hour, to CO2 limitation were the same between the WT and ΔcolA. Conversely, these photosynthetic parameters were mostly lower in the long-term response of a few days in ΔcolA than in the WT. These data suggest that ColA is required to sustain photosynthetic activity for living under ambient air in S. 7002. The unique phylogeny of ColA revealed diverse strategies to acclimate to CO2 limitation among cyanobacteria. PMID:29244744

Using variable rate models to identify genes under selection in sequence pairs: their validity and limitations for EST sequences.

PubMed

Church, Sheri A; Livingstone, Kevin; Lai, Zhao; Kozik, Alexander; Knapp, Steven J; Michelmore, Richard W; Rieseberg, Loren H

2007-02-01

Using likelihood-based variable selection models, we determined if positive selection was acting on 523 EST sequence pairs from two lineages of sunflower and lettuce. Variable rate models are generally not used for comparisons of sequence pairs due to the limited information and the inaccuracy of estimates of specific substitution rates. However, previous studies have shown that the likelihood ratio test (LRT) is reliable for detecting positive selection, even with low numbers of sequences. These analyses identified 56 genes that show a signature of selection, of which 75% were not identified by simpler models that average selection across codons. Subsequent mapping studies in sunflower show four of five of the positively selected genes identified by these methods mapped to domestication QTLs. We discuss the validity and limitations of using variable rate models for comparisons of sequence pairs, as well as the limitations of using ESTs for identification of positively selected genes.

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.

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp.

PubMed Central

Wilbrink, Maarten H.; Casabon, Israël; Stewart, Gordon R.; Liu, Jie; van der Geize, Robert; Eltis, Lindsay D.

2012-01-01

Bile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9α-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism. PMID:23024343

Bach2 Promotes Regulatory T-cell Development by Limiting Effector Differentiation | Center for Cancer Research

Cancer.gov

A number of allergic and autoimmune disorders, such as Crohn’s disease, asthma, type I diabetes and multiple sclerosis, are associated with polymorphisms in a gene encoding the transcription factor, BACH2. Despite this, the mechanism Bach2 uses to prevent immune-mediated diseases was not known. To function appropriately, the immune system relies on a delicate balance between

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

Heterogeneic dynamics of the structures of multiple gene clusters in two pathogenetically different lines originating from the same phytoplasma.

PubMed

Arashida, Ryo; Kakizawa, Shigeyuki; Hoshi, Ayaka; Ishii, Yoshiko; Jung, Hee-Young; Kagiwada, Satoshi; Yamaji, Yasuyuki; Oshima, Kenro; Namba, Shigetou

2008-04-01

Phytoplasmas are phloem-limited plant pathogens that are transmitted by insect vectors and are associated with diseases in hundreds of plant species. Despite their small sizes, phytoplasma genomes have repeat-rich sequences, which are due to several genes that are encoded as multiple copies. These multiple genes exist in a gene cluster, the potential mobile unit (PMU). PMUs are present at several distinct regions in the phytoplasma genome. The multicopy genes encoded by PMUs (herein named mobile unit genes [MUGs]) and similar genes elsewhere in the genome (herein named fundamental genes [FUGs]) are likely to have the same function based on their annotations. In this manuscript we show evidence that MUGs and FUGs do not cluster together within the same clade. Each MUG is in a cluster with a short branch length, suggesting that MUGs are recently diverged paralogs, whereas the origin of FUGs is different from that of MUGs. We also compared the genome structures around the lplA gene in two derivative lines of the 'Candidatus Phytoplasma asteris' OY strain, the severe-symptom line W (OY-W) and the mild-symptom line M (OY-M). The gene organizations of the nucleotide sequences upstream of the lplA genes of OY-W and OY-M were dramatically different. The tra5 insertion sequence, an element of PMUs, was found only in this region in OY-W. These results suggest that transposition of entire PMUs and PMU sections has occurred frequently in the OY phytoplasma genome. The difference in the pathogenicities of OY-W and OY-M might be caused by the duplication and transposition of PMUs, followed by genome rearrangement.

Programming temporal shapeshifting

NASA Astrophysics Data System (ADS)

Hu, Xiaobo; Zhou, Jing; Vatankhah-Varnosfaderani, Mohammad; Daniel, William F. M.; Li, Qiaoxi; Zhushma, Aleksandr P.; Dobrynin, Andrey V.; Sheiko, Sergei S.

2016-09-01

Shapeshifting enables a wide range of engineering and biomedical applications, but until now transformations have required external triggers. This prerequisite limits viability in closed or inert systems and puts forward the challenge of developing materials with intrinsically encoded shape evolution. Herein we demonstrate programmable shape-memory materials that perform a sequence of encoded actuations under constant environment conditions without using an external trigger. We employ dual network hydrogels: in the first network, covalent crosslinks are introduced for elastic energy storage, and in the second one, temporary hydrogen-bonds regulate the energy release rate. Through strain-induced and time-dependent reorganization of the reversible hydrogen-bonds, this dual network allows for encoding both the rate and pathway of shape transformations on timescales from seconds to hours. This generic mechanism for programming trigger-free shapeshifting opens new ways to design autonomous actuators, drug-release systems and active implants.

Jasmonate Regulates Plant Responses to Postsubmergence Reoxygenation through Transcriptional Activation of Antioxidant Synthesis1

PubMed Central

Dai, Yang-Shuo; Xie, Li-Juan; Yu, Lu-Jun; Zhou, Ying; Lai, Yong-Xia; Yang, Yi-Cong; Xu, Le; Chen, Qin-Fang

2017-01-01

Submergence induces hypoxia in plants; exposure to oxygen following submergence, termed reoxygenation, produces a burst of reactive oxygen species. The mechanisms of hypoxia sensing and signaling in plants have been well studied, but how plants respond to reoxygenation remains unclear. Here, we show that reoxygenation in Arabidopsis (Arabidopsis thaliana) involves rapid accumulation of jasmonates (JAs) and increased transcript levels of JA biosynthesis genes. Application of exogenous methyl jasmonate improved tolerance to reoxygenation in wild-type Arabidopsis; also, mutants deficient in JA biosynthesis and signaling were very sensitive to reoxygenation. Moreover, overexpression of the transcription factor gene MYC2 enhanced tolerance to posthypoxic stress, and myc2 knockout mutants showed increased sensitivity to reoxygenation, indicating that MYC2 functions as a key regulator in the JA-mediated reoxygenation response. MYC2 transcriptionally activates members of the VITAMIN C DEFECTIVE (VTC) and GLUTATHIONE SYNTHETASE (GSH) gene families, which encode rate-limiting enzymes in the ascorbate and glutathione synthesis pathways. Overexpression of VTC1 and GSH1 in the myc2-2 mutant suppressed the posthypoxic hypersensitive phenotype. The JA-inducible accumulation of antioxidants may alleviate oxidative damage caused by reoxygenation, improving plant survival after submergence. Taken together, our findings demonstrate that JA signaling interacts with the antioxidant pathway to regulate reoxygenation responses in Arabidopsis. PMID:28082717

Identification and Characterization of Alternative Promoters, Transcripts and Protein Isoforms of Zebrafish R2 Gene

PubMed Central

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5′ termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress. PMID:21887375

Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.

PubMed

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5' termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress.

Inactivation of a Pleurotus ostreatus versatile peroxidase-encoding gene (mnp2) results in reduced lignin degradation.

PubMed

Salame, Tomer M; Knop, Doriv; Levinson, Dana; Mabjeesh, Sameer J; Yarden, Oded; Hadar, Yitzhak

2014-01-01

Lignin biodegradation by white-rot fungi is pivotal to the earth's carbon cycle. Manganese peroxidases (MnPs), the most common extracellular ligninolytic peroxidases produced by white-rot fungi, are considered key in ligninolysis. Pleurotus ostreatus, the oyster mushroom, is a preferential lignin degrader occupying niches rich in lignocellulose such as decaying trees. Here, we provide direct, genetically based proof for the functional significance of MnP to P. ostreatus ligninolytic capacity under conditions mimicking its natural habitat. When grown on a natural lignocellulosic substrate of cotton stalks under solid-state culture conditions, gene and isoenzyme expression profiles of its short MnP and versatile peroxidase (VP)-encoding gene family revealed that mnp2 was predominately expressed. mnp2, encoding the versatile short MnP isoenzyme 2 was disrupted. Inactivation of mnp2 resulted in three interrelated phenotypes, relative to the wild-type strain: (i) reduction of 14% and 36% in lignin mineralization of stalks non-amended and amended with Mn(2+), respectively; (ii) marked reduction of the bioconverted lignocellulose sensitivity to subsequent bacterial hydrolyses; and (iii) decrease in fungal respiration rate. These results may serve as the basis to clarify the roles of the various types of fungal MnPs and VPs in their contribution to white-rot decay of wood and lignocellulose in various ecosystems. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

CIP1 polypeptides and their uses

DOEpatents

Foreman, Pamela [Los Altos, CA; Van Solingen, Pieter [Naaldwijk, NL; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA

2011-04-12

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

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

Relating genes to function: identifying enriched transcription factors using the ENCODE ChIP-Seq significance tool.

PubMed

Auerbach, Raymond K; Chen, Bin; Butte, Atul J

2013-08-01

Biological analysis has shifted from identifying genes and transcripts to mapping these genes and transcripts to biological functions. The ENCODE Project has generated hundreds of ChIP-Seq experiments spanning multiple transcription factors and cell lines for public use, but tools for a biomedical scientist to analyze these data are either non-existent or tailored to narrow biological questions. We present the ENCODE ChIP-Seq Significance Tool, a flexible web application leveraging public ENCODE data to identify enriched transcription factors in a gene or transcript list for comparative analyses. The ENCODE ChIP-Seq Significance Tool is written in JavaScript on the client side and has been tested on Google Chrome, Apple Safari and Mozilla Firefox browsers. Server-side scripts are written in PHP and leverage R and a MySQL database. The tool is available at http://encodeqt.stanford.edu. abutte@stanford.edu Supplementary material is available at Bioinformatics online.

The limits to growth - energetic burden of the endogenous antibiotic tropodithietic acid in Phaeobacter inhibens DSM 17395.

PubMed

Will, Sabine Eva; Neumann-Schaal, Meina; Heydorn, Raymond Leopold; Bartling, Pascal; Petersen, Jörn; Schomburg, Dietmar

2017-01-01

Phaeobacter inhibens DSM 17395, a model organism for marine Roseobacter group, was studied for its response to its own antimicrobial compound tropodithietic acid (TDA). TDA biosynthesis is encoded on the largest extrachromosomal element of P. inhibens, the 262 kb plasmid, whose curation leads to an increased growth and biomass yield. In this study, the plasmid-cured strain was compared to the wild-type strain and to transposon mutants lacking single genes of the TDA biosynthesis. The data show that the growth inhibition of the wild-type strain can be mainly attributed to the TDA produced by P. inhibens itself. Oxygen uptake rates remained constant in all strains but the growth rate dropped in the wild-type which supports the recently proposed mode of TDA action. Metabolome analysis showed no metabolic alterations that could be attributed directly to TDA. Taken together, the growth of P. inhibens is limited by its own antibacterial compound due to a partial destruction of the proton gradient which leads to a higher energetic demand. The universal presence of TDA biosynthesis in genome-sequenced isolates of the genus Phaeobacter shows that there must be a high benefit of TDA for P. inhibens in its ecological niche despite the drawback on its metabolism.

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.

Analysis and Manipulation of Aspartate Pathway Genes for l-Lysine Overproduction from Methanol by Bacillus methanolicus▿

PubMed Central

Nærdal, Ingemar; Netzer, Roman; Ellingsen, Trond E.; Brautaset, Trygve

2011-01-01

We investigated the regulation and roles of six aspartate pathway genes in l-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by l-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the l-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has—in addition to a hom-1 mutation—chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for l-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased l-lysine production levels. PMID:21724876

Analysis and manipulation of aspartate pathway genes for L-lysine overproduction from methanol by Bacillus methanolicus.

PubMed

Nærdal, Ingemar; Netzer, Roman; Ellingsen, Trond E; Brautaset, Trygve

2011-09-01

We investigated the regulation and roles of six aspartate pathway genes in L-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by L-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the L-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has-in addition to a hom-1 mutation-chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for L-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased L-lysine production levels.

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

Computer analysis of protein functional sites projection on exon structure of genes in Metazoa.

PubMed

Medvedeva, Irina V; Demenkov, Pavel S; Ivanisenko, Vladimir A

2015-01-01

Study of the relationship between the structural and functional organization of proteins and their coding genes is necessary for an understanding of the evolution of molecular systems and can provide new knowledge for many applications for designing proteins with improved medical and biological properties. It is well known that the functional properties of proteins are determined by their functional sites. Functional sites are usually represented by a small number of amino acid residues that are distantly located from each other in the amino acid sequence. They are highly conserved within their functional group and vary significantly in structure between such groups. According to this facts analysis of the general properties of the structural organization of the functional sites at the protein level and, at the level of exon-intron structure of the coding gene is still an actual problem. One approach to this analysis is the projection of amino acid residue positions of the functional sites along with the exon boundaries to the gene structure. In this paper, we examined the discontinuity of the functional sites in the exon-intron structure of genes and the distribution of lengths and phases of the functional site encoding exons in vertebrate genes. We have shown that the DNA fragments coding the functional sites were in the same exons, or in close exons. The observed tendency to cluster the exons that code functional sites which could be considered as the unit of protein evolution. We studied the characteristics of the structure of the exon boundaries that code, and do not code, functional sites in 11 Metazoa species. This is accompanied by a reduced frequency of intercodon gaps (phase 0) in exons encoding the amino acid residue functional site, which may be evidence of the existence of evolutionary limitations to the exon shuffling. These results characterize the features of the coding exon-intron structure that affect the functionality of the encoded protein and allow a better understanding of the emergence of biological diversity.

“Guilt by Association” Is the Exception Rather Than the Rule in Gene Networks

PubMed Central

Gillis, Jesse; Pavlidis, Paul

2012-01-01

Gene networks are commonly interpreted as encoding functional information in their connections. An extensively validated principle called guilt by association states that genes which are associated or interacting are more likely to share function. Guilt by association provides the central top-down principle for analyzing gene networks in functional terms or assessing their quality in encoding functional information. In this work, we show that functional information within gene networks is typically concentrated in only a very few interactions whose properties cannot be reliably related to the rest of the network. In effect, the apparent encoding of function within networks has been largely driven by outliers whose behaviour cannot even be generalized to individual genes, let alone to the network at large. While experimentalist-driven analysis of interactions may use prior expert knowledge to focus on the small fraction of critically important data, large-scale computational analyses have typically assumed that high-performance cross-validation in a network is due to a generalizable encoding of function. Because we find that gene function is not systemically encoded in networks, but dependent on specific and critical interactions, we conclude it is necessary to focus on the details of how networks encode function and what information computational analyses use to extract functional meaning. We explore a number of consequences of this and find that network structure itself provides clues as to which connections are critical and that systemic properties, such as scale-free-like behaviour, do not map onto the functional connectivity within networks. PMID:22479173

Factors Influencing the Diversity of Iron Uptake Systems in Aquatic Microorganisms

PubMed Central

Desai, Dhwani K.; Desai, Falguni D.; LaRoche, Julie

2012-01-01

Iron (Fe) is an essential micronutrient for many processes in all living cells. Dissolved Fe (dFe) concentrations in the ocean are of the order of a few nM, and Fe is often a factor limiting primary production. Bioavailability of Fe in aquatic environments is believed to be primarily controlled through chelation by Fe-binding ligands. Marine microbes have evolved different mechanisms to cope with the scarcity of bioavailable dFe. Gradients in dFe concentrations and diversity of the Fe-ligand pool from coastal to open ocean waters have presumably imposed selection pressures that should be reflected in the genomes of microbial communities inhabiting the pelagic realm. We applied a hidden Markov model (HMM)-based search for proteins related to cellular iron metabolism, and in particular those involved in Fe uptake mechanisms in 164 microbial genomes belonging to diverse taxa and occupying different aquatic niches. A multivariate statistical approach demonstrated that in phototrophic organisms, there is a clear influence of the ecological niche on the diversity of Fe uptake systems. Extending the analyses to the metagenome database from the Global Ocean Sampling expedition, we demonstrated that the Fe uptake and homeostasis mechanisms differed significantly across marine niches defined by temperatures and dFe concentrations, and that this difference was linked to the distribution of microbial taxa in these niches. Using the dN/dS ratios (which signify the rate of non-synonymous mutations) of the nucleotide sequences, we identified that genes encoding for TonB, Ferritin, Ferric reductase, IdiA, ZupT, and Fe2+ transport proteins FeoA and FeoB were evolving at a faster rate (positive selection pressure) while genes encoding ferrisiderophore, heme and Vitamin B12 uptake systems, siderophore biosynthesis, and IsiA and IsiB were under purifying selection pressure (evolving slowly). PMID:23087680

Genomic and Transcriptomic Analyses of Indole-3-Acetic Acid Biosynthesis in Diatoms

NASA Astrophysics Data System (ADS)

Lim, R.; Armbrust, V.

2016-02-01

Indole-3-acetic acid (IAA) is a major plant growth hormone and a common mediator of plant-bacterial interactions. Recently, IAA has also been found to play a role in interactions between diatoms and bacteria, with IAA production by an associated Sulfitobacter leading to increased growth rates in the marine diatom Pseudo-nitzschia multiseries. It is unclear, however, if diatoms themselves are able to synthesize IAA and whether this capability is widespread throughout Bacillariophyta. Four major tryptophan-dependent IAA biosynthesis pathways have been identified in plants and bacteria, each denoted by the first intermediate downstream of tryptophan: the indole-3-pyruvate (IPyA), tryptamine (TAM), indole-3-acetaldoxime (IAOx) and indole-3-acetamide (IAM) pathways. To investigate the possibility of IAA biosynthesis in diatoms, we first analyzed publicly available genomes of raphid pennates P. multiseries, Phaeodactylum tricornutum, Fragilariopsis cylindrus and centric Thalassiosira pseudonana for potential homologs to plant and bacterial IAA biosynthesis genes. The P. multiseries, F. cylindrus and P. tricornutum genomes encode downstream enzymes for bacterial TAM and IAM and plant IPyA pathways. The more evolutionarily ancient T. pseudonana encodes one TAM enzyme in its genome. To investigate the potential distribution of these pathways more broadly, we surveyed the transcriptomes of 11 diatom species that include representatives from all four Bacillariophyta classes. Datasets used were sequenced as part of the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) and obtained from cultures maintained axenically. Transcripts associated with the TAM pathway were most frequently detected, with potential homologs to required enzymes identified in 10 of the 11 species examined. Transcripts homologous to rate-limiting IPyA enzymes were detected in six species. Only two centric and araphid pennate species expressed transcripts associated with enzymes in the IAM and IAOx pathways. This pattern suggests multiple events of gene loss as the phylum expanded and diversified. Mass spectrometry analyses will be conducted to confirm the production of IAA in axenic cultures of P. pungens, P. multistriata, Skeletonema marinoi and F. cylindrus.

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.

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity.

PubMed

Abergel, Chantal; Blanc, Guillaume; Monchois, Vincent; Renesto, Patricia; Sigoillot, Cécile; Ogata, Hiroyuki; Raoult, Didier; Claverie, Jean-Michel

2006-11-01

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.

Inflammatory gene polymorphisms and risk of postoperative myocardial infarction after cardiac surgery.

PubMed

Podgoreanu, M V; White, W D; Morris, R W; Mathew, J P; Stafford-Smith, M; Welsby, I J; Grocott, H P; Milano, C A; Newman, M F; Schwinn, D A

2006-07-04

The inflammatory response triggered by cardiac surgery with cardiopulmonary bypass (CPB) is a primary mechanism in the pathogenesis of postoperative myocardial infarction (PMI), a multifactorial disorder with significant inter-patient variability poorly predicted by clinical and procedural factors. We tested the hypothesis that candidate gene polymorphisms in inflammatory pathways contribute to risk of PMI after cardiac surgery. We genotyped 48 polymorphisms from 23 candidate genes in a prospective cohort of 434 patients undergoing elective cardiac surgery with CPB. PMI was defined as creatine kinase-MB isoenzyme level > or = 10x upper limit of normal at 24 hours postoperatively. A 2-step analysis strategy was used: marker selection, followed by model building. To minimize false-positive associations, we adjusted for multiple testing by permutation analysis, Bonferroni correction, and controlling the false discovery rate; 52 patients (12%) experienced PMI. After adjusting for multiple comparisons and clinical risk factors, 3 polymorphisms were found to be independent predictors of PMI (adjusted P<0.05; false discovery rate <10%). These gene variants encode the proinflammatory cytokine interleukin 6 (IL6 -572G>C; odds ratio [OR], 2.47), and 2 adhesion molecules: intercellular adhesion molecule-1 (ICAM1 Lys469Glu; OR, 1.88), and E-selectin (SELE 98G>T; OR, 0.16). The inclusion of genotypic information from these polymorphisms improved prediction models for PMI based on traditional risk factors alone (C-statistic 0.764 versus 0.703). Functional genetic variants in cytokine and leukocyte-endothelial interaction pathways are independently associated with severity of myonecrosis after cardiac surgery. This may aid in preoperative identification of high-risk cardiac surgical patients and development of novel cardioprotective strategies.

Microarray-based comparative genomic profiling of reference strains and selected Canadian field isolates of Actinobacillus pleuropneumoniae

PubMed Central

Gouré, Julien; Findlay, Wendy A; Deslandes, Vincent; Bouevitch, Anne; Foote, Simon J; MacInnes, Janet I; Coulton, James W; Nash, John HE; Jacques, Mario

2009-01-01

Background Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Microarray-based comparative genomic hybridizations (M-CGH) were used to estimate whole genomic diversity of representative Actinobacillus pleuropneumoniae strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines. Results Using hierarchical clustering, our M-CGH results showed that the majority of the genes in the genome of the serovar 5 A. pleuropneumoniae L20 strain were conserved in the reference strains of all 15 serovars and in representative field isolates. Fifty-eight conserved genes predicted to encode for outer membrane proteins or lipoproteins were identified. As well, there were several clusters of diverged or absent genes including those associated with capsule biosynthesis, toxin production as well as genes typically associated with mobile elements. Conclusion Although A. pleuropneumoniae strains are essentially clonal, M-CGH analysis of the reference strains of the fifteen serovars and representative field isolates revealed several classes of genes that were divergent or absent. Not surprisingly, these included genes associated with capsule biosynthesis as the capsule is associated with sero-specificity. Several of the conserved genes were identified as candidates for vaccine development, and we conclude that M-CGH is a valuable tool for reverse vaccinology. PMID:19239696

The utility of DNA sequences of an intron from the beta-fibrinogen gene in phylogenetic analysis of woodpeckers (Aves: Picidae).

PubMed

Prychitko, T M; Moore, W S

1997-10-01

Estimating phylogenies from DNA sequence data has become the major methodology of molecular phylogenetics. To date, molecular phylogenetics of the vertebrates has been very dependent on mtDNA, but studies involving mtDNA are limited because the several genes comprising the mt-genome are inherited as a single linkage group. The only apparent solution to this problem is to sequence additional genes, each representing a distinct linkage group, so that the resultant gene trees provide independent estimates of the species tree. There exists the need to find novel gene sequences which contain enough phylogenetic information to resolve relationships between closely related species. A possible source is the nuclear-encoded introns, because they evolve more rapidly than exons. We designed primers to amplify and sequence the 7 intron from the beta-fibrinogen gene for a recently evolved group, the woodpeckers. We sequenced the entire intron for 10 specimens representing five species. Nucleotide substitutions are randomly distributed along the length of the intron, suggesting selective neutrality. A preliminary analysis indicates that the phylogenetic signal in the intron is as strong as that in the mitochondrial encoded cytochrome b (cyt b) gene. The topology of the beta-fibrinogen tree is identical to that of the cyt b tree. This analysis demonstrates the ability of the 7 intron of beta-fibrinogen to provide well resolved, independent gene trees for recently evolved groups and establishes it as a source of sequences to be used in other phylogenetic studies. Copyright 1997 Academic Press

The human TREM gene cluster at 6p21.1 encodes both activating and inhibitory single IgV domain receptors and includes NKp44.

PubMed

Allcock, Richard J N; Barrow, Alexander D; Forbes, Simon; Beck, Stephan; Trowsdale, John

2003-02-01

We have characterized a cluster of single immunoglobulin variable (IgV) domain receptors centromeric of the major histocompatibility complex (MHC) on human chromosome 6. In addition to triggering receptor expressed on myeloid cells (TREM)-1 and TREM2, the cluster contains NKp44, a triggering receptor whose expression is limited to NK cells. We identified three new related genes and two gene fragments within a cluster of approximately 200 kb. Two of the three new genes lack charged residues in their transmembrane domain tails. Further, one of the genes contains two potential immunotyrosine Inhibitory motifs in its cytoplasmic tail, suggesting that it delivers inhibitory signals. The human and mouse TREM clusters appear to have diverged such that there are unique sequences in each species. Finally, each gene in the TREM cluster was expressed in a different range of cell types.

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.

Systematic asymmetric nucleotide exchanges produce human mitochondrial RNAs cryptically encoding for overlapping protein coding genes.

PubMed

Seligmann, Hervé

2013-05-07

GenBank's EST database includes RNAs matching exactly human mitochondrial sequences assuming systematic asymmetric nucleotide exchange-transcription along exchange rules: A→G→C→U/T→A (12 ESTs), A→U/T→C→G→A (4 ESTs), C→G→U/T→C (3 ESTs), and A→C→G→U/T→A (1 EST), no RNAs correspond to other potential asymmetric exchange rules. Hypothetical polypeptides translated from nucleotide-exchanged human mitochondrial protein coding genes align with numerous GenBank proteins, predicted secondary structures resemble their putative GenBank homologue's. Two independent methods designed to detect overlapping genes (one based on nucleotide contents analyses in relation to replicative deamination gradients at third codon positions, and circular code analyses of codon contents based on frame redundancy), confirm nucleotide-exchange-encrypted overlapping genes. Methods converge on which genes are most probably active, and which not, and this for the various exchange rules. Mean EST lengths produced by different nucleotide exchanges are proportional to (a) extents that various bioinformatics analyses confirm the protein coding status of putative overlapping genes; (b) known kinetic chemistry parameters of the corresponding nucleotide substitutions by the human mitochondrial DNA polymerase gamma (nucleotide DNA misinsertion rates); (c) stop codon densities in predicted overlapping genes (stop codon readthrough and exchanging polymerization regulate gene expression by counterbalancing each other). Numerous rarely expressed proteins seem encoded within regular mitochondrial genes through asymmetric nucleotide exchange, avoiding lengthening genomes. Intersecting evidence between several independent approaches confirms the working hypothesis status of gene encryption by systematic nucleotide exchanges. Copyright © 2013 Elsevier Ltd. All rights reserved.

Analysis of complete genome sequence of Neorickettsia risticii: causative agent of Potomac horse fever

PubMed Central

Lin, Mingqun; Zhang, Chunbin; Gibson, Kathryn; Rikihisa, Yasuko

2009-01-01

Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. sennetsu showed that 758 (88.2%) of protein-coding genes are conserved between N. risticii and N. sennetsu. Four-way comparison of genes among N. risticii and other Anaplasmataceae showed that most genes are either shared among Anaplasmataceae (525 orthologs that generally associated with housekeeping functions), or specific to each genome (>200 genes that are mostly hypothetical proteins). Genes potentially involved in the pathogenesis of N. risticii were identified, including those encoding putative outer membrane proteins, two-component systems and a type IV secretion system (T4SS). The bipolar localization of T4SS pilus protein VirB2 on the bacterial surface was demonstrated for the first time in obligate intracellular bacteria. These data provide insights toward genomic potential of N. risticii and intracellular parasitism, and facilitate our understanding of PHF pathogenesis. PMID:19661282

Analysis of complete genome sequence of Neorickettsia risticii: causative agent of Potomac horse fever.

PubMed

Lin, Mingqun; Zhang, Chunbin; Gibson, Kathryn; Rikihisa, Yasuko

2009-10-01

Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. sennetsu showed that 758 (88.2%) of protein-coding genes are conserved between N. risticii and N. sennetsu. Four-way comparison of genes among N. risticii and other Anaplasmataceae showed that most genes are either shared among Anaplasmataceae (525 orthologs that generally associated with housekeeping functions), or specific to each genome (>200 genes that are mostly hypothetical proteins). Genes potentially involved in the pathogenesis of N. risticii were identified, including those encoding putative outer membrane proteins, two-component systems and a type IV secretion system (T4SS). The bipolar localization of T4SS pilus protein VirB2 on the bacterial surface was demonstrated for the first time in obligate intracellular bacteria. These data provide insights toward genomic potential of N. risticii and intracellular parasitism, and facilitate our understanding of PHF pathogenesis.

The Unique Biosynthetic Route from Lupinus β-Conglutin Gene to Blad

PubMed Central

Monteiro, Sara; Freitas, Regina; Rajasekhar, Baru T.; Teixeira, Artur R.; Ferreira, Ricardo B.

2010-01-01

Background During seed germination, β-conglutin undergoes a major cycle of limited proteolysis in which many of its constituent subunits are processed into a 20 kDa polypeptide termed blad. Blad is the main component of a glycooligomer, accumulating exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Principal Findings The sequence of the gene encoding β-conglutin precursor (1791 nucleotides) is reported. This gene, which shares 44 to 57% similarity and 20 to 37% identity with other vicilin-like protein genes, includes several features in common with these globulins, but also specific hallmarks. Most notable is the presence of an ubiquitin interacting motif (UIM), which possibly links the unique catabolic route of β-conglutin to the ubiquitin/proteasome proteolytic pathway. Significance Blad forms through a unique route from and is a stable intermediary product of its precursor, β-conglutin, the major Lupinus seed storage protein. It is composed of 173 amino acid residues, is encoded by an intron-containing, internal fragment of the gene that codes for β-conglutin precursor (nucleotides 394 to 913) and exhibits an isoelectric point of 9.6 and a molecular mass of 20,404.85 Da. Consistent with its role as a storage protein, blad contains an extremely high proportion of the nitrogen-rich amino acids. PMID:20066045

AguR, a Transmembrane Transcription Activator of the Putrescine Biosynthesis Operon in Lactococcus lactis, Acts in Response to the Agmatine Concentration.

PubMed

Linares, Daniel M; Del Rio, Beatriz; Redruello, Begoña; Ladero, Victor; Martin, M Cruz; de Jong, Anne; Kuipers, Oscar P; Fernandez, Maria; Alvarez, Miguel A

2015-09-01

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

AguR, a Transmembrane Transcription Activator of the Putrescine Biosynthesis Operon in Lactococcus lactis, Acts in Response to the Agmatine Concentration

PubMed Central

Linares, Daniel M.; del Rio, Beatriz; Redruello, Begoña; Martin, M. Cruz; de Jong, Anne; Kuipers, Oscar P.; Fernandez, Maria

2015-01-01

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins. PMID:26116671

Biodegradation analyses of trichloroethylene (TCE) by bacteria and its use for biosensing of TCE.

PubMed

Chee, Gab-Joo

2011-09-30

Trichloroethylene (TCE) is a toxic, recalcitrant groundwater pollutant. TCE-degrading microorganisms were isolated from various environments. The aerobic bacteria isolated from toluene- and tryptophan-containing media were Pseudomonas sp. strain ASA86 and Burkholderia sp. strain TAM17, respectively; these are necessary for inducing TCE biodegradation in a selective medium. The half-degradation time of TCE to a concentration of 1mg/L was 18 h for strain ASA86 and 7 days for strain TAM17. While identifying toluene/TCE degradation genes, we found that in strain ASA86, the gene was the same as the todC1 gene product encoding toluene dioxygenase identified in Pseudomonas putida F1, and that in strain TAM17, the gene was similar to the tecA1 gene product encoding chlorobenzene dioxygenase identified in Burkholderia sp. PS12. A novel TCE biosensor was developed using strain ASA86 as the inducer of toluene under aerobic conditions. The TCE biosensor exhibited a linear relationship below 3 ppm TCE. Detection limit of the biosensor was 0.05 ppm TCE. The response time of the biosensor was less than 10 min. The biosensor response displayed a constant level during a 2 day period. The TCE biosensor displayed sufficient sensitivity for monitoring TCE in environmental systems. Copyright © 2011 Elsevier B.V. All rights reserved.

Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.

PubMed

Dormeyer, Miriam; Lübke, Anastasia L; Müller, Peter; Lentes, Sabine; Reuß, Daniel R; Thürmer, Andrea; Stülke, Jörg; Daniel, Rolf; Brantl, Sabine; Commichau, Fabian M

2017-06-01

Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Genetic structure and regulation of isoprene synthase in Poplar (Populus spp.).

PubMed

Vickers, Claudia E; Possell, Malcolm; Nicholas Hewitt, C; Mullineaux, Philip M

2010-07-01

Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-D: -erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.

ISOLATION AND GENOTYPING OF CLOSTRIDIUM PERFRINGENS FROM FREE-LIVING SOUTH AMERICAN COATI (NASUA NASUA).

PubMed

Silva, Rodrigo O S; Almeida, Lara R; Oliveira Junior, Carlos A; Lima, Paula C S; Soares, Danielle F M; Pereira, Pedro L L; Silva, Israel J; Lobato, Francisco C F

2016-03-01

The importance of Clostridium perfringens for most wild animal species remains unclear. This study aimed to isolate and genotype C. perfringens in stool samples from free-living South American coati (Nasua nasua) in Brazil. Forty-six free-living N. nasua were trapped and stool samples were collected. Two different protocols for C. perfringens isolation were tested: direct plating onto selective agar and pre-enrichment in broth followed by plating in selective agar. Clostridium perfringens type A was isolated from 15 (32.6%) animals by direct plating and 36 (78.3%) animals by broth PE, and the rate of isolation was significantly different between these two methods (P < 0.01). Twelve of the 36 (33.3%) isolated strains by the PE protocol were positive for the β-2 toxin-encoding gene (cpb2) whereas the enterotoxin-encoding gene (cpe) and necrotic enteritis like-B toxin gene (netb) were not found. These results suggest that C. perfringens is commonly part of the microbiota of free-living coatis. Additionally, the use of a PE protocol appears to be essential for studies on C. perfringens in this species.

BPF-1, a pathogen-induced DNA-binding protein involved in the plant defense response.

PubMed

da Costa e Silva, O; Klein, L; Schmelzer, E; Trezzini, G F; Hahlbrock, K

1993-07-01

The mechanisms by which plants restrict the growth of pathogens include transient activation of numerous defense-related genes. Box P is a putative cis-acting element of a distinct group of such genes, including those encoding the enzyme phenylalanine ammonialyase (PAL). A DNA-binding activity to Box P was identified in nuclear extracts from cultured parsley cells and a cDNA encoding the protein BPF-1 (Box P-binding Factor) partially characterized. BPF-1 binds to this element with specificity similar to that of the binding activity in nuclear extracts. BPF-1 mRNA accumulates rapidly in elicitor-treated parsley cells and around fungal infection sites on parsley leaves. This accumulation is, at least partly, due to a rapid and transient increase in the transcription rate of BPF-1. Moreover, tight correlation between the relative amounts of BPF-1 and PAL mRNAs was observed in different organs of a parsley plant. These results are consistent with the hypothesis that BPF-1 is involved in disease resistance by modulating plant defense gene expression.

YAP1 and VGLL3, encoding two cofactors of TEAD transcription factors, are amplified and overexpressed in a subset of soft tissue sarcomas.

PubMed

Hélias-Rodzewicz, Zofia; Pérot, Gaëlle; Chibon, Frédéric; Ferreira, Céline; Lagarde, Pauline; Terrier, Philippe; Coindre, Jean-Michel; Aurias, Alain

2010-12-01

In a series of 404 adult soft tissue sarcomas, analyzed by array-CGH, we have observed in approximately 10% of them a genomic amplification of either chromosome bands 11q22 or 3p12. These two amplicons likely target the YAP1 and VGLL3 genes, respectively. Both genes encode proteins that are cofactors of the TEAD family of transcription factors. Very good correlations between amplification and expression levels were observed. Welch test analyses of transcriptome data demonstrate that tumors with amplicons share a large set of upregulated and downregulated genes. Inhibition of YAP1 and VGLL3 in cell lines with these amplifications/overexpressions leads to similar phenotypes: decrease of proliferation rate, and to a lesser extent decrease of migration properties. These data, and the fact that these amplicons are observed either in de-differentiated liposarcomas or in undifferentiated pleomorphic sarcomas, suggest that these genetics events could be involved in oncogenesis and progression of soft tissue sarcomas. © 2010 Wiley-Liss, Inc.

Draft genome sequence of a multidrug-resistant KPC-2-producing Enterobacter aerogenes isolated from a hospitalised patient in Brazil.

PubMed

Moura, Quézia; Fernandes, Miriam R; Cerdeira, Louise; Nhambe, Lúcia F; Ienne, Susan; Souza, Tiago A; Lincopan, Nilton

2017-09-01

Multidrug-resistant (MDR) Enterobacter aerogenes strains are frequently associated with nosocomial infections and high mortality rates, representing a serious public health problem. The aim of this study was to present the draft genome sequence of a MDR KPC-2-producing E. aerogenes isolated from a perineal swab of a hospitalised patient in Brazil. Genomic DNA was sequenced using an Illumina MiSeq platform. De novo genome assembly was carried out using the A5-Miseq pipeline, and whole-genome sequence analysis was performed using tools from the Center for Genomic Epidemiology. The strain harboured resistance genes to β-lactams, aminoglycosides, sulphonamides and trimethoprim in addition to genes encoding multidrug efflux system proteins, a quaternary ammonium transporter and heavy metal efflux system proteins. In addition, the strain harboured genes encoding diverse virulence factors. These data might allow a better understanding of the genetic basis of antimicrobial resistance and virulence in E. aerogenes strains. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Efficient Polar Coding of Quantum Information

NASA Astrophysics Data System (ADS)

Renes, Joseph M.; Dupuis, Frédéric; Renner, Renato

2012-08-01

Polar coding, introduced 2008 by Arıkan, is the first (very) efficiently encodable and decodable coding scheme whose information transmission rate provably achieves the Shannon bound for classical discrete memoryless channels in the asymptotic limit of large block sizes. Here, we study the use of polar codes for the transmission of quantum information. Focusing on the case of qubit Pauli channels and qubit erasure channels, we use classical polar codes to construct a coding scheme that asymptotically achieves a net transmission rate equal to the coherent information using efficient encoding and decoding operations and code construction. Our codes generally require preshared entanglement between sender and receiver, but for channels with a sufficiently low noise level we demonstrate that the rate of preshared entanglement required is zero.

The evolution of mollusc shells.

PubMed

McDougall, Carmel; Degnan, Bernard M

2018-05-01

Molluscan shells are externally fabricated by specialized epithelial cells on the dorsal mantle. Although a conserved set of regulatory genes appears to underlie specification of mantle progenitor cells, the genes that contribute to the formation of the mature shell are incredibly diverse. Recent comparative analyses of mantle transcriptomes and shell proteomes of gastropods and bivalves are consistent with shell diversity being underpinned by a rapidly evolving mantle secretome (suite of genes expressed in the mantle that encode secreted proteins) that is the product of (a) high rates of gene co-option into and loss from the mantle gene regulatory network, and (b) the rapid evolution of coding sequences, particular those encoding repetitive low complexity domains. Outside a few conserved genes, such as carbonic anhydrase, a so-called "biomineralization toolkit" has yet to be discovered. Despite this, a common suite of protein domains, which are often associated with the extracellular matrix and immunity, appear to have been independently and often uniquely co-opted into the mantle secretomes of different species. The evolvability of the mantle secretome provides a molecular explanation for the evolution and diversity of molluscan shells. These genomic processes are likely to underlie the evolution of other animal biominerals, including coral and echinoderm skeletons. This article is categorized under: Comparative Development and Evolution > Regulation of Organ Diversity Comparative Development and Evolution > Evolutionary Novelties. © 2018 Wiley Periodicals, Inc.

Identification of iron-regulated genes of Bifidobacterium breve UCC2003 as a basis for controlled gene expression

PubMed Central

Cronin, Michelle; Zomer, Aldert; Fitzgerald, Gerald; van Sinderen, Douwe

2012-01-01

Iron is an essential growth factor for virtually all organisms. However, iron is not readily available in most environments and microorganisms have evolved specialized mechanisms, such as the use of siderophores and high-affinity transport systems, to acquire iron when confronted with iron-limiting conditions. In general these systems are tightly regulated to prevent iron-induced toxicity and because they are quite costly to the microbe. Because of this tight regulation we chose to explore the response of Bifidobacterium breve UCC2003 to iron limitation. Through microarray and complementation analyses we identified and characterized a presumed ferrous iron uptake system, encoded by bfeUOB, from B. breve UCC2003 and exploited its regulated transcription to develop an inducible expression system for use in bifidobacteria. PMID:22179149

Overcoming codon-usage bias in heterologous protein expression in Streptococcus gordonii.

PubMed

Lee, Song F; Li, Yi-Jing; Halperin, Scott A

2009-11-01

One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.

Carbohydrate metabolism genes and pathways in insects: insights from the honey bee genome

PubMed Central

Kunieda, T; Fujiyuki, T; Kucharski, R; Foret, S; Ament, S A; Toth, A L; Ohashi, K; Takeuchi, H; Kamikouchi, A; Kage, E; Morioka, M; Beye, M; Kubo, T; Robinson, G E; Maleszka, R

2006-01-01

Carbohydrate-metabolizing enzymes may have particularly interesting roles in the honey bee, Apis mellifera, because this social insect has an extremely carbohydrate-rich diet, and nutrition plays important roles in caste determination and socially mediated behavioural plasticity. We annotated a total of 174 genes encoding carbohydrate-metabolizing enzymes and 28 genes encoding lipid-metabolizing enzymes, based on orthology to their counterparts in the fly, Drosophila melanogaster, and the mosquito, Anopheles gambiae. We found that the number of genes for carbohydrate metabolism appears to be more evolutionarily labile than for lipid metabolism. In particular, we identified striking changes in gene number or genomic organization for genes encoding glycolytic enzymes, cellulase, glucose oxidase and glucose dehydrogenases, glucose-methanol-choline (GMC) oxidoreductases, fucosyltransferases, and lysozymes. PMID:17069632

Developmental Regulation of Diacylglycerol Acyltransferase Family Gene Expression in Tung Tree Tissues

PubMed Central

Cao, Heping; Shockey, Jay M.; Klasson, K. Thomas; Chapital, Dorselyn C.; Mason, Catherine B.; Scheffler, Brian E.

2013-01-01

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms. PMID:24146944

Differential gene expression in response to Fusarium oxysporum infection in resistant and susceptible genotypes of flax (Linum usitatissimum L.).

PubMed

Dmitriev, Alexey A; Krasnov, George S; Rozhmina, Tatiana A; Novakovskiy, Roman O; Snezhkina, Anastasiya V; Fedorova, Maria S; Yurkevich, Olga Yu; Muravenko, Olga V; Bolsheva, Nadezhda L; Kudryavtseva, Anna V; Melnikova, Nataliya V

2017-12-28

Flax (Linum usitatissimum L.) is a crop plant used for fiber and oil production. Although potentially high-yielding flax varieties have been developed, environmental stresses markedly decrease flax production. Among biotic stresses, Fusarium oxysporum f. sp. lini is recognized as one of the most devastating flax pathogens. It causes wilt disease that is one of the major limiting factors for flax production worldwide. Breeding and cultivation of flax varieties resistant to F. oxysporum is the most effective method for controlling wilt disease. Although the mechanisms of flax response to Fusarium have been actively studied, data on the plant response to infection and resistance gene candidates are currently very limited. The transcriptomes of two resistant and two susceptible flax cultivars with respect to Fusarium wilt, as well as two resistant BC 2 F 5 populations, which were grown under control conditions or inoculated with F. oxysporum, were sequenced using the Illumina platform. Genes showing changes in expression under F. oxysporum infection were identified in both resistant and susceptible flax genotypes. We observed the predominant overexpression of numerous genes that are involved in defense response. This was more pronounced in resistant cultivars. In susceptible cultivars, significant downregulation of genes involved in cell wall organization or biogenesis was observed in response to F. oxysporum. In the resistant genotypes, upregulation of genes related to NAD(P)H oxidase activity was detected. Upregulation of a number of genes, including that encoding beta-1,3-glucanase, was significantly greater in the cultivars and BC 2 F 5 populations resistant to Fusarium wilt than in susceptible cultivars in response to F. oxysporum infection. Using high-throughput sequencing, we identified genes involved in the early defense response of L. usitatissimum against the fungus F. oxysporum. In response to F. oxysporum infection, we detected changes in the expression of pathogenesis-related protein-encoding genes and genes involved in ROS production or related to cell wall biogenesis. Furthermore, we identified genes that were upregulated specifically in flax genotypes resistant to Fusarium wilt. We suggest that the identified genes in resistant cultivars and BC 2 F 5 populations showing induced expression in response to F. oxysporum infection are the most promising resistance gene candidates.

Identification and differential induction of the expression of aquaporins by salinity in broccoli plants.

PubMed

Muries, Beatriz; Faize, Mohamed; Carvajal, Micaela; Martínez-Ballesta, María Del Carmen

2011-04-01

Plant aquaporins belong to a large superfamily of conserved proteins called the major intrinsic proteins (MIPs). There is limited information about the diversity of MIPs and their water transport capacity in broccoli (Brassica oleracea) plants. In this study, the cDNAs of isoforms of Plasma Membrane Intrinsic Proteins (PIPs), a class of aquaporins, from broccoli roots have been partially sequenced. Thus, sequencing experiments led to the identification of eight PIP1 and three PIP2 genes encoding PIPs in B. oleracea plants. The occurrence of different gene products encoding PIPs suggests that they may play different roles in plants. The screening of their expression as well as the expression of two specific PIP2 isoforms (BoPIP2;2 and BoPIP2;3), in different organs and under different salt-stress conditions in two varieties, has helped to unravel the function and the regulation of PIPs in plants. Thus, a high degree of BoPIP2;3 expression in mature leaves suggests that this BoPIP2;3 isoform plays important roles, not only in root water relations but also in the physiology and development of leaves. In addition, differences between gene and protein patterns led us to consider that mRNA synthesis is inhibited by the accumulation of the corresponding encoded protein. Therefore, transcript levels, protein abundance determination and the integrated hydraulic architecture of the roots must be considered in order to interpret the response of broccoli to salinity.

ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis.

PubMed

Larsen, Paul B; Geisler, Matt J B; Jones, Carol A; Williams, Kelly M; Cancel, Jesse D

2005-02-01

Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.

Comparative genomics analysis of Lactobacillus species associated with weight gain or weight protection.

PubMed

Drissi, F; Merhej, V; Angelakis, E; El Kaoutari, A; Carrière, F; Henrissat, B; Raoult, D

2014-02-24

Some Lactobacillus species are associated with obesity and weight gain while others are associated with weight loss. Lactobacillus spp. and bifidobacteria represent a major bacterial population of the small intestine where lipids and simple carbohydrates are absorbed, particularly in the duodenum and jejunum. The objective of this study was to identify Lactobacillus spp. proteins involved in carbohydrate and lipid metabolism associated with weight modifications. We examined a total of 13 complete genomes belonging to seven different Lactobacillus spp. previously associated with weight gain or weight protection. We combined the data obtained from the Rapid Annotation using Subsystem Technology, Batch CD-Search and Gene Ontology to classify gene function in each genome. We observed major differences between the two groups of genomes. Weight gain-associated Lactobacillus spp. appear to lack enzymes involved in the catabolism of fructose, defense against oxidative stress and the synthesis of dextrin, L-rhamnose and acetate. Weight protection-associated Lactobacillus spp. encoded a significant gene amount of glucose permease. Regarding lipid metabolism, thiolases were only encoded in the genome of weight gain-associated Lactobacillus spp. In addition, we identified 18 different types of bacteriocins in the studied genomes, and weight gain-associated Lactobacillus spp. encoded more bacteriocins than weight protection-associated Lactobacillus spp. The results of this study revealed that weight protection-associated Lactobacillus spp. have developed defense mechanisms for enhanced glycolysis and defense against oxidative stress. Weight gain-associated Lactobacillus spp. possess a limited ability to breakdown fructose or glucose and might reduce ileal brake effects.

Inversion of the chromosomal region between two mating type loci switches the mating type in Hansenula polymorpha.

PubMed

Maekawa, Hiromi; Kaneko, Yoshinobu

2014-11-01

Yeast mating type is determined by the genotype at the mating type locus (MAT). In homothallic (self-fertile) Saccharomycotina such as Saccharomyces cerevisiae and Kluveromyces lactis, high-efficiency switching between a and α mating types enables mating. Two silent mating type cassettes, in addition to an active MAT locus, are essential components of the mating type switching mechanism. In this study, we investigated the structure and functions of mating type genes in H. polymorpha (also designated as Ogataea polymorpha). The H. polymorpha genome was found to harbor two MAT loci, MAT1 and MAT2, that are ∼18 kb apart on the same chromosome. MAT1-encoded α1 specifies α cell identity, whereas none of the mating type genes were required for a identity and mating. MAT1-encoded α2 and MAT2-encoded a1 were, however, essential for meiosis. When present in the location next to SLA2 and SUI1 genes, MAT1 or MAT2 was transcriptionally active, while the other was repressed. An inversion of the MAT intervening region was induced by nutrient limitation, resulting in the swapping of the chromosomal locations of two MAT loci, and hence switching of mating type identity. Inversion-deficient mutants exhibited severe defects only in mating with each other, suggesting that this inversion is the mechanism of mating type switching and homothallism. This chromosomal inversion-based mechanism represents a novel form of mating type switching that requires only two MAT loci.

Identification of PaCOL1 and PaCOL2, two CONSTANS-like genes showing decreased transcript levels preceding short day induced growth cessation in Norway spruce.

PubMed

Holefors, Anna; Opseth, Lars; Ree Rosnes, Anne Katrine; Ripel, Linda; Snipen, Lars; Fossdal, Carl Gunnar; Olsen, Jorunn E

2009-02-01

In woody plants of the temperate zone short photoperiod (SD) leads to growth cessation. In angiosperms CONSTANS (CO) or CO-like genes play an important role in the photoperiodic control of flowering, tuberisation and shoot growth. To investigate the role of CO-like genes in photoperiodic control of shoot elongation in gymnosperms, PaCOL1 and PaCOL2 were isolated from Norway spruce. PaCOL1 encodes a 3.9kb gene with a predicted protein of 444 amino acids. PaCOL2 encodes a 1.2kb gene with a predicted protein of 385 amino acids. Both genes consist of two exons and have conserved domains found in other CO-like genes; two zinc finger domains, a CCT and a COOH domain. PaCOL1 and PaCOL2 fall into the group 1c clade of the CO-like genes, and are thus distinct from Arabidopsis CO that belongs to group 1a. Transcript levels of both PaCOL-genes appear to be light regulated, an increasing trend was observed upon transition from darkness to light, and a decreasing trend during darkness. The increasing trend at dawn was observed both in needles and shoot tips, whereas the decreasing trend in darkness was most prominent in shoot tips, and limited to the late part of the dark period in needles. The transcript levels of both genes decreased significantly in both tissues under SD prior to growth cessation and bud formation. This might suggest an involvement in photoperiodic control of shoot elongation or might be a consequence of regulation by light.

Efficient PRNP deletion in bovine genome using gene-editing technologies in bovine cells

PubMed Central

Choi, WooJae; Kim, Eunji; Yum, Soo-Young; Lee, ChoongIl; Lee, JiHyun; Moon, JoonHo; Ramachandra, Sisitha; Malaweera, Buddika Oshadi; Cho, JongKi; Kim, Jin-Soo; Kim, SeokJoong; Jang, Goo

2015-01-01

abstract Even though prion (encoded by the PRNP gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing Bmi-1 and hTert genes; 2) investigate the disruption of the PRNP in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with Bmi-1and hTert. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the PRNP gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (CSNK2A1, FAM64A, MPG and PRND) were affected after PRNP disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using Bmi-1 and hTert genes, and used TALENs to knockout the PRNP gene in these immortalized cells. The efficient PRNP KO is expected to be a useful technology to develop our understanding of in vitro prion protein functions in cattle. PMID:26217959

Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria

PubMed Central

Jackson, Christopher J; Norman, John E; Schnare, Murray N; Gray, Michael W; Keeling, Patrick J; Waller, Ross F

2007-01-01

Background Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs) within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes. Results From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression. Conclusion The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements within the genome, RNA editing, loss of stop codons, and use of trans-splicing. PMID:17897476

Genome sequence of Candidatus Riesia pediculischaeffi, endosymbiont of chimpanzee lice, and genomic comparison of recently acquired endosymbionts from human and chimpanzee lice.

PubMed

Boyd, Bret M; Allen, Julie M; de Crécy-Lagard, Valérie; Reed, David L

2014-09-11

The obligate-heritable endosymbionts of insects possess some of the smallest known bacterial genomes. This is likely due to loss of genomic material during symbiosis. The mode and rate of this erosion may change over evolutionary time: faster in newly formed associations and slower in long-established ones. The endosymbionts of human and anthropoid primate lice present a unique opportunity to study genome erosion in newly established (or young) symbionts. This is because we have a detailed phylogenetic history of these endosymbionts with divergence dates for closely related species. This allows for genome evolution to be studied in detail and rates of change to be estimated in a phylogenetic framework. Here, we sequenced the genome of the chimpanzee louse endosymbiont (Candidatus Riesia pediculischaeffi) and compared it with the closely related genome of the human body louse endosymbiont. From this comparison, we found evidence for recent genome erosion leading to gene loss in these endosymbionts. Although gene loss was detected, it was not significantly greater than in older endosymbionts from aphids and ants. Additionally, we searched for genes associated with B-vitamin synthesis in the two louse endosymbiont genomes because these endosymbionts are believed to synthesize essential B vitamins absent in the louse's diet. All of the expected genes were present, except those involved in thiamin synthesis. We failed to find genes encoding for proteins involved in the biosynthesis of thiamin or any complete exogenous means of salvaging thiamin, suggesting there is an undescribed mechanism for the salvage of thiamin. Finally, genes encoding for the pantothenate de novo biosynthesis pathway were located on a plasmid in both taxa along with a heat shock protein. Movement of these genes onto a plasmid may be functionally and evolutionarily significant, potentially increasing production and guarding against the deleterious effects of mutation. These data add to a growing resource of obligate endosymbiont genomes and to our understanding of the rate and mode of genome erosion in obligate animal-associated bacteria. Ultimately sequencing additional louse p-endosymbiont genomes will provide a model system for studying genome evolution in obligate host associated bacteria. Copyright © 2014 Boyd et al.

Novel Type V Staphylococcal Cassette Chromosome mec Driven by a Novel Cassette Chromosome Recombinase, ccrC

PubMed Central

Ito, Teruyo; Ma, Xiao Xue; Takeuchi, Fumihiko; Okuma, Keiko; Yuzawa, Harumi; Hiramatsu, Keiichi

2004-01-01

Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-ΔmecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome. PMID:15215121

Boron Toxicity Reduces Water Transport from Root to Shoot in Arabidopsis Plants. Evidence for a Reduced Transpiration Rate and Expression of Major PIP Aquaporin Genes.

PubMed

Macho-Rivero, Miguel A; Herrera-Rodríguez, M Begoña; Brejcha, Ramona; Schäffner, Anton R; Tanaka, Nobuhiro; Fujiwara, Toru; González-Fontes, Agustín; Camacho-Cristóbal, Juan J

2018-04-01

Toxic boron (B) concentrations cause impairments in several plant metabolic and physiological processes. Recently we reported that B toxicity led to a decrease in the transpiration rate of Arabidopsis plants in an ABA-dependent process within 24 h, which could indicate the occurrence of an adjustment of whole-plant water relations in response to this stress. Since plasma membrane intrinsic protein (PIP) aquaporins are key components influencing the water balance of plants because of their involvement in root water uptake and tissue hydraulic conductance, the aim of the present work was to study the effects of B toxicity on these important parameters affecting plant water status over a longer period of time. For this purpose, transpiration rate, water transport to the shoot and transcript levels of genes encoding four major PIP aquaporins were measured in Arabidopsis plants treated or not with a toxic B concentration. Our results indicate that, during the first 24 h of B toxicity, increased shoot ABA content would play a key role in reducing stomatal conductance, transpiration rate and, consequently, the water transport to the shoot. These physiological responses to B toxicity were maintained for up to 48 h of B toxicity despite shoot ABA content returning to control levels. In addition, B toxicity also caused the down-regulation of several genes encoding root and shoot aquaporins, which could reduce the cell to cell movement of water in plant tissues and, consequently, the water flux to shoot. All these changes in the water balance of plants under B toxicity could be a mechanism to prevent excess B accumulation in plant tissues.

Security proof of continuous-variable quantum key distribution using three coherent states

NASA Astrophysics Data System (ADS)

Brádler, Kamil; Weedbrook, Christian

2018-02-01

We introduce a ternary quantum key distribution (QKD) protocol and asymptotic security proof based on three coherent states and homodyne detection. Previous work had considered the binary case of two coherent states and here we nontrivially extend this to three. Our motivation is to leverage the practical benefits of both discrete and continuous (Gaussian) encoding schemes creating a best-of-both-worlds approach; namely, the postprocessing of discrete encodings and the hardware benefits of continuous ones. We present a thorough and detailed security proof in the limit of infinite signal states which allows us to lower bound the secret key rate. We calculate this is in the context of collective eavesdropping attacks and reverse reconciliation postprocessing. Finally, we compare the ternary coherent state protocol to other well-known QKD schemes (and fundamental repeaterless limits) in terms of secret key rates and loss.

Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media

PubMed Central

Hottes, Alison K.; Meewan, Maliwan; Yang, Desiree; Arana, Naomi; Romero, Pedro; McAdams, Harley H.; Stephens, Craig

2004-01-01

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation. PMID:14973021