WorldWideScience

Sample records for genes encoding proteins

  1. The Sulfolobicin Genes of Sulfolobus acidocaldarius Encode Novel Antimicrobial Proteins

    NARCIS (Netherlands)

    Ellen, Albert F.; Rohulya, Olha V.; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J. M.

    2011-01-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The acti

  2. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    Science.gov (United States)

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  3. Selection for Genes Encoding Secreted Proteins and Receptors

    Science.gov (United States)

    Klein, Robert D.; Gu, Qimin; Goddard, Audrey; Rosenthal, Arnon

    1996-07-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

  4. The sulfolobicin genes of Sulfolobus acidocaldarius encode novel antimicrobial proteins.

    Science.gov (United States)

    Ellen, Albert F; Rohulya, Olha V; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J M

    2011-09-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.

  5. Expression of genes encoding extracellular matrix proteins: a macroarray study.

    Science.gov (United States)

    Futyma, Konrad; Miotła, Paweł; Różyńska, Krystyna; Zdunek, Małgorzata; Semczuk, Andrzej; Rechberger, Tomasz; Wojcierowski, Jacek

    2014-12-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

  6. Molecular quantification of genes encoding for green-fluorescent proteins

    DEFF Research Database (Denmark)

    Felske, A; Vandieken, V; Pauling, B V

    2003-01-01

    A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is pro...

  7. Surfactant Protein-D-Encoding Gene Variant Polymorphisms Are Linked to Respiratory Outcome in Premature Infants

    DEFF Research Database (Denmark)

    Sorensen, Grith Lykke; Dahl, Marianne; Tan, Qihua

    2014-01-01

    OBJECTIVE: Associations between the genetic variation within or downstream of the surfactant protein-D-encoding gene (SFTPD), which encodes the collectin surfactant protein-D (SP-D) and may lead to respiratory distress syndrome or bronchopulmonary dysplasia, recently were reported. Our aim was to...

  8. In silicio search for genes encoding peroxisomal proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kal, A J; Hettema, E H; van den Berg, M; Koerkamp, M G; van Ijlst, L; Distel, B; Tabak, H F

    2000-01-01

    The biogenesis of peroxisomes involves the synthesis of new proteins that after, completion of translation, are targeted to the organelle by virtue of peroxisomal targeting signals (PTS). Two types of PTSs have been well characterized for import of matrix proteins (PTS1 and PTS2). Induction of the genes encoding these matrix proteins takes place in oleate-containing medium and is mediated via an oleate response element (ORE) present in the region preceding these genes. The authors have searched the yeast genome for OREs preceding open reading frames (ORFs), and for ORFs that contain either a PTS1 or PTS2. Of the ORFs containing an ORE, as well as either a PTS1 or a PTS2, many were known to encode bona fide peroxisomal matrix proteins. In addition, candidate genes were identified as encoding putative new peroxisomal proteins. For one case, subcellular location studies validated the in silicio prediction. This gene encodes a new peroxisomal thioesterase.

  9. The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus

    NARCIS (Netherlands)

    Boot, H.J.; Kolen, C.P.A.M.; Pot, B.; Kersters, K.; Pouwels, P.H.

    1996-01-01

    Previously we have shown that the type strain of Lactobacillus acidophilus possesses two S-protein-encoding genes, one of which is silent, on a chromosomal segment of 6 kb. The S-protein-encoding gene in the expression site can be exchanged for the silent S-protein-encoding gene by inversion of this

  10. The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus

    NARCIS (Netherlands)

    Boot, H.J.; Kolen, C.P.A.M.; Pot, B.; Kersters, K.; Pouwels, P.H.

    1996-01-01

    Previously we have shown that the type strain of Lactobacillus acidophilus possesses two S-protein-encoding genes, one of which is silent, on a chromosomal segment of 6 kb. The S-protein-encoding gene in the expression site can be exchanged for the silent S-protein-encoding gene by inversion of this

  11. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    Science.gov (United States)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  12. Positive selection in phytotoxic protein-encoding genes of Botrytis species

    NARCIS (Netherlands)

    Staats, M.; Baarlen, van P.; Schouten, A.; Kan, van J.A.L.; Bakker, F.T.

    2007-01-01

    Evolutionary patterns of sequence divergence were analyzed in genes from the fungal genus Botrytis (Ascomycota), encoding phytotoxic proteins homologous to a necrosis and ethylene-inducing protein from Fusarium oxysporum. Fragments of two paralogous genes (designated NEP1 and NEP2) were amplified

  13. Identification of Genes Encoding the Folate- and Thiamine-Binding Membrane Proteins in Firmicutes

    NARCIS (Netherlands)

    Eudes, Aymerick; Erkens, Guus B.; Slotboom, Dirk J.; Rodionov, Dmitry A.; Naponelli, Valeria; Hanson, Andrew D.

    2008-01-01

    Genes encoding high-affinity folate- and thiamine-binding proteins (FolT, ThiT) were identified in the Lactobacillus casei genome, expressed in Lactococcus lactis, and functionally characterized. Similar genes occur in many Firmicutes, sometimes next to folate or thiamine salvage genes. Most thiT ge

  14. Identification of Genes Encoding the Folate- and Thiamine-Binding Membrane Proteins in Firmicutes

    NARCIS (Netherlands)

    Eudes, Aymerick; Erkens, Guus B.; Slotboom, Dirk J.; Rodionov, Dmitry A.; Naponelli, Valeria; Hanson, Andrew D.

    2008-01-01

    Genes encoding high-affinity folate- and thiamine-binding proteins (FolT, ThiT) were identified in the Lactobacillus casei genome, expressed in Lactococcus lactis, and functionally characterized. Similar genes occur in many Firmicutes, sometimes next to folate or thiamine salvage genes. Most thiT ge

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Science.gov (United States)

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

    2015-12-01

    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.

  17. Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

    Science.gov (United States)

    Skazina, M A; Gorbushin, A M

    2016-07-01

    Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits.

  18. Phylogenetic Analysis of Homologous Proteins Encoded by UL2 and UL23 genes of Herpesviridae

    Institute of Scientific and Technical Information of China (English)

    Long-ding LIU; Wen-juan WU; Min HONG; Hai-jing SHI; Shao-hui MA; Jing-jing WANG; Hong-ling ZHAO; Yun LIAO; Qi-han LI

    2007-01-01

    The proteins encoded by the Herpesviridae β-gene play a critical role in the replication stage of the virus. In this paper, phylogenetic analyses provided evidence that someβ-gene products, such as UL2 and UL23 from HSV1, have their homologous genes in its family, and also exist in prokaryotic organisms, indicating that these viruses appear to have been assembled over evolutionary time by numerous independent events of horizontal gene transfer.

  19. Absence of repellents in Ustilago maydis induces genes encoding small secreted proteins.

    Science.gov (United States)

    Teertstra, Wieke R; Krijgsheld, Pauline; Wösten, Han A B

    2011-08-01

    The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae.

  20. Differences in dinucleotide frequencies of thermophilic genes encoding water soluble and membrane proteins

    Institute of Scientific and Technical Information of China (English)

    Hiroshi NAKASHIMA; Yuka KURODA

    2011-01-01

    The occurrence frequencies of the dinucleotides of genes of three thermophilic and three mesophilic species from both archaea and eubacteria were investigated in this study. The genes encoding water soluble proteins were rich in the dinucleotides of purine dimers, whereas the genes encoding membrane proteins were rich in pyrimidine dimers. The dinucleotides of purine dimers are the counterparts of pyrimidine dimers in a double-stranded DNA. The purine/pyrimidine dimers were favored in the thermophiles but not in the mesophiles, based on comparisons of observed and expected frequencies. This finding is in agreement with our previous study which showed that purine/pyrimidine dimers are positive factors that increase the thermal stability of DNA. The dinucleotides AA, AG, and GA are components of the codons of charged residues of Glu, Asp, Lys, and Arg, and the dinucleotides TT, CT, and TC are components of the codons of hydrophobic residues of Leu, He, and Phe. This is consistent with the suitabilities of the different amino acid residues for water soluble and membrane proteins. Our analysis provides a picture of how thermophilic species produce water soluble and membrane proteins with distinctive characters: the genes encoding water soluble proteins use DNA sequences rich in purine dimers, and the genes encoding membrane proteins use DNA sequences rich in pyrimidine dimers on the opposite strand.

  1. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    DEFF Research Database (Denmark)

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel;

    2015-01-01

    IP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression...... at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. Conclusions The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes...

  2. Variation in genes encoding eosinophil granule proteins in atopic dermatitis patients from Germany

    Directory of Open Access Journals (Sweden)

    Epplen Jörg T

    2008-11-01

    Full Text Available Abstract Background Atopic dermatitis (AD is believed to result from complex interactions between genetic and environmental factors. A main feature of AD as well as other allergic disorders is serum and tissue eosinophilia. Human eosinophils contain high amounts of cationic granule proteins, including eosinophil cationic protein (ECP, eosinophil-derived neurotoxin (EDN, eosinophil peroxidase (EPO and major basic protein (MBP. Recently, variation in genes encoding eosinophil granule proteins has been suggested to play a role in the pathogenesis of allergic disorders. We therefore genotyped selected single nucleotide polymorphisms within the ECP, EDN, EPO and MBP genes in a cohort of 361 German AD patients and 325 healthy controls. Results Genotype and allele frequencies did not differ between patients and controls for all polymorphisms investigated in this study. Haplotype analysis did not reveal any additional information. Conclusion We did not find evidence to support an influence of variation in genes encoding eosinophil granule proteins for AD pathogenesis in this German cohort.

  3. LEA (Late Embryogenesis Abundant proteins and their encoding genes in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Hincha Dirk K

    2008-03-01

    Full Text Available Abstract Background LEA (late embryogenesis abundant proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown. Results We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE and/or low temperature response (LTRE elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded. Conclusion The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for

  4. Cloning and expression of prion protein encoding gene of flounder ( Paralichthys olivaceus)

    Science.gov (United States)

    Zhang, Zhiwen; Sun, Xiuqin; Zhang, Jinxing; Zan, Jindong

    2008-02-01

    The prion protein (PrP) encoding gene of flounder ( Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  5. Structures of genes encoding TATA box-binding proteins from Trimeresurus gramineus and T. flavoviridis snakes.

    Science.gov (United States)

    Nakashima, K; Nobuhisa, I; Deshimaru, M; Ogawa, T; Shimohigashi, Y; Fukumaki, Y; Hattori, M; Sakaki, Y; Hattori, S; Ohno, M

    1995-01-23

    A cDNA encoding the Trimeresurus gramineus (Tg; green habu snake) TATA-box-binding protein (TgTBP) was cloned and sequenced. The cDNA encodes a 33-kDa protein with an extensive sequence similarity to those derived from other organisms, except for the N-terminal domain. Genes encoding TgTBP and Trimeresurus flavoviridis (Tf; habu snake) TBP (TfTBP) were isolated using a TgTBP cDNA and their nt sequences were determined. They are the first TBP genes entirely sequenced in higher animals. Both genes span over 15 kb and are constructed from eight exons and seven introns. Comparison of the loci of introns on the aligned amino-acid sequences of TBP from six organisms (Tg, Tf, mouse, Arabidopsis thaliana, Schizosaccharomyces pombe and Acanthamoeba castellanii) indicated that there are three highly conserved loci in the C-terminal domain.

  6. Filamentous-haemagglutinin-like protein genes encoded on a plasmid of Moraxella bovis.

    Science.gov (United States)

    Kakuda, Tsutomu; Sarataphan, Nopporn; Tanaka, Tetsuya; Takai, Shinji

    2006-11-26

    The complete nucleotide sequence of a plasmid, pMBO-1, from Moraxella bovis strain Epp63 was determined. We identified 30 open reading frames (ORFs) encoded by the 44,215bp molecule. Two large ORFs, flpA and flpB, encoding proteins with similarity to Bordetella pertussis filamentous haemagglutinin (FHA), were identified on the same plasmid. The gene for a specific accessory protein (Fap), which may play a role in the secretion of Flp protein, was also identified. Reverse transcriptase PCR analysis of total RNA isolated from M. bovis Epp63 indicated that the flpA, flpB, and fap genes are all transcribed. Southern blot analysis indicated that the flp and fap genes are present in other clinical isolates of geographically diverse M. bovis.

  7. MYB98 positively regulates a battery of synergid-expressed genes encoding filiform apparatus localized proteins.

    Science.gov (United States)

    Punwani, Jayson A; Rabiger, David S; Drews, Gary N

    2007-08-01

    The synergid cells within the female gametophyte are essential for reproduction in angiosperms. MYB98 encodes an R2R3-MYB protein required for pollen tube guidance and filiform apparatus formation by the synergid cells. To test the predicted function of MYB98 as a transcriptional regulator, we determined its subcellular localization and examined its DNA binding properties. We show that MYB98 binds to a specific DNA sequence (TAAC) and that a MYB98-green fluorescent protein fusion protein localizes to the nucleus, consistent with a role in transcriptional regulation. To identify genes regulated by MYB98, we tested previously identified synergid-expressed genes for reduced expression in myb98 female gametophytes and identified 16 such genes. We dissected the promoter of one of the downstream genes, DD11, and show that it contains a MYB98 binding site required for synergid expression, suggesting that DD11 is regulated directly by MYB98. To gain insight into the functions of the downstream genes, we chose five genes and determined the subcellular localization of the encoded proteins. We show that these five proteins are secreted into the filiform apparatus, suggesting that they play a role in either the formation or the function of this unique structure. Together, these data suggest that MYB98 functions as a transcriptional regulator in the synergid cells and activates the expression of genes required for pollen tube guidance and filiform apparatus formation.

  8. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

    Directory of Open Access Journals (Sweden)

    Nordlund Henri R

    2005-03-01

    Full Text Available Abstract Background A chicken egg contains several biotin-binding proteins (BBPs, whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  9. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family.

    Science.gov (United States)

    Niskanen, Einari A; Hytönen, Vesa P; Grapputo, Alessandro; Nordlund, Henri R; Kulomaa, Markku S; Laitinen, Olli H

    2005-03-18

    A chicken egg contains several biotin-binding proteins (BBPs), whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  10. A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis.

    Science.gov (United States)

    Town, M; Jean, G; Cherqui, S; Attard, M; Forestier, L; Whitmore, S A; Callen, D F; Gribouval, O; Broyer, M; Bates, G P; van't Hoff, W; Antignac, C

    1998-04-01

    Nephropathic cystinosis, an autosomal recessive disorder resulting from defective lysosomal transport of cystine, is the most common inherited cause of renal Fanconi syndrome. The cystinosis gene has been mapped to chromosome 17p13. We found that the locus D17S829 was homozygously deleted in 23 out of 70 patients, and identified a novel gene, CTNS, which mapped to the deletion interval. CTNS encodes an integral membrane protein, cystinosin, with features of a lysosomal membrane protein. Eleven different mutations, all predicted to cause loss of function of the protein, were found to segregate with the disorder.

  11. Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection.

    Science.gov (United States)

    Beard, Cheryl E; Court, Leon; Boets, Annemie; Mourant, Roslyn; Van Rie, Jeroen; Akhurst, Raymond J

    2008-09-01

    Of 188 Australian Bacillus thuringiensis strains screened for genes encoding soluble insecticidal proteins by polymerase chain reaction/restriction-length fragment polymorphism (RFLP) analysis, 87% showed the presence of such genes. Although 135 isolates (72%) produced an RFLP pattern identical to that expected for vip3A genes, 29 isolates possessed a novel vip-like gene. The novel vip-like gene was cloned from B. thuringiensis isolate C81, and sequence analysis demonstrated that it was 94% identical to the vip3Ba1 gene. The new gene was designated vip3Bb2. Cell-free supernatants from both the B. thuringiensis strain C81 and from Escherichia coli expressing the Vip3Bb2 protein were toxic for the cotton bollworm, Helicoverpa armigera.

  12. Cloning of human genes encoding novel G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  13. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

    Science.gov (United States)

    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.

  14. Cloning and expression analysis of a prion protein encoding gene in guppy ( Poecilia reticulata)

    Science.gov (United States)

    Wu, Suihan; Wei, Qiwei; Yang, Guanpin; Wang, Dengqiang; Zou, Guiwei; Chen, Daqing

    2008-11-01

    The full length cDNA of a prion protein (PrP) encoding gene of guppy ( Poecilia reticulata) and the corresponding genomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a protein of 515 amino acids, which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length, consisting of 2 introns and 2 exons. The 5' untranslated region of cDNA originated from the 2 exons, while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues including brain, eye, liver, intestine, muscle and tail, its transcript was most abundant in the brain. In addition, the transcription of the gene was enhanced by 5 salinity, implying that it was associated with the response of guppy to saline stress.

  15. TMC and EVER genes belong to a larger novel family, the TMC gene family encoding transmembrane proteins

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    Mutai Hideki

    2003-06-01

    Full Text Available Abstract Background Mutations in the transmembrane cochlear expressed gene 1 (TMC1 cause deafness in human and mouse. Mutations in two homologous genes, EVER1 and EVER2 increase the susceptibility to infection with certain human papillomaviruses resulting in high risk of skin carcinoma. Here we report that TMC1, EVER1 and EVER2 (now TMC6 and TMC8 belong to a larger novel gene family, which is named TMC for trans membrane channel-like gene family. Results Using a combination of iterative database searches and reverse transcriptase-polymerase chain reaction (RT-PCR experiments we assembled contigs for cDNA encoding human, murine, puffer fish, and invertebrate TMC proteins. TMC proteins of individual species can be grouped into three subfamilies A, B, and C. Vertebrates have eight TMC genes. The majority of murine TMC transcripts are expressed in most organs; some transcripts, however, in particular the three subfamily A members are rare and more restrictively expressed. Conclusion The eight vertebrate TMC genes are evolutionary conserved and encode proteins that form three subfamilies. Invertebrate TMC proteins can also be categorized into these three subfamilies. All TMC genes encode transmembrane proteins with intracellular amino- and carboxyl-termini and at least eight membrane-spanning domains. We speculate that the TMC proteins constitute a novel group of ion channels, transporters, or modifiers of such.

  16. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    DEFF Research Database (Denmark)

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel

    2015-01-01

    at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. Conclusions The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes...... and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. Results To determine the gene regulatory functions of FUS and EWS at the level of chromatin, we have performed chromatin immunoprecipitation followed by next generation sequencing (Ch......IP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression...

  17. Lipophilic proteins encoded by mitochondrial and nuclear genes in Neurospora crassa.

    Science.gov (United States)

    Küntzel, H; Pieniaźek, N J; Pieniaźek, D; Leister, D E

    1975-06-01

    Mitochondrial proteins soluble in neutral chloroform-methanol (2:1) were separated from lipids by ether precipitation and resolved by Sephadex G-200 filtration in the presence of dodecylsulfate into two major fractions eluting in the excluded region (peak I) and in a region of an apparent molecular weight 8000 (peak II). Residual phospholipids are found only in peak II. Peak I consists of several aggregated small polypeptides of molecular weights around 8000, which can be disaggregated by mild oxidation with performic acid. Cycloheximide stimulates almost two-fold incorporation of radioactive phenylalanine into peak I proteins but inhibits labelling of peak II proteins by 95%. Chloramphenicol and ethidium bromide inhibit the synthesis of peak I proteins by 70% and 95% respectively, but do not affect labelling of peak II proteins. At least 30% of the translation products of mitochondrial DNA in vitro behave like peak I proteins: they are soluble in organic solvents, they aggregate in dodecylsulfate buffer after removal of phospholipids and they contain species of molecular weights around 8000 that disaggregate upon oxidation. The data strongly suggest that the proteins of peak I are encoded by mitochondrial genes and synthesized on mitochondrial ribosomes, whereas the proteins of peak II are encoded by nuclear genes and synthesized on cytoplasmic ribosomes. Both groups of lipophilic proteins are very similar in their molecular weights, but the mitochondrially coded peak I proteins have the unique property of forming large heat-stable aggregates in the presence of dodecylsulfate.

  18. Genes encoding FAD-binding proteins in Volvariella volvacea exhibit differential expression in homokaryons and heterokaryons.

    Science.gov (United States)

    Meng, Li; Yan, Junjie; Xie, Baogui; Li, Yu; Chen, Bingzhi; Liu, Shuyan; Li, Dan; Yang, Zhiyun; Zeng, Xiancheng; Deng, Youjin; Jiang, Yuji

    2013-10-01

    Flavin adenine dinucleotide (FAD)-binding proteins play a vital role in energy transfer and utilization during fungal growth and mycelia aggregation. We sequenced the genome of Volvariella volvacea, an economically important edible fungus, and discovered 41 genes encoding FAD-binding proteins. Gene expression profiles revealed that the expression levels of four distinctly differentially expressed genes in heterokaryotic strain H1521 were higher than in homokaryotic strains PYd15 and PYd21 combined. These observations were validated by quantitative real-time PCR. The results suggest that the differential expression of FAD-binding proteins may be important in revealing the distinction between homokaryons and heterokaryons on the basis of FAD-binding protein functionality.

  19. Cloning and expression of prion protein encoding gene of flounder (Paralichthys olivaceus)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhiwen; SUN Xiuqin; ZHANG Jinxing; ZAN Jindong

    2008-01-01

    The prion protein (PrP) encoding gene of flounder (Paralichthys olivaceus) was cloned.It was not interrupted by an intron.This gene has two promoters in its 5' upstream,indicating that its transcription may be intensive,and should have an important function.It was expressed in all 14 tissues tested,demonstrating that it is a house-keeping gene.Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  20. Analysis of Genes Encoding Penicillin-Binding Proteins in Clinical Isolates of Acinetobacter baumannii ▿ †

    Science.gov (United States)

    Cayô, Rodrigo; Rodríguez, María-Cruz; Espinal, Paula; Fernández-Cuenca, Felipe; Ocampo-Sosa, Alain A.; Pascual, Álvaro; Ayala, Juan A.; Vila, Jordi; Martínez-Martínez, Luis

    2011-01-01

    There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acinetobacter baumannii to β-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes of A. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpA or mrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to β-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals. PMID:21947403

  1. Sca1, a previously undescribed paralog from autotransporter protein-encoding genes in Rickettsia species

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    Raoult Didier

    2006-02-01

    Full Text Available Abstract Background Among the 17 genes encoding autotransporter proteins of the "surface cell antigen" (sca family in the currently sequenced Rickettsia genomes, ompA, sca5 (ompB and sca4 (gene D, have been extensively used for identification and phylogenetic purposes for Rickettsia species. However, none of these genes is present in all 20 currently validated Rickettsia species. Of the remaining 14 sca genes, sca1 is the only gene to be present in all nine sequenced Rickettsia genomes. To estimate whether the sca1 gene is present in all Rickettsia species and its usefulness as an identification and phylogenetic tool, we searched for sca1genes in the four published Rickettsia genomes and amplified and sequenced this gene in the remaining 16 validated Rickettsia species. Results Sca1 is the only one of the 17 rickettsial sca genes present in all 20 Rickettsia species. R. prowazekii and R. canadensis exhibit a split sca1 gene whereas the remaining species have a complete gene. Within the sca1 gene, we identified a 488-bp variable sequence fragment that can be amplified using a pair of conserved primers. Sequences of this fragment are specific for each Rickettsia species. The phylogenetic organization of Rickettsia species inferred from the comparison of sca1 sequences strengthens the classification based on the housekeeping gene gltA and is similar to those obtained from the analyses of ompA, sca5 and sca4, thus suggesting similar evolutionary constraints. We also observed that Sca1 protein sequences have evolved under a dual selection pressure: with the exception of typhus group rickettsiae, the amino-terminal part of the protein that encompasses the predicted passenger domain, has evolved under positive selection in rickettsiae. This suggests that the Sca1 protein interacts with the host. In contrast, the C-terminal portion containing the autotransporter domain has evolved under purifying selection. In addition, sca1 is transcribed in R. conorii

  2. Overlapping protein-encoding genes in Pseudomonas fluorescens Pf0-1.

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    Mark W Silby

    2008-06-01

    Full Text Available The annotated genome sequences of prokaryotes seldom include overlapping genes encoded opposite each other by the same stretch of DNA. However, antisense transcription is becoming recognized as a widespread phenomenon in eukaryotes, and examples have been linked to important biological processes. Pseudomonas fluorescens inhabits aquatic and terrestrial environments, and can be regarded as an environmental generalist. The genetic basis for this ecological success is not well understood. In a previous search for soil-induced genes in P. fluorescens Pf0-1, ten antisense genes were discovered. These were termed 'cryptic' genes, as they had escaped detection by gene-hunting algorithms, and lacked easily recognizable promoters. In this communication, we designate such genes as 'non-predicted' or 'hidden'. Using reverse transcription PCR, we show that at each of six non-predicted gene loci chosen for study, transcription occurs from both 'sense' and 'antisense' DNA strands. Further, at least one of these hidden antisense genes, iiv14, encodes a protein, as does the sense transcript, both identified by poly-histidine tags on the C-terminus of the proteins. Mutational and complementation studies showed that this novel antisense gene was important for efficient colonization of soil, and multiple copies in the wildtype host improved the speed of soil colonization. Introduction of a stop codon early in the gene eliminated complementation, further implicating the protein in colonization of soil. We therefore designate iiv14 "cosA". These data suggest that, as is the case with eukaryotes, some bacterial genomes are more densely coded than currently recognized.

  3. Overlapping protein-encoding genes in Pseudomonas fluorescens Pf0-1.

    Science.gov (United States)

    Silby, Mark W; Levy, Stuart B

    2008-06-13

    The annotated genome sequences of prokaryotes seldom include overlapping genes encoded opposite each other by the same stretch of DNA. However, antisense transcription is becoming recognized as a widespread phenomenon in eukaryotes, and examples have been linked to important biological processes. Pseudomonas fluorescens inhabits aquatic and terrestrial environments, and can be regarded as an environmental generalist. The genetic basis for this ecological success is not well understood. In a previous search for soil-induced genes in P. fluorescens Pf0-1, ten antisense genes were discovered. These were termed 'cryptic' genes, as they had escaped detection by gene-hunting algorithms, and lacked easily recognizable promoters. In this communication, we designate such genes as 'non-predicted' or 'hidden'. Using reverse transcription PCR, we show that at each of six non-predicted gene loci chosen for study, transcription occurs from both 'sense' and 'antisense' DNA strands. Further, at least one of these hidden antisense genes, iiv14, encodes a protein, as does the sense transcript, both identified by poly-histidine tags on the C-terminus of the proteins. Mutational and complementation studies showed that this novel antisense gene was important for efficient colonization of soil, and multiple copies in the wildtype host improved the speed of soil colonization. Introduction of a stop codon early in the gene eliminated complementation, further implicating the protein in colonization of soil. We therefore designate iiv14 "cosA". These data suggest that, as is the case with eukaryotes, some bacterial genomes are more densely coded than currently recognized.

  4. Locus heterogeneity disease genes encode proteins with high interconnectivity in the human protein interaction network.

    Science.gov (United States)

    Keith, Benjamin P; Robertson, David L; Hentges, Kathryn E

    2014-01-01

    Mutations in genes potentially lead to a number of genetic diseases with differing severity. These disease genes have been the focus of research in recent years showing that the disease gene population as a whole is not homogeneous, and can be categorized according to their interactions. Locus heterogeneity describes a single disorder caused by mutations in different genes each acting individually to cause the same disease. Using datasets of experimentally derived human disease genes and protein interactions, we created a protein interaction network to investigate the relationships between the products of genes associated with a disease displaying locus heterogeneity, and use network parameters to suggest properties that distinguish these disease genes from the overall disease gene population. Through the manual curation of known causative genes of 100 diseases displaying locus heterogeneity and 397 single-gene Mendelian disorders, we use network parameters to show that our locus heterogeneity network displays distinct properties from the global disease network and a Mendelian network. Using the global human proteome, through random simulation of the network we show that heterogeneous genes display significant interconnectivity. Further topological analysis of this network revealed clustering of locus heterogeneity genes that cause identical disorders, indicating that these disease genes are involved in similar biological processes. We then use this information to suggest additional genes that may contribute to diseases with locus heterogeneity.

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

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    Meng Sun

    2014-12-01

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

  6. Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis

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    González Mauricio

    2009-09-01

    Full Text Available Abstract Background Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis. Results Microarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction. Conclusion Our data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we

  7. Expression analysis of the Theileria parva subtelomere-encoded variable secreted protein gene family.

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    Jacqueline Schmuckli-Maurer

    Full Text Available 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

  8. Identification and characterization of multiple Spidroin 1 genes encoding major ampullate silk proteins in Nephila clavipes.

    Science.gov (United States)

    Gaines, W A; Marcotte, W R

    2008-09-01

    Spider dragline silk is primarily composed of proteins called major ampullate spidroins (MaSps) that consist of a large repeat array flanked by nonrepetitive N- and C-terminal domains. Until recently, there has been little evidence for more than one gene encoding each of the two major spidroin silk proteins, MaSp1 and MaSp2. Here, we report the deduced N-terminal domain sequences for two distinct MaSp1 genes from Nephila clavipes (MaSp1A and MaSp1B) and for MaSp2. All three MaSp genes are co-expressed in the major ampullate gland. A search of the GenBank database also revealed two distinct MaSp1 C-terminal domain sequences. Sequencing confirmed that both MaSp1 genes are present in all seven Nephila clavipes spiders examined. The presence of nucleotide polymorphisms in these genes confirmed that MaSp1A and MaSp1B are distinct genetic loci and not merely alleles of the same gene. We experimentally determined the transcription start sites for all three MaSp genes and established preliminary pairing between the two MaSp1 N- and C-terminal domains. Phylogenetic analysis of these new sequences and other published MaSp N- and C-terminal domain sequences illustrated that duplications of MaSp genes may be widespread among spider species.

  9. Genes encoding Cher-TPR fusion proteins are predominantly found in gene clusters encoding chemosensory pathways with alternative cellular functions.

    Science.gov (United States)

    Muñoz-Martínez, Francisco; García-Fontana, Cristina; Rico-Jiménez, Miriam; Alfonso, Carlos; Krell, Tino

    2012-01-01

    Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be

  10. Bioinformatic identification of genes encoding C1q-domain containing proteins in zebrafish

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular (gC1q) domain similar with C1q was also found in many non-complement C1q-domain-containing (C1qDC) proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor (TNF) ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing (C1qDC) proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.

  11. Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

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    Bryan D Moyer

    Full Text Available BACKGROUND: Using fungiform (FG and circumvallate (CV taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive, sour cells (PKD2L1-positive, as well as other taste cell populations. Transmembrane protein 44 (TMEM44, a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1, a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1, a calcium-binding transmembrane protein; and anoctamin 7 (ANO7, a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B, a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins

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

    Science.gov (United States)

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

    2011-01-01

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

  13. Cloning and sequence analysis of a gene encoding polygalacturonase-inhibiting protein from cotton

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Polygalacturonase-inhibiting proteins (PGIP) play important roles in plant defense of pathogen, especially fungi. A pair of degenerated primers is designed based on the conserved sequence of 20 other known pgip genes and used to amplify Gossypium barbadense cultivation 7124 cDNA library by touch-down PCR. A 561 bp internal fragment of the pgip gene is obtained and used to design the primers for rapid amplification of cDNA ends. A composite pgip gene sequence is constructed from the products of 5′ and 3′ RACE, which are 666 bp and 906 bp respectively. Analysis of nucleic acid sequence shows 69.2% and 68.7% similarity to Citrus and Poncirus pgip genes, respectively. Its open reading frame of the gene encodes a polypeptide of 330 amino acids, in which 10 leucine-rich repeats arrange tandemly. A new set of primers is designed to the 5′ and 3′ ends of the gene, which allows amplification of the full-length gene from the cotton cDNA library. Genomic DNA analysis reveals that this gene has no intron.

  14. The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans dauer larva development.

    Science.gov (United States)

    Estevez, M; Attisano, L; Wrana, J L; Albert, P S; Massagué, J; Riddle, D L

    1993-10-14

    The bone morphogenetic protein (BMP) family is a conserved group of signalling molecules within the transforming growth factor-beta (TGF-beta) superfamily. This group, including the Drosophila decapentaplegic (dpp) protein and the mammalian BMPs, mediates cellular interactions and tissue differentiation during development. Here we show that a homologue of human BMPs controls a developmental switch in the life cycle of the free-living soil nematode Caenorhabditis elegans. Starvation and overcrowding induce C. elegans to form a developmentally arrested, third-stage dauer larva. The daf-4 gene, which acts to inhibit dauer larva formation and promote growth, encodes a receptor protein kinase similar to the daf-1, activin and TGF-beta receptor serine/threonine kinases. When expressed in monkey COS cells, the daf-4 receptor binds human BMP-2 and BMP-4. The daf-4 receptor is the first to be identified for any growth factor in the BMP family.

  15. Comparative sequence analysis of double stranded RNA binding protein encoding gene of parapoxviruses from Indian camels

    Directory of Open Access Journals (Sweden)

    G. Nagarajan

    2014-03-01

    Full Text Available The dsRNA binding protein (RBP encoding gene of parapoxviruses (PPVs from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV and PCPV (reindeer PCPV and human PCPV shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV from different geographical areas of the world shared 69.5–71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV in comparison to caprine and ovine PPV (ORFV.

  16. Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1.

    Science.gov (United States)

    Tishkoff, D X; Johnson, A W; Kolodner, R D

    1991-05-01

    Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is

  17. Cloning and Expression Analysis of a Prion Protein Encoding Gene in Guppy (Poecilia reticulata)

    Institute of Scientific and Technical Information of China (English)

    WU Suihan; WEI Qiwei; YANG Guanpin; WANG Dengqiang; ZOU Guiwei; CHEN Daqing

    2008-01-01

    The full length eDNA of a prion protein (PrP) encoding gene of guppy (Poecilia reticulata) and the corresponding ge-nomic DNA were cloned.The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a pro-tein of 515 amino acids,which held all typical structural characteristics of the functional PrP.The cloned genomic DNA fragmentcorresponding to the eDNA was 3720 bp in length,consisting of 2 introns and 2 exons.The 5' untranslated region of eDNA origi-nated from the 2 exons,while the ORF originated from the second exon.Although the gene was transcribed in diverse tissues in-cluding brain,eye,liver,intestine,muscle and tail,its transcript was most abundant in the brain.In addition,the transcription of thegene was enhanced by 5 salinity,implying that it was associated with the response of guppy to saline stress.

  18. Identification and Molecular Characterization of a Gene Encoding a Protective Leishmania amazonensis Trp-Asp (WD) Protein

    OpenAIRE

    2004-01-01

    Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates ...

  19. Gene cloning and characterization of the protein encoded by the Neospora caninum bradyzoite-specific antigen gene BAG1.

    Science.gov (United States)

    Kobayashi, T; Narabu, S; Yanai, Y; Hatano, Y; Ito, A; Imai, S; Ike, K

    2013-06-01

    Neospora caninum is an Apicomplexan parasite that causes repeated abortion and stillbirth in cattle. The aim of this study was to clone the gene encoding the N. caninum orthologue (NcBAG1) of the Toxoplasma gondii bradyzoite-specific protein TgBAG1 and characterize its expression pattern in the parasite. Isolation of the full-length 684-bp gene revealed that it shared 78.3% sequence similarity with TgBAG1. NcBAG1 encodes a predicted protein of 227 amino acids with 80.3% similarity to TgBAG1. A putative signal peptide sequence and an invariant GVL motif characteristic of small heat-shock proteins were identified in the predicted N. caninum amino acid sequence. We expressed the NcBAG1 gene as a recombinant glutathione S-transferase fusion protein (rNcBAG1) in Escherichia coli and used the purified 60 kDa protein to obtain a monoclonal antibody (Mab). rNcBAG1 reacted to Mabs specific for NcBAG1 and TgBAG1. No reaction between the NcBAG1 Mab and N. caninum tachyzoites was observed. Although the predicted molecular mass of NcBAG1 is 25 kDa, Western blot analysis of parasite lysates using the NcBAG1 Mab revealed a cross-reactive protein of approximately 30 kDa. Additionally, immunofluorescence assays using the tachyzoite-specific Mab for NcSAG1 and the bradyzoite-specific Mab for TgBAG1 or NcSAG4 revealed NcBAG1-specific expression in bradyzoites in cultures exposed to sodium nitroprusside, a reagent that increases the frequency of bradyzoites. Interestingly, the NcBAG1 protein was identified in the cytoplasm of the bradyzoite-stage parasites. This preliminary analysis of the NcBAG1 gene will assist investigations into the role of this protein in N. caninum .

  20. Hypoxia-inducible genes encoding small EF-hand proteins in rice and tomato.

    Science.gov (United States)

    Otsuka, Chie; Minami, Ikuko; Oda, Kenji

    2010-01-01

    Rice has evolved metabolic and morphological adaptations to low-oxygen stress to grow in submerged paddy fields. To characterize the molecular components that mediate the response to hypoxia in rice, we identified low-oxygen stress early response genes by microarray analysis. Among the highly responsive genes, five genes, OsHREF1 to OsHREF5, shared strong homology. They encoded small proteins harboring two EF-hands, typical Ca(2+)-binding motifs. Homologous genes were found in many land plants, including SlHREF in tomato, which is also strongly induced by hypoxia. SlHREF induction was detected in both roots and shoots of tomato plants under hypoxia. With the exception of OsHREF5, OsHREF expression was unaffected by drought, salinity, cold, or osmotic stress. Fluorescent signals of green fluorescent protein-fused OsHREFs were detected in the cytosol and nucleus. Ruthenium red, an inhibitor of intracellular Ca(2+) release, repressed induction of OsHREF1-4 under hypoxia. The HREFs may be related to the Ca(2+) response to hypoxia.

  1. Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni

    Energy Technology Data Exchange (ETDEWEB)

    Bobek, L.A.; Rekosh, D.M.; Lo Verde, P.T.

    1988-08-01

    The authors isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage map of five of the clones spanning 35 kilobase pairs (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5- end of the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotices. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.

  2. Theoretical model of the three-dimensional structure of a disease resistance gene homolog encoding resistance protein in Vigna mungo.

    Science.gov (United States)

    Basak, Jolly; Bahadur, Ranjit P

    2006-10-01

    Plant disease resistance (R) genes, the key players of innate immunity system in plants encode 'R' proteins. 'R' protein recognizes product of avirulance gene from the pathogen and activate downstream signaling responses leading to disease resistance. No three dimensional (3D) structural information of any 'R' proteins is available as yet. We have reported a 'R' gene homolog, the 'VMYR1', encoding 'R' protein in Vigna mungo. Here, we describe the homology modeling of the 'VMYR1' protein. The model was created by using the 3D structure of an ATP-binding cassette transporter protein from Vibrio cholerae as a template. The strategy for homology modeling was based on the high structural conservation in the superfamily of P-loop containing nucleoside triphosphate hydrolase in which target and template proteins belong. This is the first report of theoretical model structure of any 'R' proteins.

  3. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    Directory of Open Access Journals (Sweden)

    Linda J Savage

    Full Text Available The Chloroplast 2010 Project (http://www.plastid.msu.edu/ identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/. Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles

  4. Chromosomal localization of a novel retinoic acid induced gene RA28 and the protein distribution of its encoded protein

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Gene RA28 is a retinoic acid induced novel gene isolated in our laboratory previously. All-trans retinoic acid (ATRA) was used to induce lung adenocarcinoma cell line GLC-82, and RA28 was obtained by subtractive hybridization. Green fluorescent protein (GFP) has emerged as a unique tool for examining introcellular phenomena in living cells. GFP possesses an intrinsic fluorescence at 488 nm that does not require other co-factors. In this report, an eukaryotic expression plasmid pEGFP-C1-RA28 was constructed and transfected with parental cell line GLC-82 to analyze protein expression and its distribution in living cells. Moreover, radiation hybrid (RH) technique was used to localize RA28 to the chromosome. The results show that gene RA28 is mapped to the chromosome 19q13.1 region, its encoded protein is distributed on cell membrane. All the results further demonstrate that GFP and RH techniques are accurate, fast, repetitive, and will be powerful methods for investigating the gene and protein localization.

  5. Nuclear factor-κB regulates the expression of multiple genes encoding liver transport proteins.

    Science.gov (United States)

    Balasubramaniyan, Natarajan; Ananthanarayanan, Meenakshisundaram; Suchy, Frederick J

    2016-04-15

    In this study we identified the mechanisms underlying the inhibitory effects of NF-κB on the expression of genes encoding multiple liver transport proteins. Well-conserved NF-κB binding sites were found in the promoters of farnesoid X receptor (FXR)-target genes. An electromobility shift assay (EMSA) demonstrated the specific interaction between the NF-κB p65 protein and a (32)P-labeled BSEP NF-κB response element (NF-κBE). Chromatin immunoprecipitation (ChIP) analysis confirmed binding of NF-κB p65 to the BSEP locus but not the FXRE in vitro. NF-κB p65 overexpression in Huh-7 cells markedly repressed FXR/RXR transactivation of the BSEP, ABCG5/G8, MRP2, and FXR promoters, which was totally reversed by expression of the IκBα super-repressor. NF-κB interacted directly with FXR on coimmunoprecipitation, suggesting another level for the inhibitory effects of NF-κB on FXR-target genes. In vivo ChIP analysis with liver nuclei obtained from mice after 3 days of common bile duct ligation (BDL) or 6 h post-lipopolysaccharide (LPS) injection showed a markedly increased recruitment of NF-κB p65 to the Bsep promoter compared with controls. There was also increased recruitment of the corepressor silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and histone deacetylase (HDAC)3 and HDAC2 to the NF-κB sites. We also found that NF-κB p65 was recruited to NF-κB binding sites in the promoters of organic solute transporter, OSTα and OSTβ, and unexpectedly activated rather than repressed gene expression. In mouse liver after BDL NF-κB recruitment to Ostα and Ostβ promoters was associated with increased binding of the potent coactivator cAMP response element binding protein (CREB)-binding protein (CBP)/p300 to the NF-κBE and depletion of CBP/p300 at the FXR element. Overall, these studies demonstrate a novel role for NF-κB in adaptation to obstructive and LPS-induced cholestasis acting through recruitment to specific NF-κB binding sites in

  6. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    Science.gov (United States)

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.

  7. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

    Science.gov (United States)

    Sateriale, Adam; Miller, Peter

    2016-01-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment. PMID:26810036

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

    Science.gov (United States)

    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.

  9. [Research on the gene structure of duck hepatitis B virus and its encoding proteins].

    Science.gov (United States)

    Liu, Qiang; Jia, Ren-Yong

    2012-11-01

    Duck hepatitis B virus (DHBV) belongs to the Avihepadnavirus genus of the Hepadnaviridae, and it not only has the same replication pattern, but also has the similar genomic and antigenic structures to Hepatitis B virus (HBV). The genome of DHBV is a partially double-stranded closed circular DNA. The genome consists of three distinct open reading frames (ORFs): ORF-PreS/S, ORF-PreC/C and ORF-P, which all locate on the negative DNA strand and encode four separate proteins. The ORF-PreS/S encodes envelope proteins L and S, and the ORF-PreC/C and ORF-P encode capsid proteins C and polymerase proteins P, respectively. The characteristics of genome structure,viral proteins features and functions were described in this review in order to provide useful information for the further study of DHBV and the duck model infected by DHBV.

  10. Evidence for negative selection on the gene encoding rhoptry-associated protein 1 (RAP-1) in Plasmodium spp.

    Science.gov (United States)

    Pacheco, M Andreína; Ryan, Elizabeth M; Poe, Amanda C; Basco, Leonardo; Udhayakumar, Venkatachalam; Collins, Williams E; Escalante, Ananias A

    2010-07-01

    Assessing how natural selection, negative or positive, operates on genes with low polymorphism is challenging. We investigated the genetic diversity of orthologous genes encoding the rhoptry-associated protein 1 (RAP-1), a low polymorphic protein of malarial parasites that is involved in erythrocyte invasion. We applied evolutionary genetic methods to study the polymorphism in RAP-1 from Plasmodium falciparum (n=32) and Plasmodium vivax (n=6), the two parasites responsible for most human malaria morbidity and mortality, as well as RAP-1 orthologous in closely related malarial species found in non-human primates (NHPs). Overall, genes encoding RAP-1 are highly conserved in all Plasmodium spp. included in this investigation. We found no evidence for natural selection, positive or negative, acting on the gene encoding RAP-1 in P. falciparum or P. vivax. However, we found evidence that the orthologous genes in non-human primate parasites (Plasmodium cynomolgi, Plasmodium inui, and Plasmodium knowlesi) are under purifying (negative) selection. We discuss the importance of considering negative selection while studying genes encoding proteins with low polymorphism and how selective pressures may differ among orthologous genes in closely related malarial parasites species. Copyright 2010 Elsevier B.V. All rights reserved.

  11. The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish.

    Directory of Open Access Journals (Sweden)

    Ian G Woods

    2005-03-01

    Full Text Available Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.

  12. Genes encoding a group of related small secreted proteins from the gut of Hessian fly larvae [Mayetiola destructor (Say)

    Institute of Scientific and Technical Information of China (English)

    MING-SHUN CHEN; XIANG LIU; YU-CHENG ZHU; JOHN C. REESE; GERALD E. WILDE

    2006-01-01

    A group of related genes has been isolated and characterized from the gut of Hessian fly larvae [Mayetiola destructor (Say)]. Members in this group appear to encode proteins with secretary signal peptides at the N-terminals. The mature putative proteins are small, acidic proteins with calculated molecular masses of 14.5 to 15.3 kDa, and isoelectric points from 4.56 to 4.88. Northern blot analysis revealed that these genes are expressed predominantly in the gut of Hessian fly larvae and pupae. Two related genes, G10K1 and G10K2, were isolated as tandem repeats. Both genes contain three exons and two introns.The intron/exon boundaries were conserved in terms of amino acid encoding, suggesting that they arose by gene duplication. The fact that the frequency of this group of clones in a gut cDNA library higher than that of total cDNA clones encoding digestive enzymes suggested that this group of proteins may perform an important function in the gut physiology of this insect. However, the exact functions of these proteins are as yet known since no sequence similarity could be identified between these proteins and any known sequences in public databases using standard methods.

  13. Magnaporthe oryzae MTP1 gene encodes a type Ⅲ transmembrane protein involved in conidiation and conidial germination

    Institute of Scientific and Technical Information of China (English)

    Qin LU; Jian-ping LU; Xiao-dong LI; Xiao-hong LIU; Hang MIN; Fu-cheng LIN

    2008-01-01

    In this study the MTP1 gene, encoding a type Ⅲ integral transmembrane protein, was isolated fi'om the rice blast fungus Magnaporthe oryzae. The Mtpl protein is 520 amino acids long and is comparable to the Ytpl protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP (green fluorescent protein) fusion expression results indicate that Mtpl is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily ex-pressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for patho-genicity. The △mtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.

  14. Non-hepatic tumors change the activity of genes encoding copper trafficking proteins in the liver.

    Science.gov (United States)

    Babich, Polina S; Skvortsov, Alexey N; Rusconi, Paolo; Tsymbalenko, Nadezhda V; Mutanen, Marja; Puchkova, Ludmila V; Broggini, Massimo

    2013-07-01

    To assess the statistical relationship between tumor growth and copper metabolism, we performed a metaanalysis of studies in which patients with neoplasms were characterized according to any of the copper status indexes (atomic copper serum concentration, serum oxidase activity, ceruloplasmin protein content). Our metaanalysis shows that in the majority of cases (more than 3100 patients), tumor growth positively correlates with the copper status indexes. Nude athymic CD-1 nu/nu mice with subcutaneous tumors of human origin, C57Bl/6J mice with murine melanoma and Apc(Min) mice with spontaneously developing adenomas throughout the intestinal tract were studied to experimentally determine the relationship between tumor progression, liver copper metabolism, and copper status indexes. We showed that the copper status indexes increased significantly during tumor growth. In the liver tissue of tumor-bearing mice, ceruloplasmin gene expression, as well as the expression of genes related to ceruloplasmin metallation (CTR1 and ATP7B), increased significantly. Moreover, the presence of an mRNA splice variant encoding a form of ceruloplasmin anchored to the plasma membrane by glycosylphosphatidyl inositol, which is atypical for hepatocytes, was also detected. The ATP7A copper transporter gene, which is normally expressed in the liver only during embryonic copper metabolism, was also activated. Depletion of holo-ceruloplasmin resulted in retardation of human HCT116 colon carcinoma cell growth in nude mice and induced DNA fragmentation in tumor cells. In addition, the concentration of cytochrome c increased significantly in the cytosol, while decreasing in the mitochondria. We discuss a possible trans-effect of developing tumors on copper metabolism in the liver.

  15. Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome.

    Science.gov (United States)

    Frank, Valeska; den Hollander, Anneke I; Brüchle, Nadina Ortiz; Zonneveld, Marijke N; Nürnberg, Gudrun; Becker, Christian; Du Bois, Gabriele; Kendziorra, Heide; Roosing, Susanne; Senderek, Jan; Nürnberg, Peter; Cremers, Frans P M; Zerres, Klaus; Bergmann, Carsten

    2008-01-01

    Meckel-Gruber syndrome (MKS) is an autosomal recessive, lethal multisystemic disorder characterized by meningooccipital encephalocele, cystic kidney dysplasia, hepatobiliary ductal plate malformation, and postaxial polydactyly. Recently, genes for MKS1 and MKS3 were identified, putting MKS on the list of ciliary disorders (ciliopathies). By positional cloning in a distantly related multiplex family, we mapped a novel locus for MKS to a 3-Mb interval on 12q21. Sequencing of the CEP290 gene located in the minimal critical region showed a homozygous 1-bp deletion supposed to lead to loss of function of the encoded centrosomal protein CEP290/nephrocystin-6. CEP290 is thought to be involved in chromosome segregation and localizes to cilia, centrosomes, and the nucleus. Subsequent analysis of another consanguineous multiplex family revealed homozygous haplotypes and the same frameshift mutation. Our findings add to the increasing body of evidence that ciliopathies can cause a broad spectrum of disease phenotypes, and pleiotropic effects of CEP290 mutations range from single organ involvement with isolated Leber congenital amaurosis to Joubert syndrome and lethal early embryonic multisystemic malformations in Meckel-Gruber syndrome. We compiled clinical and genetic data of all patients with CEP290 mutations described so far. No clear-cut genotype-phenotype correlations were apparent as almost all mutations are nonsense, frameshift, or splice-site changes and scattered throughout the gene irrespective of the patients' phenotypes. Conclusively, other factors than the type and location of CEP290 mutations may underlie phenotypic variability. (c) 2007 Wiley-Liss, Inc.

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

    Science.gov (United States)

    Mokin, Maxim; Keifer, Joyce

    2005-01-01

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

  17. Cloning and functional expression in Escherichia coli of the gene encoding the di- and tripeptide transport protein of Lactobacillus helveticus

    NARCIS (Netherlands)

    Nakajima, H.; Hagting, A; Kunji, E.R S; Poolman, B.; Konings, W.N

    1997-01-01

    The gene encoding the di- and tripeptide transport protein (DtpT) of Lactobacillus helveticus (DtpT(LH)) was cloned with the aid of the inverse PCR technique and used to complement the dipeptide transport-deficient and proline-auxotrophic Escherichia coil E1772. Functional expression of the peptide

  18. Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein b-5

    Institute of Scientific and Technical Information of China (English)

    XIA; Jiahui; (夏家辉); ZHENG; Duo; (郑多),; TANG; Dongsheng; (唐冬生); DAI; Heping; (戴和平); PAN; Qian; (潘乾); LONG; Zhigao; (龙志高); LIAO; Xiaodong; (廖晓东)

    2001-01-01

    By homologous EST searching and nested PCR a new human gene GJB5 encoding gap junction protein b-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.

  19. Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein b-5

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    By homologous EST searching and nested PCR a new human gene GJB5encoding gap junction protein b-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.

  20. MYB98 Positively Regulates a Battery of Synergid-Expressed Genes Encoding Filiform Apparatus–Localized Proteins[W

    Science.gov (United States)

    Punwani, Jayson A.; Rabiger, David S.; Drews, Gary N.

    2007-01-01

    The synergid cells within the female gametophyte are essential for reproduction in angiosperms. MYB98 encodes an R2R3-MYB protein required for pollen tube guidance and filiform apparatus formation by the synergid cells. To test the predicted function of MYB98 as a transcriptional regulator, we determined its subcellular localization and examined its DNA binding properties. We show that MYB98 binds to a specific DNA sequence (TAAC) and that a MYB98–green fluorescent protein fusion protein localizes to the nucleus, consistent with a role in transcriptional regulation. To identify genes regulated by MYB98, we tested previously identified synergid-expressed genes for reduced expression in myb98 female gametophytes and identified 16 such genes. We dissected the promoter of one of the downstream genes, DD11, and show that it contains a MYB98 binding site required for synergid expression, suggesting that DD11 is regulated directly by MYB98. To gain insight into the functions of the downstream genes, we chose five genes and determined the subcellular localization of the encoded proteins. We show that these five proteins are secreted into the filiform apparatus, suggesting that they play a role in either the formation or the function of this unique structure. Together, these data suggest that MYB98 functions as a transcriptional regulator in the synergid cells and activates the expression of genes required for pollen tube guidance and filiform apparatus formation. PMID:17693534

  1. Genetic variability of Chlamydophila abortus strains assessed by PCR-RFLP analysis of polymorphic membrane protein-encoding genes.

    Science.gov (United States)

    Sait, Michelle; Clark, Ewan M; Wheelhouse, Nicholas; Spalding, Lucy; Livingstone, Morag; Sachse, Konrad; Markey, Bryan K; Magnino, Simone; Siarkou, Victoria I; Vretou, Evangelia; Caro, María R; Yaga, Raja; Lainson, F Alex; Smith, David G E; Wright, Frank; Longbottom, David

    2011-08-05

    This study used PCR-RFLP to investigate the genetic variability of pmp-encoding genes from fifty-two Chlamydophila abortus (C. abortus) strains originating from abortion cases from various geographical regions and host species. Six primer pairs were used to PCR-amplify DNA fragments encoding eighteen pmps. PCR products were digested using four restriction endonucleases and Bayesian methodologies were used to compare RFLP profiles and assign strains to a RFLP genotype. Strains could be assigned to 2 genotypes in the region encoding pmp18D, 3 genotypes in the regions encoding pmp1A-pmp2B, pmp3E-pmp6H and pmp11G-pmp15G, 4 genotypes in the region encoding pmp7G-pmp10G and 5 genotypes in the region encoding pmp16G-pmp17G. In all regions, the majority of strains (88.4-96.1%) had the same genotype as the reference strain S26/3. No correlation could be made between genotype, host species or geographical origin except for the two variant Greek strains, LLG and POS, which formed a discrete genotype in all pmp-encoding regions except pmp18D. Relative rates of evolution calculated for each pmp-encoding gene locus suggest that differing selective pressures and functional constraints may exist on C. abortus polymorphic membrane proteins. These findings suggest that although intraspecies heterogeneity of pmp-encoding genes in C. abortus is low, the sequence heterogeneity should be an important consideration when using pmps as the basis for novel diagnostics or vaccine development.

  2. The Ph-3 gene from Solanum pimpinellifolium encodes CC-NBS-LRR protein conferring resistance to Phytophthora infestans.

    Science.gov (United States)

    Zhang, Chunzhi; Liu, Lei; Wang, Xiaoxuan; Vossen, Jack; Li, Guangcun; Li, Tao; Zheng, Zheng; Gao, Jianchang; Guo, Yanmei; Visser, Richard G F; Li, Junming; Bai, Yuling; Du, Yongchen

    2014-06-01

    Ph-3 is the first cloned tomato gene for resistance to late blight and encodes a CC-NBS-LRR protein. Late blight, caused by Phytophthora infestans, is one of the most destructive diseases in tomato. The resistance (R) gene Ph-3, derived from Solanum pimpinellifolium L3708, provides resistance to multiple P. infestans isolates and has been widely used in tomato breeding programmes. In our previous study, Ph-3 was mapped into a region harbouring R gene analogues (RGA) at the distal part of long arm of chromosome 9. To further narrow down the Ph-3 interval, more recombinants were identified using the flanking markers G2-4 and M8-2, which defined the Ph-3 gene to a 26 kb region according to the Heinz1706 reference genome. To clone the Ph-3 gene, a bacterial artificial chromosome (BAC) library was constructed using L3708 and one BAC clone B25E21 containing the Ph-3 region was identified. The sequence of the BAC clone B25E21 showed that only one RGA was present in the target region. A subsequent complementation analysis demonstrated that this RGA, encoding a CC-NBS-LRR protein, was able to complement the susceptible phenotype in cultivar Moneymaker. Thus this RGA was considered the Ph-3 gene. The predicted Ph-3 protein shares high amino acid identity with the chromosome-9-derived potato resistance proteins against P. infestans (Rpi proteins).

  3. Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein

    Directory of Open Access Journals (Sweden)

    Souza Paulo R Eleutério de

    2001-01-01

    Full Text Available The zinc finger motifs (Cys2His2 are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1.

  4. Identification of antithrombin-modulating genes. Role of LARGE, a gene encoding a bifunctional glycosyltransferase, in the secretion of proteins?

    Directory of Open Access Journals (Sweden)

    María Eugenia de la Morena-Barrio

    Full Text Available The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families. Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02. Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on α1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteins.

  5. Identification of Antithrombin-Modulating Genes. Role of LARGE, a Gene Encoding a Bifunctional Glycosyltransferase, in the Secretion of Proteins?

    Science.gov (United States)

    de la Morena-Barrio, María Eugenia; Buil, Alfonso; Antón, Ana Isabel; Martínez-Martínez, Irene; Miñano, Antonia; Gutiérrez-Gallego, Ricardo; Navarro-Fernández, José; Aguila, Sonia; Souto, Juan Carlos; Vicente, Vicente; Soria, José Manuel; Corral, Javier

    2013-01-01

    The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families). Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02). Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on α1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteins. PMID:23705025

  6. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    Science.gov (United States)

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

  7. A lepidopteran-specific gene family encoding valine-rich midgut proteins.

    Directory of Open Access Journals (Sweden)

    Jothini Odman-Naresh

    Full Text Available Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM, an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps, which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran

  8. A lepidopteran-specific gene family encoding valine-rich midgut proteins.

    Science.gov (United States)

    Odman-Naresh, Jothini; Duevel, Margret; Muthukrishnan, Subbaratnam; Merzendorfer, Hans

    2013-01-01

    Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM), an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps), which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran digestive tract facing

  9. The CKH1/EER4 gene encoding a TAF12-like protein negatively regulates cytokinin sensitivity in Arabidopsis thaliana.

    Science.gov (United States)

    Kubo, Minoru; Furuta, Kaori; Demura, Taku; Fukuda, Hiroo; Liu, Yao-Guang; Shibata, Daisuke; Kakimoto, Tatsuo

    2011-04-01

    The recessive ckh1 (cytokinin hypersensitive 1) mutant of Arabidopsis thaliana shows hypersensitivity to cytokinins, which promote proliferation and greening of calli. The CKH1 gene encodes a protein resembling TAF12 (TATA BOX BINDING PROTEIN ASSOCIATED FACTOR 12), which is a component of transcription factor IID (TFIID)- and histone acetyltransferase-containing complexes in yeast and animals. Microarray analyses revealed that a substantially greater number of genes responded to a low level of cytokinins in the ckh1 mutant than in the wild type. However, expression of cytokinin primary response genes was not significantly affected by the ckh1 mutation. These results suggest that the CKH1 protein regulates a set of genes involved in late signaling processes governing a range of cytokinin responses, including cell proliferation and differentiation.

  10. Expression of the immediate-early gene-encoded protein Egr-1 (zif268) during in vitro classical conditioning.

    Science.gov (United States)

    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink classical conditioning. The results showed that Egr-1 protein expression as determined by immunocytochemistry and Western blot analysis rapidly increased during the early stages of conditioning and remained elevated during the later stages. Further, expression of Egr-1 protein required NMDA receptor activation as it was blocked by bath application of AP-5. These findings suggest that the IEG-encoded proteins such as Egr-1 are activated during relatively simple forms of learning in vertebrates. In this case, Egr-1 may have a functional role in the acquisition phase of conditioning as well as in maintaining expression of conditioned responses.

  11. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

    OpenAIRE

    Nordlund Henri R; Grapputo Alessandro; Hytönen Vesa P; Niskanen Einari A; Kulomaa Markku S; Laitinen Olli H

    2005-01-01

    Background A chicken egg contains several biotin-binding proteins (BBPs), whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Res...

  12. Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily.

    Science.gov (United States)

    Meyers, B C; Dickerman, A W; Michelmore, R W; Sivaramakrishnan, S; Sobral, B W; Young, N D

    1999-11-01

    The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.

  13. Structure and chromosomal localization of the gene encoding the human myelin protein zero (MPZ)

    Energy Technology Data Exchange (ETDEWEB)

    Hayasaka, Kiyoshi; Himoro, Masato; Takada, Goro (Akita Univ. School of Medicine, Akita (Japan)); Wang, Yimin; Takata, Mizuho; Minoshima, Shinsei; Shimizu, Nobuyoshi; Miura, Masayuki; Uyemura, Keiichi (Keio Univ. School of Medicine, Tokyo (Japan))

    1993-09-01

    The authors describe the cloning, characterization, and chromosomal mapping of the human myelin protein zero (MPZ) gene (a structural protein of myelin and an adhesive glycoprotein of the immunoglobulin superfamily). The gene is about 7 kb long and consists of six exons corresponding of the functional domains. All exon-intron junction sequences conform to the GT/AG rule. The 5[prime]-flanking region of the gene has a TA-rich element (TATA-like box), two CAAT boxes, and a single defined transcription initiation site detected by the primer extension method. The gene for human MPZ was assigned to chromosome 1q22-q23 by spot blot hybridization of flow-sorted human chromosomes and fluorescence in situ hybridization. The localization of the MPZ gene coincides with the locus for Charcot-Marie-Tooth disease type 1B, determined by linkage analysis. 20 refs., 3 figs., 1 tab.

  14. Regulatory elements in the promoter region of the rat gene encoding the acyl-CoA-binding protein

    DEFF Research Database (Denmark)

    Elholm, M; Bjerking, G; Knudsen, J

    1996-01-01

    Acyl-CoA-binding protein (ACBP) is an ubiquitously expressed 10-kDa protein which is present in high amounts in cells involved in solute transport or secretion. Rat ACBP is encoded by a gene containing the typical hallmarks of a housekeeping gene. Analysis of the promoter region of the rat ACBP...... gene by electrophoretic mobility shift assay (EMSA) revealed specific binding of proteins from rat liver nuclear extracts to potential recognition sequences of NF-1/CTF, Sp1, AP-1, C/EBP and HNF-3. In addition, specific binding to a DR-1 type element was observed. By using in vitro translated...... for the ACBP DR-1 element. Addition of peroxisome proliferators (PP) to H4IIEC3 rat hepatoma cells led to an increase in the ACBP mRNA level, indicating that the DR-1 element could be a functional peroxisome proliferator responsive element (PPRE). Analysis of the ACBP promoter by transient transfection showed...

  15. Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus.

    Science.gov (United States)

    Kanai, Akio; Oida, Hanako; Matsuura, Nana; Doi, Hirofumi

    2003-05-15

    We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic.

  16. Comparative genomics of the family Vibrionaceae reveals the wide distribution of genes encoding virulence-associated proteins

    Directory of Open Access Journals (Sweden)

    Cai Hong

    2010-06-01

    Full Text Available Abstract Background Species of the family Vibrionaceae are ubiquitous in marine environments. Several of these species are important pathogens of humans and marine species. Evidence indicates that genetic exchange plays an important role in the emergence of new pathogenic strains within this family. Data from the sequenced genomes of strains in this family could show how the genes encoded by all these strains, known as the pangenome, are distributed. Information about the core, accessory and panproteome of this family can show how, for example, genes encoding virulence-associated proteins are distributed and help us understand how virulence emerges. Results We deduced the complete set of orthologs for eleven strains from this family. The core proteome consists of 1,882 orthologous groups, which is 28% of the 6,629 orthologous groups in this family. There were 4,411 accessory orthologous groups (i.e., proteins that occurred in from 2 to 10 proteomes and 5,584 unique proteins (encoded once on only one of the eleven genomes. Proteins that have been associated with virulence in V. cholerae were widely distributed across the eleven genomes, but the majority was found only on the genomes of the two V. cholerae strains examined. Conclusions The proteomes are reflective of the differing evolutionary trajectories followed by different strains to similar phenotypes. The composition of the proteomes supports the notion that genetic exchange among species of the Vibrionaceae is widespread and that this exchange aids these species in adapting to their environments.

  17. Toward understanding the functional role of Ss-RIOK-1, a RIO protein kinase-encoding gene of Strongyloides stercoralis.

    Directory of Open Access Journals (Sweden)

    Wang Yuan

    2014-08-01

    Full Text Available Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs.The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5'-UTR, a 17 bp 3'-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3. Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis.The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.

  18. Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors

    OpenAIRE

    Jones, M.L.; Norman, J E; Morgan, N. V.; Mundell, S J; Lordkipanidze, M.; Lowe, G. C.; Daly, M E; Simpson, M.A.; Drake, S.; Watson, S P; Mumford, A D; UKGAPPS,

    2016-01-01

    Platelet responses to activating agonists are influenced by common\\ud population variants within or near G protein-coupled receptor (GPCR)\\ud genes that affect receptor activity. However, the impact of rare GPCR\\ud gene variants is unknown. We describe the rare single nucleotide variants\\ud (SNVs) in the coding and splice regions of 18 GPCR genes in\\ud 7,595 exomes from the 1,000-genomes and Exome Sequencing\\ud Project databases and in 31 cases with inherited platelet function disorders\\ud (I...

  19. Structure of the gene encoding the murine protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1995-01-01

    The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2...

  20. APUM5, encoding a Pumilio RNA binding protein, negatively regulates abiotic stress responsive gene expression

    Science.gov (United States)

    2014-01-01

    Background A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis. A Pumilio RNA binding protein-coding gene, Arabidopsis Pumilio RNA binding protein 5 (APUM5), was obtained from this screening. Results APUM5 transcriptional profiling was carried out using a bioinformatics tool. We found that APUM5 was associated with both biotic and abiotic stress responses. However, bacterial and fungal pathogen infection susceptibility was not changed in APUM5 transgenic plants compared to that in wild type plants although APUM5 expression was induced upon pathogen infection. In contrast, APUM5 was involved in the abiotic stress response. 35S-APUM5 transgenic plants showed hypersensitive phenotypes under salt and drought stresses during germination, primary root elongation at the seedling stage, and at the vegetative stage in soil. We also showed that some abiotic stress-responsive genes were negatively regulated in 35S-APUM5 transgenic plants. The APUM5-Pumilio homology domain (PHD) protein bound to the 3′ untranslated region (UTR) of the abiotic stress-responsive genes which contained putative Pumilio RNA binding motifs at the 3′ UTR. Conclusions These results suggest that APUM5 may be a new post-transcriptional regulator of the abiotic stress response by direct binding of target genes 3′ UTRs. PMID:24666827

  1. Unfolded Protein Response (UPR Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

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    Martin Hampel

    Full Text Available The unfolded protein response (UPR, a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER, coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

  2. Unfolded Protein Response (UPR) Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

    Science.gov (United States)

    Hampel, Martin; Jakobi, Mareike; Schmitz, Lara; Meyer, Ute; Finkernagel, Florian; Doehlemann, Gunther; Heimel, Kai

    2016-01-01

    The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

  3. New variants of lepidoptericidal toxin genes encoding Bacillus thuringiensis Vip3Aa proteins.

    Science.gov (United States)

    Sauka, Diego H; Rodriguez, Sonia E; Benintende, Graciela B

    2012-01-01

    Bacillus thuringiensis is an entomopathogenic bacterium characterized by producing parasporal proteinaceous insecticidal crystal inclusions during sporulation. Many strains are capable of also expressing other insecticidal proteins called Vip during the vegetative growing phase. Particularly, Vip3A proteins have activity against certain Lepidoptera species through a unique mechanism of action which emphasized their possible use in resistance management strategies against resistant pests. The aim of the work was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that can distinguish between vip3A genes from B. thuringiensis strains. In addition, 4 novel vip3Aa genes were cloned and sequenced. The method was originally based on amplification of a single PCR amplicon and the use of 2 restriction enzymes with recognition sites that facilitate simultaneous detection. Subsequently, a third restriction enzyme was used to distinguish between vip3A variants. Thirteen vip3Aa genes were identified in strains belonging to 10 different B. thuringiensis serovars. Three intra-subclass variants of vip3Aa genes could be differentiated. The presented method can serve as an invaluable tool for the investigation of known and novel vip3A genes in B. thuringiensis strains. To the best of our knowledge, this is the first report where variants of a same subclass of insecticidal genes could be distinguished following PCR-RFLP.

  4. Molecular cloning and chromosomal localization of human genes encoding three closely related G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Zhao-Hui Song; Bonner, T.I. [NIMH National Inst. of Health, Bethesda, MD (United States); Modi, W. [Frederick Cancer Research and Development Center, Frederick, MD (United States)

    1995-07-20

    Cosmids containing human genes for orphan G protein-coupled receptors, GPR12, GPR6, and GPR3, were isolated using their rat homologs as probes. Previous studies of the mouse and rat cDNAs have shown the receptors to be expressed primarily in brain but have failed to identify their ligands. The three receptor proteins of 334, 363, and 330 amino acids, respectively, are encoded by a single exon in each gene. Excluding the divergent sequences preceding the first transmembrane domain, they have {approximately}60% amino acid identity with each other. Flurorescence in situ hybridization of GPR12, GPR6, and GPR3 localized these three genes to human chromosomal regions 13q12, 6q21, and 1p34.3-p36.1, respectively. 9 refs., 2 figs.

  5. Evolutionary genomics of plant genes encoding N-terminal-TM-C2 domain proteins and the similar FAM62 genes and synaptotagmin genes of metazoans

    Directory of Open Access Journals (Sweden)

    Craxton Molly

    2007-07-01

    Full Text Available Abstract Background Synaptotagmin genes are found in animal genomes and are known to function in the nervous system. Genes with a similar domain architecture as well as sequence similarity to synaptotagmin C2 domains have also been found in plant genomes. The plant genes share an additional region of sequence similarity with a group of animal genes named FAM62. FAM62 genes also have a similar domain architecture. Little is known about the functions of the plant genes and animal FAM62 genes. Indeed, many members of the large and diverse Syt gene family await functional characterization. Understanding the evolutionary relationships among these genes will help to realize the full implications of functional studies and lead to improved genome annotation. Results I collected and compared plant Syt-like sequences from the primary nucleotide sequence databases at NCBI. The collection comprises six groups of plant genes conserved in embryophytes: NTMC2Type1 to NTMC2Type6. I collected and compared metazoan FAM62 sequences and identified some similar sequences from other eukaryotic lineages. I found evidence of RNA editing and alternative splicing. I compared the intron patterns of Syt genes. I also compared Rabphilin and Doc2 genes. Conclusion Genes encoding proteins with N-terminal-transmembrane-C2 domain architectures resembling synaptotagmins, are widespread in eukaryotes. A collection of these genes is presented here. The collection provides a resource for studies of intron evolution. I have classified the collection into homologous gene families according to distinctive patterns of sequence conservation and intron position. The evolutionary histories of these gene families are traceable through the appearance of family members in different eukaryotic lineages. Assuming an intron-rich eukaryotic ancestor, the conserved intron patterns distinctive of individual gene families, indicate independent origins of Syt, FAM62 and NTMC2 genes. Resemblances

  6. Diversity and Impact of Rare Variants in Genes Encoding the Platelet G Protein-Coupled Receptors

    Science.gov (United States)

    Jones, Matthew L.; Norman, Jane E.; Morgan, Neil V.; Mundell, Stuart J.; Lordkipanidzé, Marie; Lowe, Gillian C.; Daly, Martina E.; Simpson, Michael A.; Drake, Sian; Watson, Steve P.

    2015-01-01

    Summary Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70% had global minor allele frequency (MAF) low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes. PMID:25567036

  7. Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

    Science.gov (United States)

    Jones, Matthew L; Norman, Jane E; Morgan, Neil V; Mundell, Stuart J; Lordkipanidzé, Marie; Lowe, Gillian C; Daly, Martina E; Simpson, Michael A; Drake, Sian; Watson, Steve P; Mumford, Andrew D

    2015-04-01

    Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

  8. Ethylmalonic encephalopathy is caused by mutations in ETHE1, a gene encoding a mitochondrial matrix protein.

    Science.gov (United States)

    Tiranti, Valeria; D'Adamo, Pio; Briem, Egill; Ferrari, Gianfrancesco; Mineri, Rossana; Lamantea, Eleonora; Mandel, Hanna; Balestri, Paolo; Garcia-Silva, Maria-Teresa; Vollmer, Brigitte; Rinaldo, Piero; Hahn, Si Houn; Leonard, James; Rahman, Shamima; Dionisi-Vici, Carlo; Garavaglia, Barbara; Gasparini, Paolo; Zeviani, Massimo

    2004-02-01

    Ethylmalonic encephalopathy (EE) is a devastating infantile metabolic disorder affecting the brain, gastrointestinal tract, and peripheral vessels. High levels of ethylmalonic acid are detected in the body fluids, and cytochrome c oxidase activity is decreased in skeletal muscle. By use of a combination of homozygosity mapping, integration of physical and functional genomic data sets, and mutational screening, we identified GenBank D83198 as the gene responsible for EE. We also demonstrated that the D83198 protein product is targeted to mitochondria and internalized into the matrix after energy-dependent cleavage of a short leader peptide. The gene had previously been known as "HSCO" (for hepatoma subtracted clone one). However, given its role in EE, the name of the gene has been changed to "ETHE1." The severe consequences of its malfunctioning indicate an important role of the ETHE1 gene product in mitochondrial homeostasis and energy metabolism.

  9. Cloning and expression of gene encoding P23 protein from Cryptosporidium parvum

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    Dinh Thi Bich Lan

    2014-12-01

    Full Text Available We cloned the cp23 gene coding P23 (glycoprotein from Cryptosporidium parvum isolated from Thua Thien Hue province, Vietnam. The coding region of cp23 gene from C. parvum is 99% similar with cp23 gene deposited in NCBI (accession number: U34390. SDS-PAGE and Western blot analysis showed that the cp23 gene in E. coli BL21 StarTM (DE3 produced polypeptides with molecular weights of approximately 37, 40 and 49 kDa. These molecules may be non-glycosylated or glycosylated P23 fusion polypeptides. Recombinant P23 protein purified by GST (glutathione S-transferase affinity chromatography can be used as an antigen for C. parvum antibody production as well as to develop diagnostic kit for C. parvum.

  10. Cytorhabdovirus P3 genes encode 30K-like cell-to-cell movement proteins.

    Science.gov (United States)

    Mann, Krin S; Bejerman, Nicolas; Johnson, Karyn N; Dietzgen, Ralf G

    2016-02-01

    Plant viruses encode movement proteins (MP) to facilitate cell-to-cell transport through plasmodesmata. In this study, using trans-complementation of a movement-defective turnip vein-clearing tobamovirus (TVCV) replicon, we show for the first time for cytorhabdoviruses (lettuce necrotic yellows virus (LNYV) and alfalfa dwarf virus (ADV)) that their P3 proteins function as MP similar to the TVCV P30 protein. All three MP localized to plasmodesmata when ectopically expressed. In addition, we show that these MP belong to the 30K superfamily since movement was inhibited by mutation of an aspartic acid residue in the critical 30K-specific LxD/N50-70G motif. We also report that Nicotiana benthamiana microtubule-associated VOZ1-like transcriptional activator interacts with LNYV P3 and TVCV P30 but not with ADV P3 or any of the MP point mutants. This host protein, which is known to interact with P3 of sonchus yellow net nucleorhabdovirus, may be involved in aiding the cell-to-cell movement of LNYV and TVCV.

  11. O-demethylase from Acetobacterium dehalogenans--cloning, sequencing, and active expression of the gene encoding the corrinoid protein.

    Science.gov (United States)

    Kaufmann, F; Wohlfarth, G; Diekert, G

    1998-10-15

    The ether-cleaving O-demethylase from the strictly anaerobic homoacetogen Acetobacterium dehalogenans catalyses the methyltransfer from 4-hydroxy-3-methoxy-benzoate (vanillate) to tetrahydrofolate. In the first step a vanillate :corrinoid protein methyltransferase (methyltransferase I) mediates the methylation of a 25-kDa corrinoid protein with the cofactor reduced to cob(I)alamin. The methyl group is then transferred to tetrahydrofolate by the action of a methylcorrinoid protein:tetrahydrofolate methyltransferase (methyltransferase II). Using primers derived from the amino-terminal sequences of the corrinoid protein and the vanillate:corrinoid protein methyltransferase (methyltransferase I), a 723-bp fragment was amplified by PCR, which contained the gene odmA encoding the corrinoid protein of O-demethylase. Downstream of odmA, part of the odmB gene encoding methyltransferase I was identified. The amino acid sequence deduced from odmA showed about 60% similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli (MetH) and to corrinoid proteins of methyltransferase systems involved in methanogenesis from methanol and methylamines. The sequence contained the DXHXXG consensus sequence typical for displacement of the dimethylbenzimidazole base of the corrinoid cofactor by a histidine from the protein. Heterologous expression of odmA in E. coli yielded a colourless, oxygen-insensitive apoprotein, which was able to bind one mol cobalamin or methylcobalamin/mol protein. Both of these reconstituted forms of the protein were active in the overall O-demethylation reaction. OdmA reconstituted with hydroxocobalamin and reduced by titanium(III) citrate to the cob(I)alamin form was methylated with vanillate by methyltransferase I in an irreversible reaction. Methylcobalamin carrying OdmA served as methyl group donor for the methylation of tetrahydrofolate by methyltransferase II. This reaction was found to be reversible, since methyltranSferase II

  12. Ribosomal protein L7a is encoded by a gene (Surf-3) within the tightly clustered mouse surfeit locus.

    Science.gov (United States)

    Giallongo, A; Yon, J; Fried, M

    1989-01-01

    The mouse Surfeit locus, which contains a cluster of at least four genes (Surf-1 to Surf-4), is unusual in that adjacent genes are separated by no more than 73 base pairs (bp). The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by only 15 to 73 bp, the 3' ends of Surf-1 and Surf-3 are only 70 bp apart, and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp. This very tight clustering suggests a cis interaction between adjacent Surfeit genes. The Surf-3 gene (which could code for a basic polypeptide of 266 amino acids) is a highly expressed member of a pseudogene-containing multigene family. By use of an anti-peptide serum (against the C-terminal nine amino acids of the putative Surf-3 protein) for immunofluorescence and immunoblotting of mouse cell components and by in vitro translation of Surf-3 cDNA hybrid-selected mRNA, the Surf-3 gene product was identified as a 32-kilodalton ribosomal protein located in the 60S ribosomal subunit. From its subunit location, gel migration, and homology with a limited rat ribosomal peptide sequence, the Surf-3 gene was shown to encode the mouse L7a ribosomal protein. The Surf-3 gene is highly conserved through evolution and was detected by nucleic acid hybridization as existing in multiple copies (multigene families) in other mammals and as one or a few copies in birds, Xenopus, Drosophila, and Schizosaccharomyces pombe. The Surf-3 C-terminal anti-peptide serum detects a 32-kilodalton protein in other mammals, birds, and Xenopus but not in Drosophila and S. pombe. The possible effect of interaction of the Surf-3 ribosomal protein gene with adjacent genes in the Surfeit locus at the transcriptional or posttranscriptional level or both levels is discussed. Images PMID:2648130

  13. Expression of the gene encoding the PR-like protein PRms in germinating maize embryos.

    Science.gov (United States)

    Casacuberta, J M; Raventós, D; Puigdoménech, P; San Segundo, B

    1992-07-01

    The PRms protein is a pathogenesis-related (PR)-like protein whose mRNA accumulates during germination of maize seeds. Expression of the PRms gene is induced after infection of maize seeds with the fungus Fusarium moniliforme. To further our investigations on the expression of the PRms gene we examined the accumulation of PRms mRNA in different tissues of maize seedlings infected with F. moniliforme and studied the effect of fungal elicitors, the mycotoxin moniliformin, the hormone gibberellic acid, and specific chemical agents. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by F. moniliforme, increase the steady-state level of PRms mRNA. PRms mRNA accumulation is also stimulated by the application of the hormone gibberellic acid or by treatment with silver nitrate, whereas acetylsalicylic acid has no effect. In situ RNA hybridization in isolated germinating embryo sections demonstrates that the PRms gene is expressed in the scutellum, particularly in a group of inner cells, and in the epithelium lying at the interface of the scutellum and the endosperm. The pattern of expression of the PRms gene closely resembles that found for hydrolytic enzymes, being confined to the scutellum and the aleurone layer of the germinating maize seed. Our results suggest that the PRms protein has a function during the normal process of seed germination that has become adapted to serve among the defence mechanisms induced in response to pathogens during maize seed germination.

  14. Characterization of a ribonuclease gene and encoded protein from the reptile, Iguana iguana.

    Science.gov (United States)

    Nitto, Takeaki; Lin, Cynthia; Dyer, Kimberly D; Wagner, Robert A; Rosenberg, Helene F

    2005-06-06

    In this work we identify an intronless open reading frame encoding an RNase A ribonuclease from genomic DNA from the Iguana iguana IgH2 cell line. The iguana RNase is expressed primarily in pancreas, and represents the majority of the specific enzymatic activity in this tissue. The encoded sequence shares many features with its better-known mammalian counterparts including the crucial His12, Lys40 and His114 catalytic residues and efficient hydrolytic activity against yeast tRNA substrate (k(cat)/K(m)=6 x 10(4) M(-1) s(-1)), albeit at a reduced pH optimum (pH 6.0). Although the catalytic activity of the iguana RNase is not diminished by human placental RI, iguana RNase is not bactericidal nor is it cytotoxic even at micromolar concentrations. Phylogenetic analysis indicates moderate (46%) amino acid sequence similarity to a pancreatic RNase isolated from Chelydra serpentina (snapping turtle) although no specific relationship could be determined between these RNases and the pancreatic ribonucleases characterized among mammalian species. Further analysis of ribonucleases from non-mammalian vertebrate species is needed in order to define relationships and lineages within the larger RNase A gene superfamily.

  15. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Heiber, M.; Marchese, A.; O`Dowd, B.F. [Univ. of Toronto, Ontario (Canada)] [and others

    1996-03-05

    We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

  16. Stress tolerances of nullmutants of function-unknown genes encoding menadione stress-responsive proteins in Aspergillus nidulans.

    Science.gov (United States)

    Leiter, Éva; Bálint, Mihály; Miskei, Márton; Orosz, Erzsébet; Szabó, Zsuzsa; Pócsi, István

    2016-07-01

    A group of menadione stress-responsive function-unkown genes of Aspergillus nidulans (Locus IDs ANID_03987.1, ANID_06058.1, ANID_10219.1, and ANID_10260.1) was deleted and phenotypically characterized. Importantly, comparative and phylogenetic analyses of the tested A. nidulans genes and their orthologs shed light only on the presence of a TANGO2 domain with NRDE protein motif in the translated ANID_06058.1 gene but did not reveal any recognizable protein-encoding domains in other protein sequences. The gene deletion strains were subjected to oxidative, osmotic, and metal ion stress and, surprisingly, only the ΔANID_10219.1 mutant showed an increased sensitivity to 0.12 mmol l(-1) menadione sodium bisulfite. The gene deletions affected the stress sensitivities (tolerances) irregularly, for example, some strains grew more slowly when exposed to various oxidants and/or osmotic stress generating agents, meanwhile the ΔANID_10260.1 mutant possessed a wild-type tolerance to all stressors tested. Our results are in line with earlier studies demonstrating that the deletions of stress-responsive genes do not confer necessarily any stress-sensitivity phenotypes, which can be attributed to compensatory mechanisms based on other elements of the stress response system with overlapping functions.

  17. Human chondrocytes respond discordantly to the protein encoded by the osteoarthritis susceptibility gene GDF5.

    Directory of Open Access Journals (Sweden)

    Madhushika Ratnayake

    Full Text Available A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-β1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-β1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis

  18. Correlation of rare coding variants in the gene encoding human glucokinase regulatory protein with phenotypic, cellular, and kinetic outcomes.

    Science.gov (United States)

    Rees, Matthew G; Ng, David; Ruppert, Sarah; Turner, Clesson; Beer, Nicola L; Swift, Amy J; Morken, Mario A; Below, Jennifer E; Blech, Ilana; Mullikin, James C; McCarthy, Mark I; Biesecker, Leslie G; Gloyn, Anna L; Collins, Francis S

    2012-01-01

    Defining the genetic contribution of rare variants to common diseases is a major basic and clinical science challenge that could offer new insights into disease etiology and provide potential for directed gene- and pathway-based prevention and treatment. Common and rare nonsynonymous variants in the GCKR gene are associated with alterations in metabolic traits, most notably serum triglyceride levels. GCKR encodes glucokinase regulatory protein (GKRP), a predominantly nuclear protein that inhibits hepatic glucokinase (GCK) and plays a critical role in glucose homeostasis. The mode of action of rare GCKR variants remains unexplored. We identified 19 nonsynonymous GCKR variants among 800 individuals from the ClinSeq medical sequencing project. Excluding the previously described common missense variant p.Pro446Leu, all variants were rare in the cohort. Accordingly, we functionally characterized all variants to evaluate their potential phenotypic effects. Defects were observed for the majority of the rare variants after assessment of cellular localization, ability to interact with GCK, and kinetic activity of the encoded proteins. Comparing the individuals with functional rare variants to those without such variants showed associations with lipid phenotypes. Our findings suggest that, while nonsynonymous GCKR variants, excluding p.Pro446Leu, are rare in individuals of mixed European descent, the majority do affect protein function. In sum, this study utilizes computational, cell biological, and biochemical methods to present a model for interpreting the clinical significance of rare genetic variants in common disease.

  19. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

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    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

  20. Comparative and evolutionary studies of mammalian arylsulfatase and sterylsulfatase genes and proteins encoded on the X-chromosome.

    Science.gov (United States)

    Holmes, Roger S

    2017-06-01

    At least 19 sulfatase genes have been reported on the human genome, including four arylsulfatase (ARS) genes (ARSD; ARSE; ARSF; ARSH) and a sterylsulfatase (STS) gene located together on the X-chromosome. Bioinformatic analyses of mammalian genomes were undertaken using known human STS and ARS amino acid sequences to study the evolution of these genes and proteins encoded on eutherian and marsupial genomes. Several domain regions and key residues were conserved including signal peptides, active site residues, metal (Ca(2+)) and substrate binding sequences, transmembranes and N-glycosylation sites. Phylogenetic analyses describe the relationships and potential origins of these genes during mammalian evolution. Primate ARSH enzymes lacked signal peptide sequences which may influence their biological functions. CpG117 and CpG92 were detected within the 5' region of the human STS and ARSD genes, respectively, and miR-205 within the 3'-UTR for the human STS gene, using bioinformatic methods A proposal is described for a primordial invertebrate STS-like gene serving as an ancestor for unequal cross over events generating the gene complex on the eutherian mammalian X-chromosome. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lagasse, E.; Clerc, R.G.

    1988-06-01

    The cellular mechanisms involved in chronic inflammatory processes are poorly understood. This is especially true for the role of macrophages, which figure prominently in the inflammatory response. Two proteins, MRP8 and MRP14, which are expressed in infiltrate macrophages during inflammatory reactions but not in normal tissue macrophages, which have been characterized. Here the authors report that MRP8 and MRP14 mRNAs are specially expressed in human cells of myeloid origin and that their expression is regulated during monocycle-macrophage and granulocyte differentiation. To initiate the analysis of cis-acting elements governing the tissue-specific expression of the MRP genes, the authors cloned the human genes encoding MRP8 and MRP14. Both genes contain three exons, are single copy, and have a strikingly similar organization. They belong to a novel subfamily of highly homologous calcium-binding proteins which includes S100..cap alpha.., S100BETA, intestinal calcium-binding protein, P11, and calcyclin (2A9). A transient expression assay was devised to investigate the tissue-specific regulatory elements responsible for MRP gene expression after differentiation in leukemia HL60 cells. The results of this investigation demonstrated that the cis-acting element responsible for MRP expression are present on the cloned DNA fragment containing the MRP gene loci.

  2. Analysis of viral protein-2 encoding gene of avian encephalomyelitis virus from field specimens in Central Java region, Indonesia

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2016-01-01

    Full Text Available Aim: Avian encephalomyelitis (AE is a viral disease which can infect various types of poultry, especially chicken. In Indonesia, the incidence of AE infection in chicken has been reported since 2009, the AE incidence tends to increase from year to year. The objective of this study was to analyze viral protein 2 (VP-2 encoding gene of AE virus (AEV from various species of birds in field specimen by reverse transcription polymerase chain reaction (RT-PCR amplification using specific nucleotides primer for confirmation of AE diagnosis. Materials and Methods: A total of 13 AEV samples are isolated from various species of poultry which are serologically diagnosed infected by AEV from some areas in central Java, Indonesia. Research stage consists of virus samples collection from field specimens, extraction of AEV RNA, amplification of VP-2 protein encoding gene by RT-PCR, separation of RT-PCR product by agarose gel electrophoresis, DNA sequencing and data analysis. Results: Amplification products of the VP-2 encoding gene of AEV by RT-PCR methods of various types of poultry from field specimens showed a positive results on sample code 499/4/12 which generated DNA fragment in the size of 619 bp. Sensitivity test of RT-PCR amplification showed that the minimum concentration of RNA template is 127.75 ng/μl. The multiple alignments of DNA sequencing product indicated that positive sample with code 499/4/12 has 92% nucleotide homology compared with AEV with accession number AV1775/07 and 85% nucleotide homology with accession number ZCHP2/0912695 from Genbank database. Analysis of VP-2 gene sequence showed that it found 46 nucleotides difference between isolate 499/4/12 compared with accession number AV1775/07 and 93 nucleotides different with accession number ZCHP2/0912695. Conclusions: Analyses of the VP-2 encoding gene of AEV with RT-PCR method from 13 samples from field specimen generated the DNA fragment in the size of 619 bp from one sample with

  3. Identification of three genes encoding P(II)-like proteins in Gluconacetobacter diazotrophicus: studies of their role(s) in the control of nitrogen fixation.

    Science.gov (United States)

    Perlova, Olena; Ureta, Alejandro; Nordlund, Stefan; Meletzus, Dietmar

    2003-10-01

    In our studies on the regulation of nitrogen metabolism in Gluconacetobacter diazotrophicus, an endophytic diazotroph of sugarcane, three glnB-like genes were identified and their role(s) in the control of nitrogen fixation was studied. Sequence analysis revealed that one P(II) protein-encoding gene, glnB, was adjacent to a glnA gene (encoding glutamine synthetase) and that two other P(II) protein-encoding genes, identified as glnK1 and glnK2, were located upstream of amtB1 and amtB2, respectively, genes which in other organisms encode ammonium (or methylammonium) transporters. Single and double mutants and a triple mutant with respect to the three P(II) protein-encoding genes were constructed, and the effects of the mutations on nitrogenase expression and activity in the presence of either ammonium starvation or ammonium sufficiency were studied. Based on the results presented here, it is suggested that none of the three P(II) homologs is required for nif gene expression, that the GlnK2 protein acts primarily as an inhibitor of nif gene expression, and that GlnB and GlnK1 control the expression of nif genes in response to ammonium availability, both directly and by relieving the inhibition by GlnK2. This model includes novel regulatory features of P(II) proteins.

  4. The yeast CDP1 gene encodes a triple-helical DNA-binding protein.

    Science.gov (United States)

    Musso, M; Bianchi-Scarrà, G; Van Dyke, M W

    2000-11-01

    The formation of triple-helical DNA has been implicated in several cellular processes, including transcription, replication and recombination. While there is no direct evidence for triplexes in vivo, cellular proteins that specifically recognize triplex DNA have been described. Using a purine-motif triplex probe and southwestern library screening, we isolated five independent clones expressing the same C-terminal 210 amino acids of the Saccharomyces cerevisiae protein Cdp1p fused with beta-galactosidase. In electrophoretic mobility shift assays, recombinant Cdp1pDelta1-867 bound Pu-motif triplex DNAs with high affinity (K:(d) approximately 5 nM) and bound Py-motif triplex, duplex and single-stranded DNAs with far lower affinity (0.5-5.0 microM). Genetic analyses revealed that the CDP1 gene product was required for proper chromosome segregation. The possible involvement of triplex DNA in this process is discussed.

  5. Protein levels of genes encoded on chromosome 21 in fetal Down Syndrome brain (Part V): overexpression of phosphatidyl-inositol-glycan class P protein (DSCR5).

    Science.gov (United States)

    Ferrando-Miguel, R; Cheon, M S; Lubec, G

    2004-06-01

    Down Syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. The completed sequencing of genes encoded on chromosome 21 provides excellent basic information, however the molecular mechanisms leading to the phenotype of DS remain to be elucidated. Although overexpression of chromosome 21 encoded genes has been documented information at the protein expression level is mandatory as it is the proteins that carry out function. We therefore decided to evaluated expression level of seven proteins whose genes are encoded on chromosome 21: DSCR4, DSCR5, DSCR6; KIR4.2, GIRK2, KCNE1 and KCNE2 in fetal cortex brain of DS and controls at the early second trimester of pregnancy by Western blotting. beta-actin and neuron specific enolase (NSE) were used to normalise cell loss and neuronal loss. DSCR5 (PIG-P), a component of glycosylphosphatidylinositol- N-acetylglucosaminyltransferase (GPI-GnT), was overexpressed about twofold, even when levels were normalised with NSE. DSCR6 was overexpressed in addition but when normalised versus NSE, levels were comparable to controls. DSCR4 was not detectable in fetal brain. Potassium channels KIR4.2 and GIRK2 were comparable between DS and controls, whereas KCNE1 and KCNE2 were not detectable. Quantification of these proteins encoded on chromosome 21 revealed that not all gene products of the DS critical region are overexpressed in DS brain early in life, indicating that the DS phenotype cannot be simply explained by the gene dosage effect hypothesis. Overexpression of PIG-P (DSCR5) may lead to or represent impaired glycosylphosphatidylinositol- N-acetylglucosaminyltransferase mediated posttranslational modifications and subsequent anchoring of proteins to the plasma membrane.

  6. Mutations in Three Genes Encoding Proteins Involved in Hair Shaft Formation Cause Uncombable Hair Syndrome.

    Science.gov (United States)

    Ü Basmanav, F Buket; Cau, Laura; Tafazzoli, Aylar; Méchin, Marie-Claire; Wolf, Sabrina; Romano, Maria Teresa; Valentin, Frederic; Wiegmann, Henning; Huchenq, Anne; Kandil, Rima; Garcia Bartels, Natalie; Kilic, Arzu; George, Susannah; Ralser, Damian J; Bergner, Stefan; Ferguson, David J P; Oprisoreanu, Ana-Maria; Wehner, Maria; Thiele, Holger; Altmüller, Janine; Nürnberg, Peter; Swan, Daniel; Houniet, Darren; Büchner, Aline; Weibel, Lisa; Wagner, Nicola; Grimalt, Ramon; Bygum, Anette; Serre, Guy; Blume-Peytavi, Ulrike; Sprecher, Eli; Schoch, Susanne; Oji, Vinzenz; Hamm, Henning; Farrant, Paul; Simon, Michel; Betz, Regina C

    2016-12-01

    Uncombable hair syndrome (UHS), also known as "spun glass hair syndrome," "pili trianguli et canaliculi," or "cheveux incoiffables" is a rare anomaly of the hair shaft that occurs in children and improves with age. UHS is characterized by dry, frizzy, spangly, and often fair hair that is resistant to being combed flat. Until now, both simplex and familial UHS-affected case subjects with autosomal-dominant as well as -recessive inheritance have been reported. However, none of these case subjects were linked to a molecular genetic cause. Here, we report the identification of UHS-causative mutations located in the three genes PADI3 (peptidylarginine deiminase 3), TGM3 (transglutaminase 3), and TCHH (trichohyalin) in a total of 11 children. All of these individuals carry homozygous or compound heterozygous mutations in one of these three genes, indicating an autosomal-recessive inheritance pattern in the majority of UHS case subjects. The two enzymes PADI3 and TGM3, responsible for posttranslational protein modifications, and their target structural protein TCHH are all involved in hair shaft formation. Elucidation of the molecular outcomes of the disease-causing mutations by cell culture experiments and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild-type proteins. Scanning electron microscopy observations revealed morphological alterations in hair coat of Padi3 knockout mice. All together, these findings elucidate the molecular genetic causes of UHS and shed light on its pathophysiology and hair physiology in general. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  7. Mutation of the cytosolic ribosomal protein-encoding RPS10B gene affects shoot meristematic function in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Stirnberg Petra

    2012-09-01

    Full Text Available Abstract Background Plant cytosolic ribosomal proteins are encoded by small gene families. Mutants affecting these genes are often viable, but show growth and developmental defects, suggesting incomplete functional redundancy within the families. Dormancy to growth transitions, such as the activation of axillary buds in the shoot, are characterised by co-ordinated upregulation of ribosomal protein genes. Results A recessive mutation in RPS10B, one of three Arabidopsis genes encoding the eukaryote-specific cytoplasmic ribosomal protein S10e, was found to suppress the excessive shoot branching mutant max2-1. rps10b-1 mildly affects the formation and separation of shoot lateral organs, including the shoot axillary meristems. Axillary meristem defects are enhanced when rps10b-1 is combined with mutations in REVOLUTA, AUXIN-RESISTANT1, PINOID or another suppressor of max2-1, FAR-RED ELONGATED HYPOCOTYL3. In some of these double mutants, the maintenance of the primary shoot meristem is also affected. In contrast, mutation of ALTERED MERISTEM PROGRAMME1 suppresses the rps10b-1axillary shoot defect. Defects in both axillary shoot formation and organ separation were enhanced by combining rps10b-1 with cuc3, a mutation affecting one of three Arabidopsis NAC transcription factor genes with partially redundant roles in these processes. To assess the effect of rps10b-1 on bud activation independently from bud formation, axillary bud outgrowth on excised cauline nodes was analysed. The outgrowth rate of untreated buds was reduced only slightly by rps10b-1 in both wild-type and max2-1 backgrounds. However, rps10b-1 strongly suppressed the auxin resistant outgrowth of max2-1 buds. A developmental phenotype of rps10b-1, reduced stamen number, was complemented by the cDNA of another family member, RPS10C, under the RPS10B promoter. Conclusions RPS10B promotes shoot branching mainly by promoting axillary shoot development. It contributes to organ boundary

  8. ngs (notochord granular surface) gene encodes a novel type of intermediate filament family protein essential for notochord maintenance in zebrafish.

    Science.gov (United States)

    Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

    2013-01-25

    The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins.

  9. The divergently transcribed genes encoding yeast ribosomal proteins L46 and S24 are activated by shared RPG-boxes.

    Science.gov (United States)

    Kraakman, L S; Mager, W H; Maurer, K T; Nieuwint, R T; Planta, R J

    1989-12-11

    Transcription of the majority of the ribosomal protein (rp) genes in yeast is activated through common cis-acting elements, designated RPG-boxes. These elements have been shown to act as specific binding sites for the protein factor TUF/RAP1/GRF1 in vitro. Two such elements occur in the intergenic region separating the divergently transcribed genes encoding L46 and S24. To investigate whether the two RPG-boxes mediate transcription activation of both the L46 and S24 gene, two experimental strategies were followed: cloning of the respective genes on multicopy vectors and construction of fusion genes. Cloning of the L46 + S24 gene including the intergenic region in a multicopy yeast vector indicated that both genes are transcriptionally active. Using constructs in which only the S24 or the L46 gene is present, with or without the intergenic region, we obtained evidence that the intergenic region is indispensable for transcription activation of either gene. To demarcate the element(s) responsible for this activation, fusions of the intergenic region in either orientation to the galK reporter gene were made. Northern analysis of the levels of hybrid mRNA demonstrated that the intergenic region can serve as an heterologous promoter when it is in the 'S24-orientation'. Surprisingly, however, when fused in the reverse orientation the intergenic region did hardly confer transcription activity on the fusion gene. Furthermore, a 274 bp FnuDII-FnuDII fragment from the intergenic region that contains the RPG-boxes, could replace the naturally occurring upstream activation site (UASrpg) of the L25 rp-gene only when inserted in the 'S24-orientation'. Removal of 15 bp from the FnuDII fragment appeared to be sufficient to obtain transcription activation in the 'L46 orientation' as well. Analysis of a construct in which the RPG-boxes were selectively deleted from the promoter region of the L46 gene indicated that the RPG-boxes are needed for efficient transcriptional activation of

  10. Cloning and characterization of three genes encoding Qb-SNARE proteins in rice.

    Science.gov (United States)

    Bao, Yong-Mei; Wang, Jian-Fei; Huang, Ji; Zhang, Hong-Sheng

    2008-03-01

    Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H(2)O(2), but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H(2)O(2), but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H(2)O(2) and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses.

  11. Establishment of transgenic mice carrying gene encoding human zinc finger protein 191

    Science.gov (United States)

    Li, Jian-Zhong; Chen, Xia; Yang, Hua; Wang, Shui-Liang; Gong, Xue-Lian; Feng, Hao; Guo, Bao-Yu; Yu, Long; Wang, Zhu-Gang; Fu, Ji-Liang

    2004-01-01

    AIM: Human zinc finger protein 191 (ZNF191) was cloned and characterized as a Krüppel-like transcription factor, which might be relevant to many diseases such as liver cancer, neuropsychiatric and cardiovascular diseases. Although progress has been made recently, the biological function of ZNF191 remains largely unidentified. The aim of this study was to establish a ZNF 191 transgenic mouse model, which would promote the functional study of ZNF191. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. The offsprings were identified by PCR and Southern blot analysis. ZNF 191 gene expression was analyzed by RT-PCR. Transgenic founder mice were used to establish transgenic mouse lineages. The first generation (F1) and the second generation (F2) mice were identified by PCR analysis. Ten-week transgenic mice were used for pathological examination. RESULTS: Four mice were identified as carrying copies of ZNF191 gene. The results of RT-PCR showed that ZNF 191 gene was expressed in the liver, testis and brain in one of the transgenic mouse lineages. Genetic analysis of transgenic mice demonstrated that ZNF 191 gene was integrated into the chromosome at a single site and could be transmitted stably. Pathological analysis showed that the expression of ZNF 191 did not cause obvious pathological changes in multiple tissues of transgenic mice. CONCLUSION: ZNF 191 transgenic mouse model would facilitate the investigation of biological functions of ZNF191 in vivo. PMID:14716836

  12. Characteristics and phylogeny of light-harvesting complex gene encoded proteins from marine red alga Griffithsia japonica

    Institute of Scientific and Technical Information of China (English)

    LIU Chenlin; HUANG Xiaohang; LEE Yookyung; LEE Hongkum; LI Guangyou

    2005-01-01

    Six genes encoding light-harvesting complex (LHC) protein have been characterized in the multicellular red alga Griffithsia japonica EST analysis. Three of them were full sequences while others were partial sequences with 3'-UTRs. The cleavage sites between signal peptide and mature LHC protein were analyzed on these three full sequences. The sequence characteristics, calculated molecular weights and isoelectric point (pI) values and hydrophobieity of the mature proteins were deduced and analyzed. Comparing the LHC sequences of G. japonica with higher plant, Chlorophyta, chromophytes and other red algae, the high conservation of the chlorophyll (Chl) binding site among chromophytes and red algae were revealed. Phylogenetic analysis on LHC proteins from higher plant, green algae, euglena, brown algae, diatom, cryptomonad, Raphidophyte and red algae reveals that (1) there are two distinct groups of Chl a/b and Chl a/c -binding LHC; (2) Chl a binding proteins of red algae share greater similarities with the Chl a/c-binding proteins of the chromophytes and dinoflagellate than with the Chl a/b - binding proteins of the green algae and higher plants; (3) chromophyte' s LHC is supposed to be evolved from red algae LHC.

  13. The rice HGW gene encodes a ubiquitin-associated (UBA domain protein that regulates heading date and grain weight.

    Directory of Open Access Journals (Sweden)

    Juan Li

    Full Text Available Heading date and grain weight are two determining agronomic traits of crop yield. To date, molecular factors controlling both heading date and grain weight have not been identified. Here we report the isolation of a hemizygous mutation, heading and grain weight (hgw, which delays heading and reduces grain weight in rice. Analysis of hgw mutant phenotypes indicate that the hemizygous hgw mutation decreases latitudinal cell number in the lemma and palea, both composing the spikelet hull that is known to determine the size and shape of brown grain. Molecular cloning and characterization of the HGW gene showed that it encodes a novel plant-specific ubiquitin-associated (UBA domain protein localized in the cytoplasm and nucleus, and functions as a key upstream regulator to promote expressions of heading date- and grain weight-related genes. Moreover, co-expression analysis in rice and Arabidopsis indicated that HGW and its Arabidopsis homolog are co-expressed with genes encoding various components of ubiquitination machinery, implying a fundamental role for the ubiquitination pathway in heading date and grain weight control.

  14. Optically encoded microspheres for high-throughput analysis of genes and proteins

    Science.gov (United States)

    Gao, Xiaohu; Han, Mingyong; Nie, Shuming

    2002-06-01

    We have developed a novel optical coding technology for massively parallel and high-throughput analysis of biological molecules. Its unprecedented multiplexing capability is based on the unique optical properties of semiconductor quantum dots (QDs) and the ability to incorporate multicolor QQs into small polymer beads at precisely controlled ratios. The use of 10 intensity levels and 6 colors could theoretically code one million nucleic acid or protein sequences. Imaging and spectroscopic studies indicate that the QD tagged beads are highly uniform and reproducible, yielding bead identification accuracies as high as 99.99 percent under favorable conditions. DNA hybridization results demonstrate that the coding and target signals can be simultaneously read at the single-bead level. This spectral coding technology is expected to open new opportunities in gene expression studies, high-throughput screening, and medical diagnosis.

  15. Inactivation of genes encoding plastoglobuli-like proteins in Synechocystis sp. PCC 6803 leads to a light-sensitive phenotype.

    Science.gov (United States)

    Cunningham, Francis X; Tice, Ashley B; Pham, Christina; Gantt, Elisabeth

    2010-03-01

    Plastoglobulins (PGL) are the predominant proteins of lipid globules in the plastids of flowering plants. Genes encoding proteins similar to plant PGL are also present in algae and cyanobacteria but in no other organisms, suggesting an important role for these proteins in oxygenic photosynthesis. To gain an understanding of the core and fundamental function of PGL, the two genes that encode PGL-like polypeptides in the cyanobacterium Synechocystis sp. PCC 6803 (pgl1 and pgl2) were inactivated individually and in combination. The resulting mutants were able to grow under photoautotrophic conditions, dividing at rates that were comparable to that of the wild-type (WT) under low-light (LL) conditions (10 microeinsteins x m(-2) x s(-1)) but lower than that of the WT under moderately high-irradiance (HL) conditions (150 microeinsteins x m(-2) x s(-1)). Under HL, each Deltapgl mutant had less chlorophyll, a lower photosystem I (PSI)/PSII ratio, more carotenoid per unit of chlorophyll, and very much more myxoxanthophyll (a carotenoid symptomatic of high light stress) per unit of chlorophyll than the WT. Large, heterogeneous inclusion bodies were observed in cells of mutants inactivated in pgl2 or both pgl2 and pgl1 under both LL and HL conditions. The mutant inactivated in both pgl genes was especially sensitive to the light environment, with alterations in pigmentation, heterogeneous inclusion bodies, and a lower PSI/PSII ratio than the WT even for cultures grown under LL conditions. The WT cultures grown under HL contained 2- to 3-fold more PGL1 and PGL2 per cell than cultures grown under LL conditions. These and other observations led us to conclude that the PGL-like polypeptides of Synechocystis play similar but not identical roles in some process relevant to the repair of photooxidative damage.

  16. Brain transcriptional stability upon prion protein-encoding gene invalidation in zygotic or adult mouse

    Directory of Open Access Journals (Sweden)

    Béringue Vincent

    2010-07-01

    Full Text Available Abstract Background The physiological function of the prion protein remains largely elusive while its key role in prion infection has been expansively documented. To potentially assess this conundrum, we performed a comparative transcriptomic analysis of the brain of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic or at the adult stages. Results Only subtle transcriptomic differences resulting from the Prnp knockout could be evidenced, beside Prnp itself, in the analyzed adult brains following microarray analysis of 24 109 mouse genes and QPCR assessment of some of the putatively marginally modulated loci. When performed at the adult stage, neuronal Prnp disruption appeared to sequentially induce a response to an oxidative stress and a remodeling of the nervous system. However, these events involved only a limited number of genes, expression levels of which were only slightly modified and not always confirmed by RT-qPCR. If not, the qPCR obtained data suggested even less pronounced differences. Conclusions These results suggest that the physiological function of PrP is redundant at the adult stage or important for only a small subset of the brain cell population under classical breeding conditions. Following its early reported embryonic developmental regulation, this lack of response could also imply that PrP has a more detrimental role during mouse embryogenesis and that potential transient compensatory mechanisms have to be searched for at the time this locus becomes transcriptionally activated.

  17. Cloning, expression analysis and recombinant expression of a gene encoding a polygalacturonase-inhibiting protein from tobacco, Nicotiana tabacum

    Directory of Open Access Journals (Sweden)

    Chengsheng Zhang

    2016-05-01

    Full Text Available Polygalacturonase inhibiting proteins (PGIPs are major defensive proteins produced by plant cell walls that play a crucial role in pathogen resistance by reducing polygalacturonase (PG activity. In the present study, a novel PGIP gene was isolated from tobacco (Nicotiana tabacum, hereafter referred as NtPGIP. A full-length NtPGIP cDNA of 1,412 bp with a 186 bp 5′-untranslated region (UTR, and 209 bp 3′-UTR was cloned from tobacco, NtPGIP is predicted to encode a protein of 338 amino acids. The NtPGIP sequence from genomic DNA showed no introns and sequence alignments of NtPGIP’s deduced amino acid sequence showed high homology with known PGIPs from other plant species. Moreover, the putative NtPGIP protein was closely clustered with several Solanaceae PGIPs. Further, the expression profile of NtPGIP was examined in tobacco leaves following stimulation with the oomycete Phytophthora nicotianae and other stressors, including salicylic acid (SA, abscisic acid (ABA, salt, and cold treatment. The results showed that all of the treatments up-regulated the expression of NtPGIP at different times. To understand the biochemical activity of NtPGIP gene, a full-length NtPGIP cDNA sequence was subcloned into a pET28a vector and transformed into E. coli BL21 (DE3. Recombinant proteins were successfully induced by 1.0 nmol/L IPTG and the purified proteins effectively inhibited Phytophthora capsici PG activity. The results of this study suggest that NtPGIP may be a new candidate gene with properties that could be exploited in plant breeding.

  18. Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum

    Indian Academy of Sciences (India)

    Xiuhong Yang; Chao Sun; Yuanlei Hun; Zhongping Lin

    2008-03-01

    A RING zinc finger ankyrin protein gene, designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE. Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4-type RING finger domain was found at the C-terminal region of the AdZFP1 protein, and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869. Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root, stem and leaf of the plant. Semi-quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity, cold and heat to some extent. Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

  19. A comparative analysis of green fluorescent protein and -glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

    Indian Academy of Sciences (India)

    P Kavita; Pradeep Kumar Burma

    2008-09-01

    The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, -glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

  20. Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from Chenopodium ficifolium.

    Science.gov (United States)

    Takahashi, Shigekazu; Abe, Eriko; Nakayama, Katsumi; Satoh, Hiroyuki

    2015-01-01

    Photoconvertible water-soluble chlorophyll-binding proteins, called Class I WSCPs, have been detected in Chenopodiaceae, Amaranthaceae and Polygonaceae plant species. To date, Chenopodium album WSCP (CaWSCP) is the only cloned gene encoding a Class I WSCP. In this study, we identified two cDNAs encoding Chenopodium ficifolium Class I WSCPs, CfWSCP1, and CfWSCP2. Sequence analyses revealed that the open reading frames of CfWSCP1 and CfWSCP2 were 585 and 588 bp, respectively. Furthermore, both CfWSCPs contain cystein2 and cystein30, which are essential for the chlorophyll-binding ability of CaWSCP. Recombinant CfWSCP1 and CfWSCP2, expressed in Escherichia coli as hexa-histidine fusion proteins (CfWSCP1-His and CfWSCP2-His), formed inclusion bodies; however, we were able to solubilize these using a buffer containing 8 M urea and then refold them by dialysis. The refolded CfWSCP1-His and CfWSCP2-His could bind chlorophylls and exhibited photoconvertibility, confirming that the cloned CfWSCPs are further examples of Class I WSCPs.

  1. Chromosome locations of genes encoding human signal transduction adapter proteins, Nck (NCK), Shc (SHC1), and Grb2 (GRB2)

    DEFF Research Database (Denmark)

    Huebner, K; Kastury, K; Druck, T;

    1994-01-01

    Abnormalities due to chromosomal aberration or point mutation in gene products of growth factor receptors or in ras gene products, which lie on the same signaling pathway, can cause disease in animals and humans. Thus, it can be important to determine chromosomal map positions of genes encoding "...

  2. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase

    Science.gov (United States)

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 famil...

  3. Identification and regulation of expression of a gene encoding a filamentous hemagglutinin-related protein in Bordetella holmesii

    Directory of Open Access Journals (Sweden)

    Gross Roy

    2007-11-01

    Full Text Available Abstract Background Bordetella holmesii is a human pathogen closely related to B. pertussis, the etiological agent of whooping cough. It is able to cause disease in immunocompromised patients, but also whooping cough-like symptoms in otherwise healthy individuals. However, virtually nothing was known so far about the underlying virulence mechanisms and previous attempts to identify virulence factors related to those of B. pertussis were not successful. Results By use of a PCR approach we were able to identify a B. holmesii gene encoding a protein with significant sequence similarities to the filamentous hemagglutinin (FHA of B. avium and to a lesser extent to the FHA proteins of B. pertussis, B. parapertussis, and B. bronchiseptica. For these human and animal pathogens FHA is a crucial virulence factor required for successful colonization of the host. Interestingly, the B. holmesii protein shows a relatively high overall sequence similarity with the B. avium protein, while sequence conservation with the FHA proteins of the human and mammalian pathogens is quite limited and is most prominent in signal sequences required for their export to the cell surface. In the other Bordetellae expression of the fhaB gene encoding FHA was shown to be regulated by the master regulator of virulence, the BvgAS two-component system. Recently, we identified orthologs of BvgAS in B. holmesii, and here we show that this system also contributes to regulation of fhaB expression in B. holmesii. Accordingly, the purified BvgA response regulator of B. holmesii was shown to bind specifically in the upstream region of the fhaB promoter in vitro in a manner similar to that previously described for the BvgA protein of B. pertussis. Moreover, by deletion analysis of the fhaB promoter region we show that the BvgA binding sites are relevant for in vivo transcription from this promoter in B. holmesii. Conclusion The data reported here show that B. holmesii is endowed with a

  4. Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria.

    Science.gov (United States)

    Spelbrink, J N; Li, F Y; Tiranti, V; Nikali, K; Yuan, Q P; Tariq, M; Wanrooij, S; Garrido, N; Comi, G; Morandi, L; Santoro, L; Toscano, A; Fabrizi, G M; Somer, H; Croxen, R; Beeson, D; Poulton, J; Suomalainen, A; Jacobs, H T; Zeviani, M; Larsson, C

    2001-07-01

    The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

  5. Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein.

    Science.gov (United States)

    Pogue-Geile, K; Geiser, J R; Shu, M; Miller, C; Wool, I G; Meisler, A I; Pipas, J M

    1991-08-01

    We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library. The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa. Increased levels of the S3 transcript were present in the tumors of all eight patients examined. Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma. Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps. These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia.

  6. Meta-analysis: a functional polymorphism in the gene encoding for activity of the serotonin transporter protein is not associated with the irritable bowel syndrome.

    NARCIS (Netherlands)

    Kerkhoven, L.A.S. van; Laheij, R.J.F.; Jansen, J.B.M.J.

    2007-01-01

    BACKGROUND: Serotonin is associated with symptoms of the irritable bowel syndrome, its action is terminated by the serotonin transporter protein. AIM: To assess the association between a functional polymorphism in the gene encoding for activity of the serotonin transporter protein and the irritable

  7. Identification of two novel genes encoding 97- to 99-kilodalton outer membrane proteins of Chlamydia pneumoniae.Infect Immun. 1999 Jan;67(1):375-83

    DEFF Research Database (Denmark)

    Knudsen, K; Madsen, AS; Mygind, P

    1999-01-01

    of putative outer membrane proteins encoded by the Chlamydia psittaci and Chlamydia trachomatis gene families. By use of a monospecific polyclonal antibody against purified recombinant Omp4, it was shown that without heating, the protein migrated at 65 to 75 kDa in sodium dodecyl sulfate-polyacrylamide gel...

  8. Identification of the gene encoding Brain Cell Membrane Protein 1 (BCMP1, a putative four-transmembrane protein distantly related to the Peripheral Myelin Protein 22 / Epithelial Membrane Proteins and the Claudins

    Directory of Open Access Journals (Sweden)

    Christophe Daniel

    2001-07-01

    Full Text Available Abstract Background A partial cDNA clone from dog thyroid presenting a very significant similarity with an uncharacterized mouse EST sequence was isolated fortuitously. We report here the identification of the complete mRNA and of the gene, the product of which was termed "brain cell membrane protein 1" (BCMP1. Results The 4 kb-long mRNA sequence exhibited an open-reading frame of only 543 b followed by a 3.2 kb-long 3' untranslated region containing several AUUUA instability motifs. Analysis of the encoded protein sequence identified the presence of four putative transmembrane domains. Similarity searches in protein domain databases identified partial sequence conservations with peripheral myelin protein 22 (PMP22/ epithelial membrane proteins (EMPs and Claudins, defining the encoded protein as representative of the existence of a novel subclass in this protein family. Northern-blot analysis of the expression of the corresponding mRNA in adult dog tissues revealed the presence of a huge amount of the 4 kb transcript in the brain. An EGFP-BCMP1 fusion protein expressed in transfected COS-7 cells exhibited a membranous localization as expected. The sequences encoding BCMP1 were assigned to chromosome X in dog, man and rat using radiation hybrid panels and were partly localized in the currently available human genome sequence. Conclusions We have identified the existence in several mammalian species of a gene encoding a putative four-transmembrane protein, BCMP1, wich defines a novel subclass in this family of proteins. In dog at least, the corresponding mRNA is highly present in brain cells. The chromosomal localization of the gene in man makes of it a likely candidate gene for X-linked mental retardation.

  9. Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

    Science.gov (United States)

    Mitchison, A

    1997-01-01

    In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.

  10. NMR Structure of the hypothetical protein encoded by the YjbJ gene from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Pineda-Lucena, Antonio; Liao, Jack; Wu, Bin; Yee, Adelinda; Cort, John R.; Kennedy, Michael A.; Edwards, Aled M.; Arrowsmith, Cheryl H.

    2002-06-01

    Here we describe the solution structure of YjbJ (gil418541) as part of a structural proteomics project on the feasibility of the high-throughput generation of samples from Escherichia coli for structural studies. YjbJ is a hypothetical protein from Escherichia coli protein of unknown function. It is conserved, showing significant sequence identity to four predicted prokaryotic proteins, also of unknown function (Figure 1A). These include gil16762921 from Salmonella enterica (S. typhi), gil17938413 from Agrobacterium tumefaciens, gil16265654 from Sinorizhobium meliloti, and gil15599932 from Pseudomona aeruginosa. The structure of YjbJ reveals a new variation of a common motif (four-helix bundle) that could not be predicted from the protein sequence. Although the biochemical function is unknown, the existence of patterns of conserved residues on the protein surface suggest that the fold and function of all these proteins could be similar.

  11. liver-enriched gene 1a and 1b encode novel secretory proteins essential for normal liver development in zebrafish.

    Directory of Open Access Journals (Sweden)

    Changqing Chang

    Full Text Available liver-enriched gene 1 (leg1 is a liver-enriched gene in zebrafish and encodes a novel protein. Our preliminary data suggested that Leg1 is probably involved in early liver development. However, no detailed characterization of Leg1 has been reported thus far. We undertook both bioinformatic and experimental approaches to study leg1 gene structure and its role in early liver development. We found that Leg1 identifies a new conserved protein superfamily featured by the presence of domain of unknown function 781 (DUF781. There are two copies of leg1 in zebrafish, namely leg1a and leg1b. Both leg1a and leg1b are expressed in the larvae and adult liver with leg1a being the predominant form. Knockdown of Leg1a or Leg1b by their respective morpholinos specifically targeting their 5'-UTR each resulted in a small liver phenotype, demonstrating that both Leg1a and Leg1b are important for early liver development. Meanwhile, we found that injection of leg1-ATG(MO, a morpholino which can simultaneously block the translation of Leg1a and Leg1b, caused not only a small liver phenotype but hypoplastic exocrine pancreas and intestinal tube as well. Further examination of leg1-ATG(MO morphants with early endoderm markers and early hepatic markers revealed that although depletion of total Leg1 does not alter the hepatic and pancreatic fate of the endoderm cells, it leads to cell cycle arrest that results in growth retardation of liver, exocrine pancreas and intestine. Finally, we proved that Leg1 is a secretory protein. This intrigued us to propose that Leg1 might act as a novel secreted regulator that is essential for liver and other digestive organ development in zebrafish.

  12. Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

    Science.gov (United States)

    Tandon, Ritesh; LePage, Keith T; Kaplan, Ray M

    2006-11-01

    The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

  13. Mushroom body defect, a gene involved in the control of neuroblast proliferation in Drosophila, encodes a coiled–coil protein

    Science.gov (United States)

    Guan, Zhonghui; Prado, Antonio; Melzig, Jörg; Heisenberg, Martin; Nash, Howard A.; Raabe, Thomas

    2000-01-01

    Neurogenesis relies on the establishment of the proper number and precisely controlled proliferation of neuroblasts, the neuronal precursor cells. A role for the mushroom body defect (mud) gene in both of these aspects of neuroblast behavior, as well as possible roles in other aspects of fruit fly biology, is implied by phenotypes associated with mud mutations. We have localized mud by determining the sequence change in one point mutant, identifying a predicted ORF affected by the mutation, and showing that an appropriate segment of the genome rescues mud mutant phenotypes. An analysis of mud cDNAs and a survey of mud transcripts by Northern blotting indicate that the gene is subject to differential splicing and is expressed primarily during embryogenesis but also, at lower levels, during subsequent developmental stages in a sexually dimorphic manner. The gene is predicted to encode a polypeptide without obvious homologs but with two prominent structural features, a long coiled coil that constitutes the central core of the protein and a carboxyl-terminal transmembrane domain. PMID:10884435

  14. Cloning and characterization of a gene encoding phage-related tail protein (PrTP) of endosymbiont Wolbachia

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Wolbachia is an obligatory, maternally inherited intracellular bacterium, known to infect a wide range of arthropods. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, the feminization of genetic males and male-killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these reproductive abnormalities have not yet been determined. In this study, we report on the cloning and characterization of the gene encoding phage-related tail protein (PrTP) from Wolbachia in Drosophila melanogaster CantonS (wMelCS) and from Wolbachia in Drosophila melanogaster yw67c23 (wMel) by representational difference analysis (RDA) and ligation-mediated PCR (LM-PCR). The functionality of a bipartite nuclear localization signal sequence (NLS) of the gene was also successfully tested in Drosophila S2 cells. PrTP expression in various strains of Wolbachia was investigated. Our results suggest that PrTP may not induce CI directly. However, the existence of prtp provided direct evidence of phage-mediated horizontal gene transfer (HGT) that might play an important role in a variety of reproductive abnormalities of Wolbachia.

  15. Transformation of Coat Protein Encoding Gene from Soil-Borne Mosaic Virus into Wheat

    Institute of Scientific and Technical Information of China (English)

    PANG Jun-lan; XU Hui-jun; DU Li-pu; YE Xing-guo; LI Lian-cheng; XIN Zhi-yong; MA You-zhi; DIAO Ai-po; Adams M J

    2003-01-01

    CWMV-CP1 target gene and bar selection gene were co-transferred into commercial wheat vari-ety of Yangmai158 by particle bombardment. In total, 145 resistant plants to 3 - 5 mg L-1 Bialaphos were ob-tained, 21 plants were identified to be positive in T0 generation by PCR-Southern test, and the transformationfrequency had 0. 99%. T1 plants were further tested by PCR and Southern hybridization. Results demonstra-ted that the alien resistance gene had been integrated into the wheat genome. The segregation ratio of CP1+ toCP1- in T1 generation was 1.0 to 1.3, and didn't agree with Mendelian rule. RT-PCR result from T2 plantsshowed that the alien gene CWMV-CP1 had stable expression in wheat genetic background.

  16. Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome.

    NARCIS (Netherlands)

    Frank, V.; Hollander, A.I. den; Bruchle, N.O.; Zonneveld, M.N.; Nurnberg, G.; Becker, C.; Bois, G. Du; Kendziorra, H.; Roosing, S.; Senderek, J.; Nurnberg, P.; Cremers, F.P.M.; Zerres, K.; Bergmann, C.

    2008-01-01

    Meckel-Gruber syndrome (MKS) is an autosomal recessive, lethal multisystemic disorder characterized by meningooccipital encephalocele, cystic kidney dysplasia, hepatobiliary ductal plate malformation, and postaxial polydactyly. Recently, genes for MKS1 and MKS3 were identified, putting MKS on the

  17. Sequence analysis of a Molluscum contagiosum virus DNA region which includes the gene encoding protein kinase 2 and other genes with unique organization.

    Science.gov (United States)

    Martin-Gallardo, A; Moratilla, M; Funes, J M; Agromayor, M; Nuñez, A; Varas, A J; Collado, M; Valencia, A; Lopez-Estebaranz, J L; Esteban, M

    1996-01-01

    The nucleotide sequence of a near left-terminal region from the genome of Molluscum contagiosum virus subtype I (MCVI) was determined. This region was contained within three adjacent BamHI fragments, designated L (2.4 kilobases (kb)), M (1.8 kb), and N (1.6 kb). BamHI cleavage of MCVI DNA produced another 1.6-kb fragment (N'), which had been mapped 30-50 kb from the L,M region. The MCVI restriction fragments were cloned and end-sequenced. The N fragment that maps at the L,M region was identified by the polymerase chain reaction, using primers devised from the sequence of each fragment. The results from this analysis led to establish the relative position of these fragments within the MCVI genome. The analysis of 3.6 kb of DNA sequence revealed the presence of ten open reading frames (ORFs). Comparison of the amino acid sequence of these ORFs to the amino acid sequence of vaccinia virus (VAC) proteins revealed that two complete MCVI ORFs, termed N1L and L1L, showed high degree of homology with VAC F9 and F10 genes, respectively. The F10 gene encodes a 52-kDa serine/threonine protein kinase (protein kinase 2), an essential protein involved in virus morphogenesis. The MCVI homologue (L1L) encoded a putative polypeptide of 443 aa, with a calculated molecular mass of 53 kDa, and 60.5/30.2% sequence identity/similarity to VAC F10. The MCV N1L (213 aa, 24 kDa) showed 42.6/40.6% amino acid sequence identity/similarity to VAC F9, a gene of unknown function encoding a 24-kDa protein with a hydrophobic C-terminal domain, which was conserved in MCVI. The genomic arrangement of MCVI N1L and L1L was equivalent to that of the vaccinia and variola virus homologues. However, the ORFs contained within MCVI fragment M (leftward) showed no homology, neither similarity in genetic organization, to the genes encoded by the corresponding regions of vaccinia and variola viruses.

  18. Obesity risk gene TMEM18 encodes a sequence-specific DNA-binding protein.

    Directory of Open Access Journals (Sweden)

    Jaana M Jurvansuu

    Full Text Available Transmembrane protein 18 (TMEM18 has previously been connected to cell migration and obesity. However, the molecular function of the protein has not yet been described. Here we show that TMEM18 localises to the nuclear membrane and binds to DNA in a sequence-specific manner. The protein binds DNA with its positively charged C-terminus that contains also a nuclear localisation signal. Increase in the amount of TMEM18 in cells suppresses expression from a reporter vector with the TMEM18 target sequence. TMEM18 is a small protein of 140 residues and is predicted to be mostly alpha-helical with three transmembrane parts. As a consequence the DNA binding by TMEM18 would bring the chromatin very near to nuclear membrane. We speculate that this closed perinuclear localisation of TMEM18-bound DNA might repress transcription from it.

  19. Mutations in Three Genes Encoding Proteins Involved in Hair Shaft Formation Cause Uncombable Hair Syndrome

    DEFF Research Database (Denmark)

    Ü Basmanav, F Buket; Cau, Laura; Tafazzoli, Aylar

    2016-01-01

    Uncombable hair syndrome (UHS), also known as "spun glass hair syndrome," "pili trianguli et canaliculi," or "cheveux incoiffables" is a rare anomaly of the hair shaft that occurs in children and improves with age. UHS is characterized by dry, frizzy, spangly, and often fair hair that is resistant...... in the majority of UHS case subjects. The two enzymes PADI3 and TGM3, responsible for posttranslational protein modifications, and their target structural protein TCHH are all involved in hair shaft formation. Elucidation of the molecular outcomes of the disease-causing mutations by cell culture experiments...... and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild-type proteins. Scanning electron microscopy observations revealed morphological alterations in hair coat of Padi3 knockout mice. All together, these findings elucidate the molecular genetic...

  20. Molecular Characterization and Sequencing of a Gene Encoding Mannose Binding Protein in an Iranian Isolate of Acanthamoeba castellanii as a Major Agent of Acanthamoeba Keratitis

    Directory of Open Access Journals (Sweden)

    SH Farnia

    2008-07-01

    Full Text Available Background: Acanthamoeba castellanii is the important cause of amoebic keratitis in Iran. The key molecule in pathogenesis of Acanthamoeba keratitis is Mannose Binding Protein (MBP led to adhesion of amoeba to corneal epithelium. Subsequent to adhesion other cytopathic effects occur. The goal of this study was to identify the molecular characterization of a gene encoding MBP in an Iranian isolate of A.castellanii in order to pave the way for further investigations such as new therapeutic advances or immunization. Methods: A.castellanii was cultured on non nutrient agar. Extraction of DNA was performed by phenol-chloroform method. After designing a pair of primer for the gene encoding MBP, PCR analysis was performed. Finally, the PCR product has been sequenced and the result submitted to the gene data banks. Results: An MBP gene of 1081 nucleotides was sequenced. This fragment contained three introns and encodes a protein with 194 amino acids. Homology search by Blast program showed a significant homology with the MBP gene in gene data banks (96%. Besides, the identity of amino acids with the other MBPs in gene data banks was about 86%. Conclusion: We isolated and sequenced a gene fragment encoding MBP in an Iranian isolate of A.castellanii. Molecular characterization of this important gene is the first step in pursuing researches such as developing better therapeutic agents, immunization of population at risk or even developing a diagnostic tool by PCR techniques.

  1. Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome.

    NARCIS (Netherlands)

    Frank, V.; Hollander, A.I. den; Bruchle, N.O.; Zonneveld, M.N.; Nurnberg, G.; Becker, C.; Bois, G. Du; Kendziorra, H.; Roosing, S.; Senderek, J.; Nurnberg, P.; Cremers, F.P.M.; Zerres, K.; Bergmann, C.

    2008-01-01

    Meckel-Gruber syndrome (MKS) is an autosomal recessive, lethal multisystemic disorder characterized by meningooccipital encephalocele, cystic kidney dysplasia, hepatobiliary ductal plate malformation, and postaxial polydactyly. Recently, genes for MKS1 and MKS3 were identified, putting MKS on the li

  2. Identification and characterization of a gene encoding a UBX domain-containing protein in the migratory locust, Locusta migratoria manilensis.

    Science.gov (United States)

    He, Zheng-Bo; Xie, Yu; Si, Feng-Ling; Chen, Bin

    2013-08-01

    Ubiquitin regulatory X (UBX) domain-containing proteins are believed to function as cofactors for p97/CDC48, an adenosine triphosphatase shown to be involved in multiple cellular processes. In the present study, a full-length complementary DNA (cDNA) of UBX domain-containing gene, termed LmUBX1, was cloned from Locusta migratoria manilensis and characterized, using random amplification of cDNA ends polymerase chain reaction (RACE PCR), sequence analysis and quantitative real-time PCR. LmUBX1, 1 600 bp in length, is predicted to encode a 446-amino acid protein with a predicted molecular weight of 51.18 kDa that contains a central PUB domain and a carboxy-terminal UBX domain. Homology analysis revealed that LmUBX1 has higher similarity to the known UBX domain-containing proteins from insects than from other species. Moreover, based on sequence characteristics and phylogenetic relationships, it is suggested that LmUBX1 can be classified into the UBXD1 subfamily. Expression analysis founded that LmUBX1 exhibited significant expression variations at different developmental stages and in different tissues, suggesting that the expression of LmUBX1 was highly regulated. Interestingly, its messenger RNA transcript was more abundant in ovary and testis than in other tissues examined, suggesting that it may have more important roles in the reproductive system. In addition, LmUBX1 was differentially expressed in gregarious and solitary locusts and was significantly up-regulated in third and fifth instars of gregarious locusts, implying that LmUBX1 was also likely involved in the phase polyphenisms in L. migratoria manilensis. To our knowledge, this is the first report of cloning of a full-length cDNA of UBX domain-containing gene from L. migratoria manilensis.

  3. Ethylmalonic Encephalopathy Is Caused by Mutations in ETHE1, a Gene Encoding a Mitochondrial Matrix Protein

    OpenAIRE

    2004-01-01

    Ethylmalonic encephalopathy (EE) is a devastating infantile metabolic disorder affecting the brain, gastrointestinal tract, and peripheral vessels. High levels of ethylmalonic acid are detected in the body fluids, and cytochrome c oxidase activity is decreased in skeletal muscle. By use of a combination of homozygosity mapping, integration of physical and functional genomic data sets, and mutational screening, we identified GenBank D83198 as the gene responsible for EE. We also demonstrated t...

  4. Molecular adaptation within the coat protein-encoding gene of Tunisian almond isolates of Prunus necrotic ringspot virus

    Indian Academy of Sciences (India)

    Moncef Boulila; Sawssen Ben Tiba; Saoussen Jilani

    2013-04-01

    The sequence alignments of five Tunisian isolates of Prunus necrotic ringspot virus (PNRSV) were searched for evidence of recombination and diversifying selection. Since failing to account for recombination can elevate the false positive error rate in positive selection inference, a genetic algorithm (GARD) was used first and led to the detection of potential recombination events in the coat protein-encoding gene of that virus. The Recco algorithm confirmed these results by identifying, additionally, the potential recombinants. For neutrality testing and evaluation of nucleotide polymorphism in PNRSV CP gene, Tajima’s , and Fu and Li’s and statistical tests were used. About selection inference, eight algorithms (SLAC, FEL, IFEL, REL, FUBAR, MEME, PARRIS, and GA branch) incorporated in HyPhy package were utilized to assess the selection pressure exerted on the expression of PNRSV capsid. Inferred phylogenies pointed out, in addition to the three classical groups (PE-5, PV-32, and PV-96), the delineation of a fourth cluster having the new proposed designation SW6, and a fifth clade comprising four Tunisian PNRSV isolates which underwent recombination and selective pressure and to which the name Tunisian outgroup was allocated.

  5. Isolation and characterization of two genes encoding polygalacturonase-inhibiting protein from Populus deltoides

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Polygalacturonase-inhibiting proteins (PGIPs) are extracellular proteins that belong to the leucine-rich repeat (LRR) protein superfamily. PGIPs inhibit fungal polygalacturonases (PGs) and promote accumulation of oligogalacturonides, which activate plant defense responses. PGIPs play important roles in resistance to infection of pathogens. In this study, reverse transcriptase-polymerase chain reaction (RT-PCR) and RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) were used to isolate the full-length PGIP cDNA from Populus deltoides (GenBank accession no. Of PdPGIP2 and PdPGIP4:EF684913 and EF684912). Domain analysis revealed that the deduced amino acid sequences of PdPGIP2 and PdPGIP4 had a typical POIP topology. Phylogenetic analysis of known PGIPs indicated that the two PdPGIPs were clustered to the defense-related PGIP clade. Using real-time RT-PCR, the expression patterns of the two PdPGIPs following treatment with a fungal pathogen and defense-related signaling molecules were studied. The expression levels of PdPGIP2 and PdPGIP4 were both up-regulated when inoculated with the phytopathogenic fungus Marssonina brunnea. Therefore, it was proposed that the two PGIPs might be involved in the resistance to Marssonina brunnea in P. Deltoides.

  6. Coat protein gene sequence of tobacco mosaic virus encodes a host response determinant.

    OpenAIRE

    Saito, T.; Meshi, T; Takamatsu, N; Okada, Y.

    1987-01-01

    The common strain and tomato strain of tobacco mosaic virus (TMV) are known to be closely related to each other. However, plants with the N' gene, such as Nicotiana sylvestris and Nicotiana tabacum L. cv. Bright Yellow, respond differently to infections by these viruses. In the N' plants, TMV-OM (common strain) spreads systemically with mosaic symptoms, whereas TMV-L (tomato strain) induces the necrotic response of plants, causing local lesions. To reveal the viral factor of TMV-L inducing th...

  7. Mutational analysis of the coding regions of the genes encoding protein kinase B-alpha and -beta, phosphoinositide-dependent protein kinase-1, phosphatase targeting to glycogen, protein phosphatase inhibitor-1, and glycogenin

    DEFF Research Database (Denmark)

    Hansen, L; Fjordvang, H; Rasmussen, S K

    1999-01-01

    be caused by genetic variability in the genes encoding proteins shown by biochemical evidence to be involved in insulin-stimulated glycogen synthesis in skeletal muscle. In 70 insulin-resistant Danish NIDDM patients, mutational analysis by reverse transcription-polymerase chain reaction-single strand...... conformation polymorphism-heteroduplex analysis was performed on genomic DNA or skeletal muscle-derived cDNAs encoding glycogenin, protein phosphatase inhibitor-1, phophatase targeting to glycogen, protein kinase B-alpha and -beta, and the phosphoinositide-dependent protein kinase-1. Although a number...

  8. Identification of human microRNA-like sequences embedded within the protein-encoding genes of the human immunodeficiency virus.

    Directory of Open Access Journals (Sweden)

    Bryan Holland

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are highly conserved, short (18-22 nts, non-coding RNA molecules that regulate gene expression by binding to the 3' untranslated regions (3'UTRs of mRNAs. While numerous cellular microRNAs have been associated with the progression of various diseases including cancer, miRNAs associated with retroviruses have not been well characterized. Herein we report identification of microRNA-like sequences in coding regions of several HIV-1 genomes. RESULTS: Based on our earlier proteomics and bioinformatics studies, we have identified 8 cellular miRNAs that are predicted to bind to the mRNAs of multiple proteins that are dysregulated during HIV-infection of CD4+ T-cells in vitro. In silico analysis of the full length and mature sequences of these 8 miRNAs and comparisons with all the genomic and subgenomic sequences of HIV-1 strains in global databases revealed that the first 18/18 sequences of the mature hsa-miR-195 sequence (including the short seed sequence, matched perfectly (100%, or with one nucleotide mismatch, within the envelope (env genes of five HIV-1 genomes from Africa. In addition, we have identified 4 other miRNA-like sequences (hsa-miR-30d, hsa-miR-30e, hsa-miR-374a and hsa-miR-424 within the env and the gag-pol encoding regions of several HIV-1 strains, albeit with reduced homology. Mapping of the miRNA-homologues of env within HIV-1 genomes localized these sequence to the functionally significant variable regions of the env glycoprotein gp120 designated V1, V2, V4 and V5. CONCLUSIONS: We conclude that microRNA-like sequences are embedded within the protein-encoding regions of several HIV-1 genomes. Given that the V1 to V5 regions of HIV-1 envelopes contain specific, well-characterized domains that are critical for immune responses, virus neutralization and disease progression, we propose that the newly discovered miRNA-like sequences within the HIV-1 genomes may have evolved to self-regulate survival of the

  9. [Point mutations of genes encoding proteins involvedin RNA splicing in patients with myelodysplastic syndromes].

    Science.gov (United States)

    Barańska, Marta; Czerwińska-Rybak, Joanna; Gil, Lidia; Komarnicki, Mieczysław

    2015-01-01

    The myelodysplastic syndromes (MDS) constitute heterogeneous group of clonal disorders, characterized by ineffective hematopoiesis, peripheral cytopenia and increased risk of acute myeloid leukemia development. Molecular mechanisms behind MDS have not been fully explained, however recent studies based on new technologies confirmed that epigenetic abnormalities and somatic mutation in the spliceasome machinery are crucial in pathogenesis of these diseases. Abnormal mRNA splicing (excision of intronic sequences from mRNA) has been found in over half of all MDS patients and resulted in accumulation of cytogenetical and molecular changes. The biological impact of splicing factor genes mutations has been evaluated only in a limited extend and current studies concentrate on analysis of MDS transcriptome. Molecular characteristic of classical and alternative splicing is presented in the paper, according to current knowledge. We review the most prominent findings from recent years concerning mutation in the spliceasome machinery with respect to MDS phenotype and disease prognosis. Perspectives in applying of novel diagnostic and therapeutic possibilities for myelodysplasia, based on spliceosome mutations identification are also presented.

  10. Characterization and phylogenetic analysis of environmental stress-responsive SAP gene family encoding A20/AN1 zinc finger proteins in tomato.

    Science.gov (United States)

    Solanke, Amolkumar U; Sharma, Manoj K; Tyagi, Akhilesh K; Sharma, Arun Kumar

    2009-08-01

    Characterization of genes responsive to stress is important for efforts on improving stress tolerance of plants. To address components involved in stress tolerance of tomato (Solanum lycopersicum), a stress-responsive gene family encoding A20/AN1 zinc finger proteins was characterized. In the present study, 13 members of this gene family were cloned from tomato cultivar Pusa Ruby and named as Stress Associated Protein (SAP) genes. Out of 13 genes, 12 have been mapped on their respective chromosomes. Expression of these genes in response to cold, heat, salt, desiccation, wounding, abscisic acid, oxidative and submergence stresses was analysed. All tomato SAP genes were found to be responsive to one or other type of environmental stress. The phylogenetic analysis of these genes, along with their orthologs from Solanaceae species suggests the presence of a common set of SAP genes in the studied Solanaceae species. The present study characterizes a SAP gene family, which encodes A20/AN1 zinc finger containing proteins from tomato for the first time. Genes showing high expression in response to a particular stress can be exploited for improving stress tolerance of tomato and other Solanaceae members.

  11. Nucleotide sequence analysis of the Legionella micdadei mip gene, encoding a 30-kilodalton analog of the Legionella pneumophila Mip protein

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Cianciotto, N P; Hindersson, P

    1991-01-01

    After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid p...... homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins)....

  12. An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription.

    Directory of Open Access Journals (Sweden)

    Reza Djavadian

    2016-06-01

    Full Text Available Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit "true" late kinetics-their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other β- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these "βγ" genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes.

  13. The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton

    OpenAIRE

    Folkers, U; Kirik, V.; Schöbinger, U.; Falk, S; Krishnakumar, S; Pollock, M A; Oppenheimer, D.G.; Day, I.; Reddy, A.R.; Jürgens, G; Hülskamp, M

    2002-01-01

    The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant...

  14. Molecular cloning, modeling and differential expression of a gene encoding a silent information regulator-like protein from Sporothrix schenckii.

    Science.gov (United States)

    Hou, Binbin; Liu, Xiaoming; Zheng, Fangliang; Xu, Xuezhu; Zhang, Zhenying

    2014-06-01

    Sporothrix schenckii (S. schenckii) is a dimorphic fungus that produces lymphocutaneous lesions. The signature characteristic of S. schenckii is a temperature-induced phase transition. Silent information regulator (Sir) has been proven to be involved in phenotypic switching in Saccharomyces cerevisiae (S. cerevisiae) and Candida albicans (C. albicans) by organizing chromatin structure. In this study, we isolated and characterized a Sir homologue gene, designated as SsSir2, from the yeast form of S. schenckii. The full-length SsSir2 cDNA sequence is 1753 bp in size and contains an open reading frame of 1329 bp encoding 442 amino acids. The predicted molecular mass of SsSir2 is 48.1 kDa with an estimated theoretical isoelectric point of 4.6. The SsSir2 kinase domain shows a 78% identity with that of Hst2, a Sir2 Ib gene from S. cerevisiae. Three exons and two introns were identified within the 1472‑bp SsSir2 genomic DNA sequence of S. schenckii. A three-dimensional model of SsSir2 was constructed using a homology modeling method, and its reliability was evaluated. The active site of SsSir2 was identified by docking simulation, which indicated that several important residues, such as Asn127 and Asp129, play an important role in the histone deacetylase activity of Sir2 family proteins. The differential expression of the SsSir2 in two stages was demonstrated by real-time RT-PCR. The expression of SsSir2 was higher in the yeast stage compared with that in the mycelial one, which indicated that SsSir2 may be involved in the phenotypic switching and morphogenesis of the yeast phase in S. schenckii.

  15. Distinct functional specificities are associated with protein isoforms encoded by the Drosophila dorsal-ventral patterning gene pipe.

    Science.gov (United States)

    Zhang, Zhenyu; Zhu, Xianjun; Stevens, Leslie M; Stein, David

    2009-08-01

    Spatially regulated transcription of the pipe gene in ventral cells of the Drosophila ovary follicle cell epithelium is a key event that specifies progeny embryo dorsal-ventral (DV) polarity. pipe encodes ten putative protein isoforms, all of which exhibit similarity to vertebrate glycosaminoglycan-modifying enzymes. Expression of one of the isoforms, Pipe-ST2, in follicle cells has previously been shown to be essential for DV patterning. pipe is also expressed in the embryonic salivary gland and its expression there is required for normal viability. Here, we show that in addition to Pipe-ST2, seven of the other Pipe isoforms are expressed in the ovary, whereas all Pipe isoforms are abundantly expressed in the embryo. Of the ten isoforms, only Pipe-ST2 can restore ventral and lateral pattern elements to the progeny of otherwise pipe-null mutant females. By contrast, three Pipe isoforms, but not Pipe-ST2, support the production of a novel pipe-dependent epitope present in the embryonic salivary gland. These data indicate that differences in functional specificity, and presumably enzymatic specificity, are associated with several of the Pipe isoforms. In addition, we show that uniform expression of the Pipe-ST2 isoform in the follicle cell layer of females otherwise lacking pipe expression leads to the formation of embryos with a DV axis that is appropriately oriented with respect to the intrinsic polarity of the eggshell. This suggests the existence of a second mechanism that polarizes the Drosophila embryo, in addition to the ventrally restricted transcription of the pipe gene.

  16. Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana.

    Science.gov (United States)

    Kaido, Masanori; Inoue, Yosuke; Takeda, Yoshika; Sugiyama, Kazuhiko; Takeda, Atsushi; Mori, Masashi; Tamai, Atsushi; Meshi, Tetsuo; Okuno, Tetsuro; Mise, Kazuyuki

    2007-06-01

    The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.

  17. Mapping of two genes encoding isoforms of the actin binding protein ABP-280, a dystrophin like protein, to Xq28 and to chromosome 7.

    Science.gov (United States)

    Maestrini, E; Patrosso, C; Mancini, M; Rivella, S; Rocchi, M; Repetto, M; Villa, A; Frattini, A; Zoppè, M; Vezzoni, P

    1993-06-01

    ABP-280 is a ubiquitous actin binding protein present in the cytoskeleton of many different cell types. ABP-280 was mapped to distal Xq28, 50-60 kb downstream of the Green Colour Pigment (GCP) genes. To establish if ABP-280 may be a candidate for one of the muscle disease localized by linkage analysis to distal Xq28 we looked for alternative forms of ABP-280 mRNA. Several different ABP-280 mRNAs were indeed identified: two are X-linked and are produced by alternative splicing of a small exon of 24 nucleotides. At least one additional gene encoding a RNA more than 70% identical to ABP-280 in the 1700 bp sequenced has also been found. It was mapped to chromosome 7. While both forms of the X-linked ABP-280 are ubiquitous, the gene on chromosome 7 is highly expressed only in skeletal muscle and heart. The two genes were therefore excellent candidates for the X-linked and for the autosomal dominant form of the Emery-Dreifuss Muscular Dystrophy (EDMD) both of which have been described. So far, however we were unable to demonstrate mutations in the coding region or affecting the alternative splicing of the X-linked form of ABP-280, in several patients studied, and we think that it is quite unlikely that this is the gene responsible for EDMD.

  18. Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila. Evidence for differential codon usage in highly expressed genes

    DEFF Research Database (Denmark)

    Larsen, L K; Andreasen, P H; Dreisig, H

    1999-01-01

    We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features...

  19. Genes encoding chimeras of Neurospora crassa erg-3 and human TM7SF2 proteins fail to complement Neurospora and yeast sterol C-14 reductase mutants

    Indian Academy of Sciences (India)

    A Prakash; Durgadas P Kasbekar

    2002-03-01

    The human gene TM7SF2 encodes a polypeptide (SR-1) with high sequence similarity to sterol C-14 reductase, a key sterol biosynthetic enzyme in fungi, plants and mammals. In Neurospora and yeast this enzyme is encoded by the erg-3 and erg24 genes respectively. In an effort to demonstrate sterol C-14 reductase activity for SR-1 we constructed six recombinant genes coding for chimeras of the Neurospora erg-3 and SR-1 protein sequences and tested them for complementation of the Neurospora erg-3 mutant. To our surprise, all the chimeras failed to complement erg-3. A few of the chimeric proteins were also tested against the yeast erg24 mutant, but again there was no complementation. We discuss some reasons that might account for these unexpected findings.

  20. Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria

    Directory of Open Access Journals (Sweden)

    Muscariello Lidia

    2006-05-01

    Full Text Available Abstract Background Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components. Results A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters. Conclusion We propose that the CscA, CscB, CscC and Csc

  1. IRE1 KNOCKDOWN MODIFIES THE GLUTAMINE AND GLUCOSE DEPRIVATION EFFECT ON THE EXPRESSION OF NUCLEAR GENES ENCODING MITOCHONDRIAL PROTEINS IN U87 GLIOMA CELLS

    Directory of Open Access Journals (Sweden)

    O. O.

    2016-04-01

    Full Text Available We have studied the glucose and glutamine deprivation effect on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells in relation to inhibition of inositol requiring enzyme-1 (IRE1. It was shown that glutamine deprivation down-regulated the expression of mitochondrial (NADP+-dependent isocitrate dehydrogenase 2 (IDH2, malic enzyme 2 (ME2, mitochondrial aspartate aminotransferase (GOT2, and subunit B of succinate dehydrogenase (SDHB genes in control glioma cells in gene specific manner. At the same time, the expression level of malate dehydrogenase 2 (MDH2 and subunit D of succinate dehydrogenase (SDHD genes in these cells was not changed upon glutamine deprivation. It was also shown that inhibition of ІRE1 signaling enzyme function in U87 glioma cells modified the glutamine deprivation effect on the expression of all studied genes. Furthermore, the expression of the majority of studied genes was resistant to glucose deprivation, except IDH2 and SDHB genes, which expression levels were slightly down-regulated. Inhibition of IRE1 modified the effect of glucose deprivation on ME2, SDHB, SDHD, and GOT2 genes expression. Therefore, glucose and glutamine deprivation affected the expression level of the majority of nuclear genes encoding mitochondrial proteins in relation to the functional activity of IRE1 enzyme, which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth.

  2. Comparative analysis of Phytophthora genes encoding secreted proteins reveals conserved synteny and lineage-specific gene duplications and deletions

    NARCIS (Netherlands)

    Jiang, R.H.Y.; Tyler, B.M.; Govers, F.

    2006-01-01

    Comparative analysis of two Phytophthora genomes revealed overall colinearity in four genomic regions consisting of a 1.5-Mb sequence of Phytophthora sojae and a 0.9-Mb sequence of R ramorum. In these regions with conserved synteny, the gene order is largely similar; however, genome rearrangements a

  3. Characterization and evolution of a gene encoding a Trimeresurus flavoviridis serum protein that inhibits basic phospholipase A2 isozymes in the snake's venom.

    Science.gov (United States)

    Nobuhisa, I; Inamasu, S; Nakai, M; Tatsui, A; Mimori, T; Ogawa, T; Shimohigashi, Y; Fukumaki, Y; Hattori, S; Kihara, H; Ohno, M

    1997-11-01

    The proteins that bind phospholipase A2 (PLA2) isozymes of Trimeresurus flavoviridis (habu snake, crotalinae) venom were fractionated from sera on four columns, each conjugated with one of four PLA2 isozymes. Five proteins, termed PLA2 inhibitors (PLI) I-V, were obtained as the binding components. The combinations of the binding components differed depending on the PLA2 isozymes. PLI-IV and PLI-V correspond to PLI-A and PLI-B, respectively, which were known to bind to a major [Asp49]PLA2, PLA2, and contained a segment similar to the carbohydrate-recognition domain of C-type lectins. PLI-I, which is a major component of inhibitory proteins against three basic PLA2 isozymes, PLA-B (a basic [Asp49]PLA2) and basic proteins I and II (both [Lys49]PLA2s), has been isolated, and its partial amino acid sequence has been determined. A cDNA encoding PLI-I was isolated from a T. flavoviridis liver cDNA library and sequenced. PLI-I cDNA encoded 200 amino acid residues, including a signal peptide of 19 amino acid residues. One sugar chain was predicted to occur at position 157. A gene coding for PLI-I was isolated. It is 9.6-kb long and consists of five exons and four introns. Comparison of the exon-intron structure of the PLI-I gene with those of genes encoding urokinase-type-plasminogen-activator receptor (uPAR), Ly-6, CD59 and neurotoxins showed that they have characteristic unit encoding approximately 90 amino acid residues, which is divided over two exons. This strongly suggests that the PLI-I gene belongs to the uPAR, Ly-6, CD59 and neurotoxin gene family. There are two types of structurally different inhibitors against PLA2 isozymes in T. flavoviridis serum with different evolutionary origins.

  4. The Bartonella vinsonii subsp. arupensis Immunodominant Surface Antigen BrpA Gene, Encoding a 382-Kilodalton Protein Composed of Repetitive Sequences, Is a Member of a Multigene Family Conserved among Bartonella Species

    OpenAIRE

    Gilmore, Robert D.; Bellville, Travis M.; Sviat, Steven L.; Frace, Michael

    2005-01-01

    Bartonella proteins that elicit an antibody response during an infection are poorly defined; therefore, to characterize antigens recognized by the host, a Bartonella genomic expression library was screened with serum from an infected mouse. This process led to the discovery of a Bartonella vinsonii subsp. arupensis gene encoding a 382-kDa protein, part of a gene family encoding large proteins, each containing multiple regions of repetitive segments. The genes were termed brpA to -C (bartonell...

  5. Inferring Gene Networks for Strains of Dehalococcoides Highlights Conserved Relationships between Genes Encoding Core Catabolic and Cell-Wall Structural Proteins.

    Science.gov (United States)

    Mansfeldt, Cresten B; Heavner, Gretchen W; Rowe, Annette R; Hayete, Boris; Church, Bruce W; Richardson, Ruth E

    2016-01-01

    The interpretation of high-throughput gene expression data for non-model microorganisms remains obscured because of the high fraction of hypothetical genes and the limited number of methods for the robust inference of gene networks. Therefore, to elucidate gene-gene and gene-condition linkages in the bioremediation-important genus Dehalococcoides, we applied a Bayesian inference strategy called Reverse Engineering/Forward Simulation (REFS™) on transcriptomic data collected from two organohalide-respiring communities containing different Dehalococcoides mccartyi strains: the Cornell University mixed community D2 and the commercially available KB-1® bioaugmentation culture. In total, 49 and 24 microarray datasets were included in the REFS™ analysis to generate an ensemble of 1,000 networks for the Dehalococcoides population in the Cornell D2 and KB-1® culture, respectively. Considering only linkages that appeared in the consensus network for each culture (exceeding the determined frequency cutoff of ≥ 60%), the resulting Cornell D2 and KB-1® consensus networks maintained 1,105 nodes (genes or conditions) with 974 edges and 1,714 nodes with 1,455 edges, respectively. These consensus networks captured multiple strong and biologically informative relationships. One of the main highlighted relationships shared between these two cultures was a direct edge between the transcript encoding for the major reductive dehalogenase (tceA (D2) or vcrA (KB-1®)) and the transcript for the putative S-layer cell wall protein (DET1407 (D2) or KB1_1396 (KB-1®)). Additionally, transcripts for two key oxidoreductases (a [Ni Fe] hydrogenase, Hup, and a protein with similarity to a formate dehydrogenase, "Fdh") were strongly linked, generalizing a strong relationship noted previously for Dehalococcoides mccartyi strain 195 to multiple strains of Dehalococcoides. Notably, the pangenome array utilized when monitoring the KB-1® culture was capable of resolving signals from multiple

  6. Inferring Gene Networks for Strains of Dehalococcoides Highlights Conserved Relationships between Genes Encoding Core Catabolic and Cell-Wall Structural Proteins

    Science.gov (United States)

    Mansfeldt, Cresten B.; Heavner, Gretchen W.; Rowe, Annette R.; Hayete, Boris; Church, Bruce W.; Richardson, Ruth E.

    2016-01-01

    The interpretation of high-throughput gene expression data for non-model microorganisms remains obscured because of the high fraction of hypothetical genes and the limited number of methods for the robust inference of gene networks. Therefore, to elucidate gene-gene and gene-condition linkages in the bioremediation-important genus Dehalococcoides, we applied a Bayesian inference strategy called Reverse Engineering/Forward Simulation (REFS™) on transcriptomic data collected from two organohalide-respiring communities containing different Dehalococcoides mccartyi strains: the Cornell University mixed community D2 and the commercially available KB-1® bioaugmentation culture. In total, 49 and 24 microarray datasets were included in the REFS™ analysis to generate an ensemble of 1,000 networks for the Dehalococcoides population in the Cornell D2 and KB-1® culture, respectively. Considering only linkages that appeared in the consensus network for each culture (exceeding the determined frequency cutoff of ≥ 60%), the resulting Cornell D2 and KB-1® consensus networks maintained 1,105 nodes (genes or conditions) with 974 edges and 1,714 nodes with 1,455 edges, respectively. These consensus networks captured multiple strong and biologically informative relationships. One of the main highlighted relationships shared between these two cultures was a direct edge between the transcript encoding for the major reductive dehalogenase (tceA (D2) or vcrA (KB-1®)) and the transcript for the putative S-layer cell wall protein (DET1407 (D2) or KB1_1396 (KB-1®)). Additionally, transcripts for two key oxidoreductases (a [Ni Fe] hydrogenase, Hup, and a protein with similarity to a formate dehydrogenase, “Fdh”) were strongly linked, generalizing a strong relationship noted previously for Dehalococcoides mccartyi strain 195 to multiple strains of Dehalococcoides. Notably, the pangenome array utilized when monitoring the KB-1® culture was capable of resolving signals from

  7. Cloning and Heterologous Expression of Insecticidal-Protein-Encoding Genes from Photorhabdus luminescens TT01 in Enterobacter cloacae for Termite Control▿

    OpenAIRE

    Zhao, Ruihua; Han, Richou; Qiu, Xuehong; Yan, Xun; Cao, Li; Liu, Xiuling

    2008-01-01

    Enterobacter cloacae, one of the indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus), was genetically modified with a transposon Tn5 vector containing genes (tcdA1 and tcdB1) encoding orally insecticidal proteins from the entomopathogenic bacterium Photorhabdus luminescens subsp. laumondii TT01, a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora, for termite control. In the laboratory, termites were fed filter paper inoculated with the ...

  8. Light-intensity-dependent expression of Lhc gene family encoding light-harvesting chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Teramoto, Haruhiko; Nakamori, Akira; Minagawa, Jun; Ono, Taka-aki

    2002-09-01

    Excessive light conditions repressed the levels of mRNAs accumulation of multiple Lhc genes encoding light-harvesting chlorophyll-a/b (LHC) proteins of photosystem (PS)II in the unicellular green alga, Chlamydomonas reinhardtii. The light intensity required for the repression tended to decrease with lowering temperature or CO(2) concentration. The responses of six LhcII genes encoding the major LHC (LHCII) proteins and two genes (Lhcb4 and Lhcb5) encoding the minor LHC proteins of PSII (CP29 and CP26) were similar. The results indicate that the expression of these Lhc genes is coordinately repressed when the energy input through the antenna systems exceeds the requirement for CO(2) assimilation. The Lhc mRNA level repressed under high-light conditions was partially recovered by adding the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting that redox signaling via photosynthetic electron carriers is involved in the gene regulation. However, the mRNA level was still considerably lower under high-light than under low-light conditions even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Repression of the Lhc genes by high light was prominent even in the mutants deficient in the reaction center(s) of PSII or both PSI and PSII. The results indicate that two alternative processes are involved in the repression of Lhc genes under high-light conditions, one of which is independent of the photosynthetic reaction centers and electron transport events.

  9. A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER

    Directory of Open Access Journals (Sweden)

    Haga Christopher L

    2007-09-01

    Full Text Available Abstract Background In mouse the cytokine interleukin-7 (IL-7 is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER. The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR, a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules.

  10. The highly abundant chlorophyll-protein complex of iron-deficient Synechococcus sp. PCC7942 (CP43') is encoded by the isiA gene.

    Science.gov (United States)

    Burnap, R L; Troyan, T; Sherman, L A

    1993-11-01

    A chlorophyll (Chl)-protein complex designated CPVI-4 becomes the major pigment-protein complex in Synechococcus sp. PCC7942 cells grown under conditions of iron limitation. Work by Laudenbach et al. (J Bacteriol [1988] 170: 5018-5026) has identified an iron-repressible operon, designated isiAB, containing the flavodoxin gene and a gene predicted to encode a Chl-binding protein resembling CP43 of photosystem II. To test the hypothesis that the CP43-like protein is a component of the CPVI-4 complex, we have inactivated the isiAB operon in Synechococcus sp. PCC7942 using directed insertional mutagenesis. Mutant cells grown under conditions of iron limitation exhibit pronounced changes in their spectroscopic and photosynthetic properties relative to similarly grown wild-type cells. Notably, the strong 77 K fluorescence emission at 685 nm, which dominates the spectrum of iron-deficient wild-type cells, is dramatically reduced in the mutant. The loss of this emission appears to unmask the otherwise obscured photosystem II emissions at 685 and 695 nm. Most importantly, mildly denaturing gel electrophoresis shows that mutant cells no longer express the CPVI-4 complex, indicating that the isiA gene encodes a component of this abundant Chl-protein complex.

  11. The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton.

    Science.gov (United States)

    Folkers, U; Kirik, V; Schöbinger, U; Falk, S; Krishnakumar, S; Pollock, M A; Oppenheimer, D G; Day, I; Reddy, A S M; Jürgens, G; Hülskamp, M; Reddy, A R

    2002-03-15

    The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant. A role of AN in the control of the microtubule cytoskeleton is further supported by the finding that AN genetically and physically interacts with ZWICHEL, a kinesin motor molecule involved in trichome branching. Our data suggest that CtBP/BARS-like protein function in plants is directly associated with the microtubule cytoskeleton.

  12. windbeutel, a gene required for dorsoventral patterning in Drosophila, encodes a protein that has homologies to vertebrate proteins of the endoplasmic reticulum.

    Science.gov (United States)

    Konsolaki, M; Schüpbach, T

    1998-01-01

    The formation of the dorsoventral axis of the Drosophila embryo depends on cell-cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The gene windbeutel functions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilization, when the egg has already been laid. We have performed a molecular analysis of windbeutel. We have found that windbeutel encodes a putative resident protein of the endoplasmic reticulum, and has homologs in rats and humans. The gene is expressed for a brief period of time in the follicle cells of the ovary, at around the time when the dorsoventral axis of the egg chamber is first established. We propose that Windbeutel is responsible for the folding and/or modification of a specific factor that is secreted from the follicle cells and participates in the activation of the ventralizing signal.

  13. Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation

    Institute of Scientific and Technical Information of China (English)

    DUN Baoqing; ZHAO Zhonglin; LIANG Aimin; HOU Songna; XU Ming-Qun; LIN Min; LU Wei; ZHANG Wei; PING Shuzhen; WANG Xujing; CHEN Ming; XU Yuquan; JIN Dan; WANG Jin

    2006-01-01

    A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS proteins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media.12 unique sites, which can tolerate a 5-aa insertion,were identified. In all of the 12 sites, only F295/T296site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799.The G2-EPSPS gene was then divided into N-terminal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp. DnaE intein, respectively, creating two plasmids pMEPSN295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescued growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Reconsituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.

  14. batman Interacts with polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor.

    Science.gov (United States)

    Faucheux, M; Roignant, J-Y; Netter, S; Charollais, J; Antoniewski, C; Théodore, L

    2003-02-01

    Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.

  15. A temperature-responsive gene in sorghum encodes a glycine-rich protein that interacts with calmodulin.

    Science.gov (United States)

    Singh, Supreet; Virdi, Amardeep Singh; Jaswal, Rajdeep; Chawla, Mrinalini; Kapoor, Sanjay; Mohapatra, Samar B; Manoj, Narayanan; Pareek, Ashwani; Kumar, Sanjay; Singh, Prabhjeet

    2017-06-01

    Imposition of different biotic and abiotic stress conditions results in an increase in intracellular levels of Ca(2+) which is sensed by various sensor proteins. Calmodulin (CaM) is one of the best studied transducers of Ca(2+) signals. CaM undergoes conformational changes upon binding to Ca(2+) and interacts with different types of proteins, thereby, regulating their activities. The present study reports the cloning and characterization of a sorghum cDNA encoding a protein (SbGRBP) that shows homology to glycine-rich RNA-binding proteins. The expression of SbGRBP in the sorghum seedlings is modulated by heat stress. The SbGRBP protein is localized in the nucleus as well as in cytosol, and shows interaction with CaM that requires the presence of Ca(2+). SbGRBP depicts binding to single- and also double-stranded DNA. Fluorescence spectroscopic analyses suggest that interaction of SbGRBP with nucleic acids may be modulated after binding with CaM. To our knowledge, this is the first study to provide evidence for interaction of a stress regulated glycine-rich RNA-binding protein with CaM. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  16. Exploration of Csp genes from temperate and glacier soils of the Indian Himalayas and in silico analysis of encoding proteins.

    Science.gov (United States)

    Latha, Prema K; Soni, Ravindra; Khan, Mahejibin; Marla, Soma S; Goel, Reeta

    2009-04-01

    The metagenomic Csp library was constructed from the temperate and glacier soils of central Himalaya, India followed by polymerase chain reaction (PCR) amplification. The library was further screened for low-temperature adaptation, and the positive recombinants were sorted out by determining changes in the melting temperature (Tm). A homology search of cloned sequence showed their identity with the Csp genes of Pseudomonas fluorescens, Psychrobacter cryohalolentis K5, and Shewanella spp MR-4. Amino acid sequence analysis annotated the presence of conserved aromatic and basic amino acids as well as RNA binding motifs from the cold shock domain. Furthermore, a PROSITE scan showed a moderate identity of less than 60% with the known cold shock-inducible proteins (ribosomal proteins, rbfA, DEAD-box helicases), cold acclimation protein, and temperature-induced protein (SRP1/TIP1). This study highlighted the prevalence of Csp genes from cold Himalayan environments that can be explored for tailor-made crop constructions in future.

  17. A gene encoding a vicilin-like protein is specifically expressed in fern spores. Evolutionary pathway of seed storage globulins.

    Science.gov (United States)

    Shutov, A D; Braun, H; Chesnokov, Y V; Bäumlein, H

    1998-02-15

    The isolation and characterisation of a cDNA coding for a vicilin-like protein of the fern Matteuccia struthiopteris is described. The corresponding gene is specifically expressed during late stages of spore development. Extensive sequence comparisons suggest that the fern protein can be considered as a molecular missing link between single-domain germin/spherulin-like proteins and two-domain seed storage globulins of gymnosperms and angiosperms. Further, evidence is provided for the existence of a superfamily of structurally related, functionally different proteins which includes storage globulins of the vicilin and legumin families, a membrane-associated sucrose-binding protein of soybean, a Forssman antigen-binding lectin of velvet bean, the precursor of the vacuolar membrane bound proteins MP27/MP32 of pumpkin, the embryogenesis-specific protein Gea8 of carrot, the fern-spore-specific protein described here as well as the functionally diverse family of germins/germin-like proteins and the spherulins of myxomycetes. We propose that seed storage globulins of spermatophytes evolved from desiccation-related single-domain proteins of prokaryotes via a duplicated two-domain ancestor that is best represented by the extant fern spore-specific vicilin-like protein.

  18. Distribution, structure and diversity of “bacterial” genes encoding two-component proteins in the Euryarchaeota

    Directory of Open Access Journals (Sweden)

    Mark K. Ashby

    2006-01-01

    Full Text Available The publicly available annotated archaeal genome sequences (23 complete and three partial annotations, October 2005 were searched for the presence of potential two-component open reading frames (ORFs using gene category lists and BLASTP. A total of 489 potential two-component genes were identified from the gene category lists and BLASTP. Two-component genes were found in 14 of the 21 Euryarchaeal sequences (October 2005 and in neither the Crenarchaeota nor the Nanoarchaeota. A total of 20 predicted protein domains were identified in the putative two-component ORFs that, in addition to the histidine kinase and receiver domains, also includes sensor and signalling domains. The detailed structure of these putative proteins is shown, as is the distribution of each class of two-component genes in each species. Potential members of orthologous groups have been identified, as have any potential operons containing two or more two-component genes. The number of two-component genes in those Euryarchaeal species which have them seems to be linked more to lifestyle and habitat than to genome complexity, with most examples being found in Methanospirillum hungatei, Haloarcula marismortui, Methanococcoides burtonii and the mesophilic Methanosarcinales group. The large numbers of two-component genes in these species may reflect a greater requirement for internal regulation. Phylogenetic analysis of orthologous groups of five different protein classes, three probably involved in regulating taxis, suggests that most of these ORFs have been inherited vertically from an ancestral Euryarchaeal species and point to a limited number of key horizontal gene transfer events.

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

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    Lang Daniel

    2005-03-01

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

  20. The active gene that encodes human High Mobility Group 1 protein (HMG1) contains introns and maps to chromosome 13

    Energy Technology Data Exchange (ETDEWEB)

    Ferrari, S. [Dipartimento di Genetica e di Biologia dei Microrganismi, Milan (Italy); Finelli, P.; Rocchi, M. [Istituto di Genetica, Bari (Italy)] [and others

    1996-07-15

    The human genome contains a large number of sequences related to the cDNA for High Mobility Group 1 protein (HMG1), which so far has hampered the cloning and mapping of the active HMG1 gene. We show that the human HMG1 gene contains introns, while the HMG1-related sequences do not and most likely are retrotransposed pseudogenes. We identified eight YACs from the ICI and CEPH libraries that contain the human HMG1 gene. The HMG1 gene is similar in structure to the previously characterized murine homologue and maps to human chromosome 13 and q12, as determined by in situ hybridization. The mouse Hmg1 gene maps to the telomeric region of murine Chromosome 5, which is syntenic to the human 13q12 band. 18 refs., 3 figs.

  1. Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.

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    Bharat Bhusan Patnaik

    Full Text Available BACKGROUND: Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. METHODOLOGY/PRINCIPAL FINDINGS: Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4, at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC. SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC. The activity of the purified protein was tested against selected Gram +/- bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp. In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5'- and 3'-rapid amplification of cDNA ends (RACE-PCR. The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps involved in plant development and defense. CONCLUSIONS/SIGNIFICANCE: The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was

  2. Expression profiling of two stress-inducible genes encoding for miraculin-like proteins in citrus plants under insect infestation or salinity stress.

    Science.gov (United States)

    Podda, A; Simili, M; Del Carratore, R; Mouhaya, W; Morillon, R; Maserti, B E

    2014-01-01

    The expression of two genes, namely Mir1 and Mir3 and the abundance of their encoded proteins, the putative miraculin-like proteins, MLP1 and MLP3, showing similarity to the Kunitz family of protease inhibitors, were monitored in the leaves of the citrus variety, 'Clementine' after Tetranychus urticae infestation and elicitor treatments, or in the leaves of three other diploid citrus: 'Willow leaf', 'Cleopatra' mandarins and 'Trifoliate' orange, as well as their respective doubled diploids and the allotetraploid somatic hybrid 'FLHORAG1' under salt stress. RT-PCR and 2-DE indicated that Mir1 and Mir3 and their products were present at low-basal expression in all citrus genotypes. Both genes and products were induced in the 'Clementine' leaves infested by T. urticae, but a contrasting profile was observed under elicitor treatments. Under salt stress, the two genes showed an expression pattern contrasting each other and depending on the genotypes. 'Cleopatra' mandarin, 'Trifoliate' orange and 'FLHORAG1' presented overexpression of Mir3 and MLP3 and decreased levels of Mir1 and MPL1. The opposite behaviour was found in 'Willow leaf' mandarin. The positive correlation of the expression profile of the two genes with that of a gene encoding a putative apoplastic cysteine protease (CysP) might suggest a possible interaction of the respective encoded proteins during the response to biotic stress. Under salt stress, CysP and Mir 1 showed a similar expression pattern but only at transcript level. The possible occurrence of post-translational CysP regulation is discussed.

  3. Characterization of promoter region and genomic structure of the murine and human genes encoding Src like adapter protein.

    Science.gov (United States)

    Kratchmarova, I; Sosinowski, T; Weiss, A; Witter, K; Vincenz, C; Pandey, A

    2001-01-10

    Src-like adapter protein (SLAP) was identified as a signaling molecule in a yeast two-hybrid system using the cytoplasmic domain of EphA2, a receptor protein tyrosine kinase (Pandey et al., 1995. Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase. J. Biol. Chem. 270, 19201-19204). It is very similar to members of the Src family of cytoplasmic tyrosine kinases in that it contains very homologous SH3 and SH2 domains (Abram and Courtneidge, 2000. Src family tyrosine kinases and growth factor signaling. Exp. Cell. Res. 254, 1-13.). However, instead of a kinase domain at the C-terminus, it contains a unique C-terminal region. In order to exclude the possibility that an alternative form exists, we have isolated genomic clones containing the murine Slap gene as well as the human SLA gene. The coding regions of murine Slap and human SLA genes contain seven exons and six introns. Absence of any kinase domain in the genomic region confirm its designation as an adapter protein. Additionally, we have cloned and sequenced approximately 2.6 kb of the region 5' to the initiator methionine of the murine Slap gene. When subcloned upstream of a luciferase gene, this fragment increased the transcriptional activity about 6-fold in a human Jurkat T cell line and approximately 52-fold in a murine T cell line indicating that this region contains promoter elements that dictate SLAP expression. We have also cloned the promoter region of the human SLA gene. Since SLAP is transcriptionally regulated by retinoic acid and by activation of B cells, the cloning of its promoter region will permit a detailed analysis of the elements required for its transcriptional regulation.

  4. Screening of candidate genes encoding proteins expressed in pectoral fins of fugu Takifugu rubripes, in relation to habitat site of parasitic copepod Caligus fugu, using suppression subtractive hybridization.

    Science.gov (United States)

    Tasumi, Satoshi; Norshida, Ismail; Boxshall, Geoffrey A; Kikuchi, Kiyoshi; Suzuki, Yuzuru; Ohtsuka, Susumu

    2015-05-01

    Caligus fugu is a parasitic copepod specific to the tetraodontid genus Takifugu including the commercially important Takifugu rubripes. Despite the rapid accumulation of knowledge on other aspects of its biology, the host and settlement-site recognition mechanisms of this parasite are not yet well understood. Since the infective copepodid stage shows preferential site selection in attaching to the fins, we considered it likely that the copepodid recognizes chemical cues released or leaking from the fins, and/or transmembrane protein present on the fins. To isolate molecules potentially related to attachment site specificity, we applied suppression subtractive hybridization (SSH) PCR by identifying genes expressed more highly in pectoral fins of T. rubripes than in the body surface skin. We sequenced plasmid DNA from 392 clones in a SSH library. The number of non-redundant sequences was 276, which included 135 sequences located on 117 annotated genes and 141 located in positions where no genes had been annotated. We characterized those annotated genes on the basis of gene ontology terms, and found that 46 of the identified genes encode secreted proteins, enzymes or membrane proteins. Among them nine showed higher expression in the pectoral fins than in the skin. These could be candidate genes for involvement in behavioral mechanisms related to the site specificity shown by the infective copepodids of C. fugu.

  5. RNA-Seq-based analysis of cold shock response in Thermoanaerobacter tengcongensis, a bacterium harboring a single cold shock protein encoding gene.

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    Bo Liu

    Full Text Available BACKGROUND: Although cold shock responses and the roles of cold shock proteins in microorganisms containing multiple cold shock protein genes have been well characterized, related studies on bacteria possessing a single cold shock protein gene have not been reported. Thermoanaerobacter tengcongensis MB4, a thermophile harboring only one known cold shock protein gene (TtescpC, can survive from 50° to 80 °C, but has poor natural competence under cold shock at 50 °C. We therefore examined cold shock responses and their effect on natural competence in this bacterium. RESULTS: The transcriptomes of T. tengcongensis before and after cold shock were analyzed by RNA-seq and over 1200 differentially expressed genes were successfully identified. These genes were involved in a wide range of biological processes, including modulation of DNA replication, recombination, and repair; energy metabolism; production of cold shock protein; synthesis of branched amino acids and branched-chain fatty acids; and sporulation. RNA-seq analysis also suggested that T. tengcongensis initiates cell wall and membrane remodeling processes, flagellar assembly, and sporulation in response to low temperature. Expression profiles of TtecspC and failed attempts to produce a TtecspC knockout strain confirmed the essential role of TteCspC in the cold shock response, and also suggested a role of this protein in survival at optimum growth temperature. Repression of genes encoding ComEA and ComEC and low energy metabolism levels in cold-shocked cells are the likely basis of poor natural competence at low temperature. CONCLUSION: Our study demonstrated changes in global gene expression under cold shock and identified several candidate genes related to cold shock in T. tengcongensis. At the same time, the relationship between cold shock response and poor natural competence at low temperature was preliminarily elucidated. These findings provide a foundation for future studies on genetic

  6. Molecular mechanisms for protein-encoded inheritance.

    Science.gov (United States)

    Wiltzius, Jed J W; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R; Apostol, Marcin I; Goldschmidt, Lukasz; Soriaga, Angela B; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

    2009-09-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of beta-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct beta-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  7. Molecular mechanisms for protein-encoded inheritance

    Energy Technology Data Exchange (ETDEWEB)

    Wiltzius, Jed J.W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David; (Cornell); (HHMI)

    2009-12-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of {beta}-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct {beta}-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  8. DNA-encoded antibody libraries: a unified platform for multiplexed cell sorting and detection of genes and proteins.

    Science.gov (United States)

    Bailey, Ryan C; Kwong, Gabriel A; Radu, Caius G; Witte, Owen N; Heath, James R

    2007-02-21

    Whether for pathological examination or for fundamental biology studies, different classes of biomaterials and biomolecules are each measured from a different region of a typically heterogeneous tissue sample, thus introducing unavoidable sources of noise that are hard to quantitate. We describe the method of DNA-encoded antibody libraries (DEAL) for spatially multiplexed detection of ssDNAs and proteins as well as for cell sorting, all on the same diagnostic platform. DEAL is based upon the coupling of ssDNA oligomers onto antibodies which are then combined with the biological sample of interest. Spotted DNA arrays, which are found to inhibit biofouling, are utilized to spatially stratify the biomolecules or cells of interest. We demonstrate the DEAL technique for (1) the rapid detection of multiple proteins within a single microfluidic channel, and, with the additional step of electroless amplification of gold-nanoparticle labeled secondary antibodies, we establish a detection limit of 10 fM for the protein IL-2, 150 times more sensitive than the analogue ELISA; (2) the multiplexed, on-chip sorting of both immortalized cell lines and primary immune cells with an efficiency that exceeds surface-confined panning approaches; and (3) the co-detection of ssDNAs, proteins, and cell populations on the same platform.

  9. The Vacuolar ATPase from Entamoeba histolytica: Molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein

    Directory of Open Access Journals (Sweden)

    Luna-Arias Juan

    2008-12-01

    Full Text Available Abstract Background Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. Results We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. Conclusion We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits

  10. Modular evolution of glutathione peroxidase genes in association with different biochemical properties of their encoded proteins in invertebrate animals

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    Zo Young-Gun

    2009-04-01

    Full Text Available Abstract Background Phospholipid hydroperoxide glutathione peroxidases (PHGPx, the most abundant isoforms of GPx families, interfere directly with hydroperoxidation of lipids. Biochemical properties of these proteins vary along with their donor organisms, which has complicated the phylogenetic classification of diverse PHGPx-like proteins. Despite efforts for comprehensive analyses, the evolutionary aspects of GPx genes in invertebrates remain largely unknown. Results We isolated GPx homologs via in silico screening of genomic and/or expressed sequence tag databases of eukaryotic organisms including protostomian species. Genes showing strong similarity to the mammalian PHGPx genes were commonly found in all genomes examined. GPx3- and GPx7-like genes were additionally detected from nematodes and platyhelminths, respectively. The overall distribution of the PHGPx-like proteins with different biochemical properties was biased across taxa; selenium- and glutathione (GSH-dependent proteins were exclusively detected in platyhelminth and deuterostomian species, whereas selenium-independent and thioredoxin (Trx-dependent enzymes were isolated in the other taxa. In comparison of genomic organization, the GSH-dependent PHGPx genes showed a conserved architectural pattern, while their Trx-dependent counterparts displayed complex exon-intron structures. A codon for the resolving Cys engaged in reductant binding was found to be substituted in a series of genes. Selection pressure to maintain the selenocysteine codon in GSH-dependent genes also appeared to be relaxed during their evolution. With the dichotomized fashion in genomic organizations, a highly polytomic topology of their phylogenetic trees implied that the GPx genes have multiple evolutionary intermediate forms. Conclusion Comparative analysis of invertebrate GPx genes provides informative evidence to support the modular pathways of GPx evolution, which have been accompanied with sporadic

  11. NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3β resulting in the stabilization of MYCN in human neuroblastomas.

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    Yusuke Suenaga

    2014-01-01

    Full Text Available The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

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

    Science.gov (United States)

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

    2014-06-01

    Insect cuticle pigmentation and sclerotization (tanning) are vital physiological processes for insect growth, development and survival. We have previously identified several colorless precursor molecules as well as enzymes involved in their biosynthesis and processing to yield the mature intensely colored body cuticle pigments. A recent study indicated that the Bombyx mori (silkmoth) gene, BmMucK, which encodes a protein orthologous to a Culex pipiens quiquefasciatus (Southern house mosquito) cis,cis, muconate transporter, is a member of the "Major Facilitator Superfamily" (MFS) of transporter proteins and is associated with the appearance of pigmented body segments of naturally occurring body color mutants of B. mori. While RNA interference of the BmMucK gene failed to result in any observable phenotype, RNAi using a dsRNA for an orthologous gene from the red flour beetle, Tribolium castaneum, was reported to result in molting defects and darkening of the cuticle and some body parts, leading to the suggestion that orthologs of MucK genes may differ in their functions among insects. To verify the role and essentiality of the ortholog of this gene in development and body pigmentation function in T. castaneum we obtained cDNAs for the orthologous gene (TcMucK) from RNA isolated from the GA-1 wild-type strain of T. castaneum. The sequence of a 1524 nucleotides-long cDNA for TcMucK which encodes the putatively full-length protein, was assembled from two overlapping RT-PCR fragments and the expression profile of this gene during development was analyzed by real-time PCR. This cDNA encodes a 55.8 kDa protein consisting of 507 amino acid residues and includes 11 putative transmembrane segments. Transcripts of TcMucK were detected throughout all of the developmental stages analyzed. The function of this gene was explored by injection of two different double-stranded RNAs targeting different regions of the TcMucK gene (dsTcMucKs) into young larvae to down

  13. Protein Product Encoded by a Human Novel Gene E9730 Enhances AP-1 Activity through Interacting with Jab1

    Institute of Scientific and Technical Information of China (English)

    Zhao-Qing WANG; Han-Dong WEI; Fu-Chu HE

    2004-01-01

    A novel human gene, named E9730 (a clone number of fetal liver cDNA library), has been identified from more than 14,000 expressed sequence tags (ESTs) based on our large scale sequencing of human fetal liver cDNA libraries. Although sequencing of this novel human gene indicates that it is a leucine zipper protein, the function of E9730 and its homologous genes among species is unknown yet. To find out physiological functional clue of E9730, the yeast two-hybrid system was used to screen the E9730-interacting protein(s), and one clone containing a cDNA insert with almost the entire coding sequence (amino acids 39-335) of human Jab1 (Jun-activating domain binding protein 1) that interacted specifically with E9730 was identified. A specific association between Jab1 and E9730 was shown by co-immunoprecipitation and co-localization experiments. Furthermore, E9730 appeared to enhance Jab1-induced AP-1 activity in a concentration-dependent manner and Jab1 may be involved in the intracellular signaling transduction from E9730 to AP-1.

  14. Protein Product Encoded by a Human Novel Gene E9730 Enhances AP-1 Activity through Interacting with Jab1

    Institute of Scientific and Technical Information of China (English)

    Zhao-QingWANG; Han-DongWEI; Fu-ChuHE

    2004-01-01

    A novel human gene, named E9730 (a clone number of fetal liver cDNA library), has been identified from more than 14,000 expressed sequence tags (ESTs) based on our large scale sequencing of human fetal liver cDNA libraries. Although sequencing of this novel human gene indicates that it is a leucine zipper protein, the function of E9730 and its homologous genes among species is unknown yet. To find out physiological functional clue of E9730, the yeast two-hybrid system was used to screen the E9730-interacting protein(s), and one clone containing a cDNA insert with almost the entire coding sequence (amino acids 39-335) of human Jabl (Jun-activating domain binding protein 1) that interacted specifically with E9730 was identified. A specific association between Jab1 and E9730 was shown by co-immunoprecipitation and co-localization experiments. Furthermore, E9730 appeared to enhance Jabl-induced AP-1 activity in a concentration-dependent manner and Jabl may be involved in the intracellular signaling tra.nsduction from E9730 to AP-1.

  15. Investigation of genes encoding calcineurin B-like protein family in legumes and their expression analyses in chickpea (Cicer arietinum L..

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    Mukesh Kumar Meena

    Full Text Available Calcium ion (Ca2+ is a ubiquitous second messenger that transmits various internal and external signals including stresses and, therefore, is important for plants' response process. Calcineurin B-like proteins (CBLs are one of the plant calcium sensors, which sense and convey the changes in cytosolic Ca2+-concentration for response process. A search in four leguminous plant (soybean, Medicago truncatula, common bean and chickpea genomes identified 9 to 15 genes in each species that encode CBL proteins. Sequence analyses of CBL peptides and coding sequences (CDS suggested that there are nine original CBL genes in these legumes and some of them were multiplied during whole genome or local gene duplication. Coding sequences of chickpea CBL genes (CaCBL were cloned from their cDNAs and sequenced, and their annotations in the genome assemblies were corrected accordingly. Analyses of protein sequences and gene structures of CBL family in plant kingdom indicated its diverse origin but showed a remarkable conservation in overall protein structure with appearance of complex gene structure in the course of evolution. Expression of CaCBL genes in different tissues and in response to different stress and hormone treatment were studied. Most of the CaCBL genes exhibited high expression in flowers. Expression profile of CaCBL genes in response to different abiotic stresses and hormones related to development and stresses (ABA, auxin, cytokinin, SA and JA at different time intervals suggests their diverse roles in development and plant defence in addition to abiotic stress tolerance. These data not only contribute to a better understanding of the complex regulation of chickpea CBL gene family, but also provide valuable information for further research in chickpea functional genomics.

  16. Investigation of genes encoding calcineurin B-like protein family in legumes and their expression analyses in chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Meena, Mukesh Kumar; Ghawana, Sanjay; Sardar, Atish; Dwivedi, Vikas; Khandal, Hitaishi; Roy, Riti; Chattopadhyay, Debasis

    2015-01-01

    Calcium ion (Ca2+) is a ubiquitous second messenger that transmits various internal and external signals including stresses and, therefore, is important for plants' response process. Calcineurin B-like proteins (CBLs) are one of the plant calcium sensors, which sense and convey the changes in cytosolic Ca2+-concentration for response process. A search in four leguminous plant (soybean, Medicago truncatula, common bean and chickpea) genomes identified 9 to 15 genes in each species that encode CBL proteins. Sequence analyses of CBL peptides and coding sequences (CDS) suggested that there are nine original CBL genes in these legumes and some of them were multiplied during whole genome or local gene duplication. Coding sequences of chickpea CBL genes (CaCBL) were cloned from their cDNAs and sequenced, and their annotations in the genome assemblies were corrected accordingly. Analyses of protein sequences and gene structures of CBL family in plant kingdom indicated its diverse origin but showed a remarkable conservation in overall protein structure with appearance of complex gene structure in the course of evolution. Expression of CaCBL genes in different tissues and in response to different stress and hormone treatment were studied. Most of the CaCBL genes exhibited high expression in flowers. Expression profile of CaCBL genes in response to different abiotic stresses and hormones related to development and stresses (ABA, auxin, cytokinin, SA and JA) at different time intervals suggests their diverse roles in development and plant defence in addition to abiotic stress tolerance. These data not only contribute to a better understanding of the complex regulation of chickpea CBL gene family, but also provide valuable information for further research in chickpea functional genomics.

  17. Pelizaeus-Merzbacher disease: an X-linked neurologic disorder of myelin metabolism with a novel mutation in the gene encoding proteolipid protein.

    Science.gov (United States)

    Gencic, S; Abuelo, D; Ambler, M; Hudson, L D

    1989-01-01

    The nosology of the inborn errors of myelin metabolism has been stymied by the lack of molecular genetic analysis. Historically, Pelizaeus-Merzbacher disease has encompassed a host of neurologic disorders that present with a deficit of myelin, the membrane elaborated by glial cells that encircles and successively enwraps axons. We describe here a Pelizaeus-Merzbacher pedigree of the classical type, with X-linked inheritance, a typical clinical progression, and a pathologic loss of myelinating cells and myelin in the central nervous system. To discriminate variants of Pelizaeus-Merzbacher disease, a set of oligonucleotide primers was constructed to polymerase-chain-reaction (PCR) amplify and sequence the gene encoding proteolipid protein (PLP), a structural protein that comprises half of the protein of the myelin sheath. The PLP gene in one of two affected males and the carrier mother of this family exhibited a single base difference in the more than 2 kb of the PLP gene sequenced, a C----T transition that would create a serine substitution for proline at the carboxy end of the protein. Our results delineate the clinical features of Pelizaeus-Merzbacher disease, define the possible molecular pathology of this dysmyelinating disorder, and address the molecular classification of inborn errors of myelin metabolism. Patients with the classical form (type I) and the more severely affected, connatal variant of Pelizaeus-Merzbacher disease (type II) would be predicted to display mutation at the PLP locus. The other variants (types III-VI), which have sometimes been categorized as Pelizaeus-Merzbacher disease, may represent mutations in genes encoding other structural myelin proteins or proteins critical to myelination. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:2773936

  18. Characterization of Genes Encoding Protein Disulfide Isomerase in Wheat Including the Specific Role of PDI in the Formation of Gluten.

    Science.gov (United States)

    Filip, Ewa; Demska, Katarzyna

    2016-01-01

    The results of phylogenetic analyses revealed that the family of plant PDI may comprise of at least eight different subfamilies with varying numbers and positions of active centers while retention signals in the endoplasmic reticulum may be present or absent. At least one gene has been cloned for each phylogenetic group. Other phylogenetic analyses have indicated that the family of PDIlike proteins consists of ten classes, the first five of which include proteins equipped with two thioredoxin domains. These results indicate complexity and diversity of the family of protein disulfide isomerase in plants. The study of molecular characteristics of PDI in some cereal species have shown that this enzyme participates in the maturation of secretory proteins and also in the formation of albuminous substances in endosperm, in the mechanism of formation of disulfide bonds and polymerization of gluten polypeptides in wheat. The mechanism of formation of disulfide linkage was tested through in vitro experiments. However, it is not entirely certain whether it reflects their formation in vivo. The results of researches suggest that protein folding and disulfide bond formation occurs in the endoplasmic reticulum. PDI plays a prominent role among enzymes involved in posttranslational modification of proteins. The main goal of this work is to present research data on protein disulfide isomerase, which may be a leading research objective in the area of wheat gluten and the impact of PDI on the baking quality of wheat flour.

  19. The Escherichia coli MntR Miniregulon Includes Genes Encoding a Small Protein and an Efflux Pump Required for Manganese Homeostasis ▿†

    Science.gov (United States)

    Waters, Lauren S.; Sandoval, Melissa; Storz, Gisela

    2011-01-01

    Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA) is repressed by manganese through MntR and encodes an unannotated 42-amino-acid protein. Overproduction of MntS causes manganese sensitivity, while a lack of MntS perturbs proper manganese-dependent repression of mntH. We also provide evidence that mntP (formerly yebN), which encodes a putative efflux pump, is positively regulated by MntR. Deletion of mntP leads to profound manganese sensitivity and to elevated intracellular manganese levels. This work thus defines two new proteins involved in manganese homeostasis and suggests mechanisms for their action. PMID:21908668

  20. The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis.

    Science.gov (United States)

    Waters, Lauren S; Sandoval, Melissa; Storz, Gisela

    2011-11-01

    Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA) is repressed by manganese through MntR and encodes an unannotated 42-amino-acid protein. Overproduction of MntS causes manganese sensitivity, while a lack of MntS perturbs proper manganese-dependent repression of mntH. We also provide evidence that mntP (formerly yebN), which encodes a putative efflux pump, is positively regulated by MntR. Deletion of mntP leads to profound manganese sensitivity and to elevated intracellular manganese levels. This work thus defines two new proteins involved in manganese homeostasis and suggests mechanisms for their action.

  1. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  2. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Science.gov (United States)

    Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

    2015-01-01

    Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were

  3. Structural analysis of the wheat genes encoding NADH-dependent glutamine-2-oxoglutarate amidotransferases and correlation with grain protein content.

    Directory of Open Access Journals (Sweden)

    Domenica Nigro

    Full Text Available BACKGROUND: Nitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT. Together with glutamine synthetase (GS, GOGAT maintains the flow of nitrogen from NH4 (+ into glutamine and glutamate, which are then used for several aminotransferase reactions during amino acid synthesis. RESULTS: The aim of the present work was to identify and analyse the structure of wheat NADH-GOGAT genomic sequences, and study the expression in two durum wheat cultivars characterized by low and high kernel protein content. The genomic sequences of the three homoeologous A, B and D NADH-GOGAT genes were obtained for hexaploid Triticum aestivum and the tetraploid A and B genes of Triticum turgidum ssp. durum. Analysis of the gene sequences indicates that all wheat NADH-GOGAT genes are composed of 22 exons and 21 introns. The three hexaploid wheat homoeologous genes have high conservation of sequence except intron 13 which shows differences in both length and sequence. A comparative analysis of sequences among di- and mono-cotyledonous plants shows both regions of high conservation and of divergence. qRT-PCR performed with the two durum wheat cvs Svevo and Ciccio (characterized by high and low protein content, respectively indicates different expression levels of the two NADH-GOGAT-3A and NADH-GOGAT-3B genes. CONCLUSION: The three hexaploid wheat homoeologous NADH-GOGAT gene sequences are highly conserved - consistent with the key metabolic role of this gene. However, the dicot and monocot amino acid sequences show distinctive patterns, particularly in the transit peptide, the exon 16-17 junction, and the C-terminus. The lack of conservation in the transit peptide may indicate subcellular differences between the two plant divisions - while the sequence

  4. The milkweed pod1 gene encodes a KANADI protein that is required for abaxial/adaxial patterning in maize leaves.

    Science.gov (United States)

    Candela, Héctor; Johnston, Robyn; Gerhold, Abigail; Foster, Toshi; Hake, Sarah

    2008-08-01

    Leaf primordia initiate from the shoot apical meristem with inherent polarity; the adaxial side faces the meristem, while the abaxial side faces away from the meristem. Adaxial/abaxial polarity is thought to be necessary for laminar growth of leaves, as mutants lacking either adaxial or abaxial cell types often develop radially symmetric lateral organs. The milkweed pod1 (mwp1) mutant of maize (Zea mays) has adaxialized sectors in the sheath, the proximal part of the leaf. Ectopic leaf flaps develop where adaxial and abaxial cell types juxtapose. Ectopic expression of the HD-ZIPIII gene rolled leaf1 (rld1) correlates with the adaxialized regions. Cloning of mwp1 showed that it encodes a KANADI transcription factor. Double mutants of mwp1-R with a microRNA-resistant allele of rld1, Rld1-N1990, show a synergistic phenotype with polarity defects in sheath and blade and a failure to differentiate vascular and photosynthetic cell types in the adaxialized sectors. The sectored phenotype and timing of the defect suggest that mwp1 is required late in leaf development to maintain abaxial cell fate. The phenotype of mwp1; Rld1 double mutants shows that both genes are also required early in leaf development to delineate leaf margins as well as to initiate vascular and photosynthetic tissues.

  5. The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer

    Directory of Open Access Journals (Sweden)

    Natsume Hiroko

    2012-07-01

    Full Text Available Abstract Background Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer. Methods and Results A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2 in five highly amplified chromosome regions (with a copy number of more than 6 were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3 domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH, and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360 of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. Conclusion These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

  6. Molecular Characterization of Trypanosoma cruzi Tc8.2 Gene Indicates Two Differential Locations for the Encoded Protein in Epimastigote and Trypomastigote Forms.

    Science.gov (United States)

    Kian, Danielle; Lancheros, César Armando Contreras; Damiani, Igor Alexandre Campos; Fernandes, Tamiris Zanforlin Olmos; Pinge-Filho, Phileno; Santos, Márcia Regina Machado dos; Silveira, José Franco da; Nakamura, Celso Vataru; Silva, João Santana da; Yamada-Ogatta, Sueli Fumie; Yamauchi, Lucy Megumi

    2015-08-01

    This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.

  7. Construction of a quinoa (Chenopodium quinoa Willd.) BAC library and its use in identifying genes encoding seed storage proteins.

    Science.gov (United States)

    Stevens, M R; Coleman, C E; Parkinson, S E; Maughan, P J; Zhang, H-B; Balzotti, M R; Kooyman, D L; Arumuganathan, K; Bonifacio, A; Fairbanks, D J; Jellen, E N; Stevens, J J

    2006-05-01

    Quinoa (Chenopodium quinoa Willd.) is adapted to the harsh environments of the Andean Altiplano region. Its seeds have a well-balanced amino acid composition and exceptionally high protein content with respect to human nutrition. Quinoa grain is a staple in the diet of some of the most impoverished people in the world. The plant is an allotetraploid displaying disomic inheritance (2n=4x=36) with a di-haploid genome of 967 Mbp (megabase pair), or 2C=2.01 pg. We constructed two quinoa BAC libraries using BamHI (26,880 clones) and EcoRI (48,000 clones) restriction endonucleases. Cloned inserts in the BamHI library average 113 kb (kilobase) with approximately 2% of the clones lacking inserts, whereas cloned inserts in the EcoRI library average 130 kb and approximately 1% lack inserts. Three plastid genes used as probes of high-density arrayed blots of 73,728 BACs identified approximately 2.8% of the clones as containing plastid DNA inserts. We estimate that the combined quinoa libraries represent at least 9.0 di-haploid nuclear genome equivalents. An average of 12.2 positive clones per probe were identified with 13 quinoa single-copy ESTs as probes of the high-density arrayed blots, suggesting that the estimate of 9.0x coverage of the genome is conservative. Utility of the BAC libraries for gene identification was demonstrated by probing the library with a partial sequence of the 11S globulin seed storage protein gene and identifying multiple positive clones. The presence of the 11S globulin gene in four of the clones was verified by direct comparison with quinoa genomic DNA on a Southern blot. Besides serving as a useful tool for gene identification, the quinoa BAC libraries will be an important resource for physical mapping of the quinoa genome.

  8. Characterization of big bang, a novel gene encoding for PDZ domain-containing proteins that are dynamically expressed throughout Drosophila development.

    Science.gov (United States)

    Kim, Sabrina Y; Renihan, Maia K; Boulianne, Gabrielle L

    2006-06-01

    PDZ (PSD-95, Discs-large, ZO-1) domain proteins often function as scaffolding proteins and have been shown to play important roles in diverse cellular processes such as the establishment and maintenance of cell polarity, and signal transduction. Here, we report the identification and cloning of a novel Drosophila melanogaster gene that is predicted to produce several different PDZ domain-containing proteins through alternative promoter usage and alternative splicing. This gene, that we have named big bang (bbg), was first identified as C96-GAL4, a GAL4 enhancer trap line that was generated in our lab. To further characterize bbg, its expression pattern was examined in ovaries, embryos, and late third instar larvae using UAS reporter gene constructs, in situ hybridization, or immunocytochemistry. In addition, the expression of alternatively spliced transcripts was examined in more detail using in situ hybridization. We find that during embryogenesis bbg is predominantly expressed in the developing gut, but it is also expressed in external sensory organs found in the epidermis. In the late third instar larva, bbg is expressed along the presumptive wing margin in the wing disc, broadly in the eye disc, and in other imaginal discs as well as in the brain. The expression patterns observed are dynamic and specific during development, suggesting that like other genes that encode for several different PDZ domain protein isoforms, bbg likely plays important roles in multiple developmental processes.

  9. Targeted disruption of the mouse Csrp2 gene encoding the cysteine- and glycine-rich LIM domain protein CRP2 result in subtle alteration of cardiac ultrastructure

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    Stoll Doris

    2008-08-01

    Full Text Available Abstract Background The cysteine and glycine rich protein 2 (CRP2 encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 in vivo, we disrupted the Csrp2 gene in mice and analysed the resulting phenotype. Results A ~17.3 kbp fragment of the murine Csrp2 gene containing exon 3 through 6 was isolated. Using this construct we confirmed the recently determined chromosomal localization (Chromosome 10, best fit location between markers D10Mit203 proximal and D10Mit150 central. A gene disruption cassette was cloned into exon 4 and a mouse strain lacking functional Csrp2 was generated. Mice lacking CRP2 are viable and fertile and have no obvious deficits in reproduction and survival. However, detailed histological and electron microscopic studies reveal that CRP2-deficient mice have subtle alterations in their cardiac ultrastructure. In these mice, the cardiomyocytes display a slight increase in their thickness, indicating moderate hypertrophy at the cellular level. Although the expression of several intercalated disc-associated proteins such as β-catenin, N-RAP and connexin-43 were not affected in these mice, the distribution of respective proteins was changed within heart tissue. Conclusion We conclude that the lack of CRP2 is associated with alterations in cardiomyocyte thickness and hypertrophy.

  10. Identification and Characterization of hmr19 Gene Encoding a Multidrug Resistance Efflux Protein from Streptomyces hygroscopicus subsp.yingchengensis Strain 10-22

    Institute of Scientific and Technical Information of China (English)

    Lei QIN; Heng-An WANG; Zhong-Qin WU; Xiao-Feng ZHANG; Mei-Lei JIN; Zi-Xin DENG; Guo-Ping ZHAO

    2004-01-01

    The hmr19 gene was cloned from Streptomyces hygroscopicus subsp.yingchengensis strain 10-22,a bacterium strain producing agricultural antibiotics.Sequence similarity comparison indicates that hmr19gene may encode a predicted protein with 14 putative transmembrane α-helical spanners,belonging to the drug:H+ antiporter-2 family of the major facilitator superfamily.The expression ofhmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis.Gene replacement technology was employed to construct an hmr19 disruption mutant.The growth inhibition test against different antibiotics indicated that the mutant strain was 5-20 fold more susceptible to tetracycline,vancomycin and mitomycin C than the parental wild type strain.The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22.While with the addition of an energy uncoupler,carbonyl cyanide mchlorophenylhydrazone,the characteristics of the accumulation of [3H]tetracycline in these two strains were almost the same.It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

  11. The GmFWL1 (FW2-2-like) nodulation gene encodes a plasma membrane microdomain-associated protein.

    Science.gov (United States)

    Qiao, Zhenzhen; Brechenmacher, Laurent; Smith, Benjamin; Strout, Gregory W; Mangin, William; Taylor, Christopher; Russell, Scott D; Stacey, Gary; Libault, Marc

    2017-08-01

    The soybean gene GmFWL1 (FW2-2-like1) belongs to a plant-specific family that includes the tomato FW2-2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2-2-like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain-associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process. © 2017 John Wiley & Sons Ltd.

  12. cmdABCDEF, a cluster of genes encoding membrane proteins for differentiation and antibiotic production in Streptomyces coelicolor A3(2

    Directory of Open Access Journals (Sweden)

    Qin Zhongjun

    2009-08-01

    Full Text Available Abstract Background Streptomyces coelicolor is the most studied Streptomyces species and an excellent model for studying differentiation and antibiotic production. To date, many genes have been identified to be required for its differentiation (e.g. bld genes for aerial growth and whi genes for sporulation and antibiotics production (including actII-orf4, redD, cdaR as pathway-specific regulatory genes and afsR, absA1/A2 as pleiotropic regulatory genes. Results A gene cluster containing six genes (SCO4126-4131 was proved to be co-transcribed in S. coelicolor. Deletions of cmdABCDEF (SCO4126-4131 displayed defective sporulation including formation of aberrant branches, and abnormalities in chromosome segregation and spore septation. Disruption mutants of apparently orthologous genes of S. lividans and S. avermitilis also showed defective sporulation, implying that the role of these genes is similar among Streptomyces. Transcription of cmdB, and therefore presumably of the whole operon, was regulated developmentally. Five of the encoded proteins (CmdA, C, D, E, F were predicted membrane proteins. The other, CmdB, a predicted ATP/GTP-binding protein with an ABC-transporter-ATPase domain shown here to be essential for its function, was also located on the cell membrane. These results indicate that CmdABCDEF proteins mainly affect Streptomyces differentiation at an early stage of aerial hyphae formation, and suggest that these proteins may form a complex on cell membrane for proper segregation of chromosomes. In addition, deletions of cmdABCDEF also revealed over-production of blue-pigmented actinorhodin (Act via activation of transcription of the pathway-specific regulatory gene actII-orf4 of actinorhodin biosynthesis. Conclusion In this study, six co-transcribed genes cmdABCDEF were identified by their effects on differentiation and antibiotic production in Streptomyces coelicolor A3(2. These six membrane-located proteins are possibly assembled

  13. The p10 gene of Bombyx mori nucleopolyhedrosis virus encodes a 7.5-kDa protein and is hypertranscribed from a TAAG motif

    Indian Academy of Sciences (India)

    Vikas B. Palhan; Karumathil P. Gopinathan

    2000-08-01

    In baculovirus-based high-level expression of cloned foreign genes, the viral very late gene promoters of polyhedrin (polh) and p10 are extensively exploited. Here we report the cloning and characterization of the p10 gene from a local isolate of Bombyx mori nucleopolyhedrosis virus (BmNPV). The gene harbours a 213-bp open reading frame encoding a protein of 70 amino acids with a predicted molecular mass of 7.5 kDa. The BmNPV p10 showed deletion of a single A at +210 nucleotide compared to the prototype baculovirus, Autographa californica multinucleocapsid nucleopolyhedrosis virus (AcMNPV), p10 gene, resulting in a translational frameshift to generate a termination codon and consequently a truncated polypeptide instead of the 10-kDa protein. This protein P7.5 from BmNPV has a putative leucine zipper dimerization motif towards the N-terminal end and the central nuclear disintegration domain but the carboxy-terminal domain implicated in protein association for fibrillar structure formation is absent. Phylogenetic analysis revealed that p10 is highly conserved among baculoviruses and the BmNPV strains are more closely related to AcMNPV than other baculoviruses. The transcription of p10 is regulated in a temporal manner, reaching maximal levels by 72 h post-infection. RNAase protection and primer extension analysis mapped the transcription start sites at $-70$ and $-71$ nt with respect to the ATG, within the conserved baculovirus late gene motif T\\underline{AA}G. The upstream region showed complete homology to the strong promoter of the AcMNPV p10, suggesting that this promoter from BmNPV could also be exploited for high-level expression of cloned foreign genes in silkworm cells or larvae.

  14. Smoking and polymorphisms of genes encoding mannose-binding lectin and surfactant protein-D in patients with rheumatoid arthritis

    DEFF Research Database (Denmark)

    Kristiansen, Malthe; Frisch, Morten; Madsen, Hans Ole;

    2014-01-01

    To investigate whether polymorphisms in genes coding for mannose-binding lectin (MBL) and surfactant protein-D (SP-D) are associated directly or by interaction with smoking with rheumatoid arthritis (RA), anti-citrullinated peptide antibody (ACPA) positive RA, and erosive RA. MBL2 genotypes, SFTPD...... genotype at codon 11, and HLA-shared epitope were determined in 456 patients with rheumatoid arthritis and 533 sex- and age-matched controls. Patients were grouped according to the presence of ACPA antibodies and RA-associated bone erosions and sub-stratified according to smoking status as never or ever...

  15. Nitrogen availability and electron transport control the expression of glnB gene (encoding PII protein) in the cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    García-Domínguez, M; Florencio, F J

    1997-12-01

    The glnB gene from Synechocystis sp. PCC 6803 that encodes the PII protein has been cloned by heterologous hybridization using the corresponding glnB gene from Synechococcus sp. PCC 7942. An ORF of 336 nucleotides appeared that potentially coded for a protein of 112 amino acid residues (M(r) 12,397). The deduced amino acid sequence revealed a high identity (higher than 80%) with its cyanobacterial counterparts and a basal level of identity (close to 60%) with other PII proteins. A single mRNA of about 680 nucleotides was found under all growth conditions studied. glnB gene expression was specifically activated under nitrogen deprivation (a 10-fold increase respect to nitrogen-replete conditions). No differences in glnB mRNA levels were observed when using nitrate or ammonium as nitrogen sources. Amount of glnB mRNA decreased to undetectable levels when transferring cells to the dark, but effect was avoided by adding glucose to the culture medium. Primer extension analysis and band-shift assays indicated that expression of the glnB gene, elevated under nitrogen deprivation, might lie under the control of the nitrogen transcriptional regulator NtcA, although constitutive levels of expression were also detected from a sigma 70-dependent Escherichia coli-like promoter.

  16. Isolation of a Latimeria menadoensis heat shock protein 70 (Lmhsp70) that has all the features of an inducible gene and encodes a functional molecular chaperone.

    Science.gov (United States)

    Modisakeng, Keoagile W; Jiwaji, Meesbah; Pesce, Eva-Rachele; Robert, Jacques; Amemiya, Chris T; Dorrington, Rosemary A; Blatch, Gregory L

    2009-08-01

    Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system.

  17. Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana.

    Science.gov (United States)

    Carretero-Paulet, Lorenzo; Cairó, Albert; Talavera, David; Saura, Andreu; Imperial, Santiago; Rodríguez-Concepción, Manuel; Campos, Narciso; Boronat, Albert

    2013-07-15

    The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.

  18. The cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1 encodes an ICE-like protein in apple

    Science.gov (United States)

    2012-01-01

    Background Plant growth is greatly affected by low temperatures, and the expression of a number of genes is induced by cold stress. Although many genes in the cold signaling pathway have been identified in Arabidopsis, little is known about the transcription factors involved in the cold stress response in apple. Results Here, we show that the apple bHLH (basic helix-loop-helix) gene MdCIbHLH1 (Cold-Induced bHLH1), which encodes an ICE-like protein, was noticeably induced in response to cold stress. The MdCIbHLH1 protein specifically bound to the MYC recognition sequences in the AtCBF3 promoter, and MdCIbHLH1 overexpression enhanced cold tolerance in transgenic Arabidopsis. In addition, the MdCIbHLH1 protein bound to the promoters of MdCBF2 and favorably contributed to cold tolerance in transgenic apple plants by upregulating the expression of MdCBF2 through the CBF (C-repeat-binding factor) pathway. Our findings indicate that MdCIbHLH1 functions in stress tolerance in different species. For example, ectopic MdCIbHLH1 expression conferred enhanced chilling tolerance in transgenic tobacco. Finally, we observed that cold induces the degradation of the MdCIbHLH1 protein in apple and that this degradation was potentially mediated by ubiquitination and sumoylation. Conclusions Based on these findings, MdCIbHLH1 encodes a transcription factor that is important for the cold tolerance response in apple. PMID:22336381

  19. Over-expression of gene encoding heat shock protein 70 from Mycobacterium tuberculosis and its evaluation as vaccine adjuvant

    Directory of Open Access Journals (Sweden)

    J Dhakal

    2013-01-01

    Full Text Available Background: Heat shock proteins (Hsps are evolutionary ancient and highly conserved molecular chaperons found in prokaryotes as well as eukaryotes. Hsp70 is a predominant member of Hsp family. Microbial Hsp70s (mHsp70s have acquired special significance in immunity since they have been shown to be potent activators of the innate immune system and generate specific immune responses against tumours and infectious agents. Objectives: The present study was aimed to clone express and purify recombinant Hsp70 from the Mycobacterium tuberculosis and characterise it immunologically. The study also aimed at determining the potential of recombinant M. tuberculosis heat shock protein (rMTB-Hsp70 as adjuvant or antigen carrier. Materials and Methods: Cloning of M. tuberculosis heat shock protein (MTB-Hsp70 amplicon was carried out using the pGEMT-Easy vector although for expression, pProExHTb prokaryotic expression vector was used. Purification of recombinant Hsp70 was carried out by nickel-nitrilotriacetic acid (Ni-NTA affinity chromatography. For immunological characterization and determining the adjuvant effect of MTB-Hsp70, BALB/c mice were used. The data obtained was statistically analysed. Results: Hsp70 gene was cloned, sequenced and the sequence data were submitted to National Center for Biotechnology Information (NCBI. Recombinant MTB-Hsp70 was successfully over-expressed using the prokaryotic expression system and purified to homogeneity. The protein was found to be immunodominant. Significant adjuvant effect was produced by the rMTB-Hsp70 when inoculated with recombinant outer membrane protein 31; however, effect was less than the conventionally used the Freund′s adjuvant. Conclusion: Protocol standardised can be followed for bulk production of rHsp70 in a cost-effective manner. Significant adjuvant effect was produced by rMTB-Hsp70; however, the effect was than Freund′s adjuvant. Further, studies need to be carried out to explore its

  20. Normalization with genes encoding ribosomal proteins but not GAPDH provides an accurate quantification of gene expressions in neuronal differentiation of PC12 cells

    Directory of Open Access Journals (Sweden)

    Lim Qing-En

    2010-01-01

    Full Text Available Abstract Background Gene regulation at transcript level can provide a good indication of the complex signaling mechanisms underlying physiological and pathological processes. Transcriptomic methods such as microarray and quantitative real-time PCR require stable reference genes for accurate normalization of gene expression. Some but not all studies have shown that housekeeping genes (HGKs, β-actin (ACTB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which are routinely used for normalization, may vary significantly depending on the cell/tissue type and experimental conditions. It is currently unclear if these genes are stably expressed in cells undergoing drastic morphological changes during neuronal differentiation. Recent meta-analysis of microarray datasets showed that some but not all of the ribosomal protein genes are stably expressed. To test the hypothesis that some ribosomal protein genes can serve as reference genes for neuronal differentiation, a genome-wide analysis was performed and putative reference genes were identified based on stability of expressions. The stabilities of these potential reference genes were then analyzed by reverse transcription quantitative real-time PCR in six differentiation conditions. Results Twenty stably expressed genes, including thirteen ribosomal protein genes, were selected from microarray analysis of the gene expression profiles of GDNF and NGF induced differentiation of PC12 cells. The expression levels of these candidate genes as well as ACTB and GAPDH were further analyzed by reverse transcription quantitative real-time PCR in PC12 cells differentiated with a variety of stimuli including NGF, GDNF, Forskolin, KCl and ROCK inhibitor, Y27632. The performances of these candidate genes as stable reference genes were evaluated with two independent statistical approaches, geNorm and NormFinder. Conclusions The ribosomal protein genes, RPL19 and RPL29, were identified as suitable reference genes

  1. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.

    Science.gov (United States)

    Hensel, M; Shea, J E; Waterman, S R; Mundy, R; Nikolaus, T; Banks, G; Vazquez-Torres, A; Gleeson, C; Fang, F C; Holden, D W

    1998-10-01

    The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.

  2. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase.

    Science.gov (United States)

    Lu, Shunwen; Faris, Justin D; Edwards, Michael C

    2017-04-01

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here, we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 family. The two TaPr-1-rk genes are located on homoeologous chromosomes 3D and 3A, respectively, and each contains a large open reading frame (7385 or 6060 bp) that is interrupted by seven introns and subjected to alternative splicing (AS) with five or six isoforms of mRNA transcripts. The deduced full-length TaPR-1-RK1 and TaPR-1-RK2 proteins (95% identity) contain two repeat PR-1 domains, the second of which is fused via a transmembrane helix to a serine/threonine kinase catalytic (STKc) domain characteristic of receptor-like protein kinases. Phylogenetic analysis indicated that the two PR-1 domains of the TaPR-1-RK proteins form sister clades with their homologues identified in other monocot plants and are well separated from stand-alone PR-1 proteins, whereas the STKc domains may have originated from cysteine-rich receptor-like kinases (CRKs). Reverse-transcriptase-PCR analysis revealed that the TaPr-1-rk genes are predominantly expressed in wheat leaves and their expression levels are elevated in response to pathogen attack, such as infection by barley stripe mosaic virus (BSMV), and also to stress conditions, most obviously, to soil salinity. This is the first report of PR-1-CRK hybrid proteins in wheat. The data may shed new insights into the function/evolutionary origin of the PR-1 family and the STKc-mediated defense/stress response pathways in plants.

  3. Transgenic Mouse Studies to Understand the Regulation, Expression and Function of the Testis-Specific Protein Y-Encoded (TSPY Gene

    Directory of Open Access Journals (Sweden)

    Stephanie Schubert

    2010-08-01

    Full Text Available The TSPY gene, which encodes the testis-specific protein, Y-encoded, was first discovered and characterized in humans, but orthologous genes were subsequently identified on the Y chromosome of many other placental mammals. TSPY is expressed in the testis and to a much lesser extent in the prostate gland, and it is assumed that TSPY serves function in spermatogonial proliferation and/or differentiation. It is further supposed that TSPY is involved in male infertility and exerts oncogenic effects in gonadal and prostate tumor formation. As a member of the TSPY/SET/NAP protein family, TSPY is able to bind cyclin B types, and stimulates the cyclin B1-CDK1 kinase activity, thereby accelerating the G2/M phase transition of the cell cycle of target cells. Because the laboratory mouse carries only a nonfunctional Y-chromosomal Tspy-ps pseudogene, a knockout mouse model for functional research analyses is not a feasible approach. In the last decade, three classical transgenic mouse models have been developed to contribute to our understanding of TSPY regulation, expression and function. The different transgenic mouse approaches and their relevance for studying TSPY regulation, expression and function are discussed in this review.

  4. The carriage of the serine-aspartate repeat protein-encoding sdr genes among Staphylococcus aureus lineages.

    Science.gov (United States)

    Liu, Huanle; Lv, Jingnan; Qi, Xiuqin; Ding, Yu; Li, Dan; Hu, Longhua; Wang, Liangxing; Yu, Fangyou

    2015-01-01

    The serine-aspartate repeat proteins (Sdr) are members of a family of surface proteins and contribute to the pathogenicity of Staphylococcus aureus. Among 288 S. aureus isolates including 158 and 130 associated with skin and soft tissue infections and bloodstream infection, respectively; 275 (95.5%) were positive for at least one of three sdr genes tested. The positivity rates for sdrC, sdrD, and sdrE among S. aureus isolates were 87.8% (253/288), 63.9% (184/288), and 68.1% (196/288), respectively. 224 (77.8%) of 288 isolates were concomitantly positive for two or three sdr genes. There was an association between carriage of sdrE and methicillin-resistant S. aureus (MRSA) isolates, while the carriage rates of sdrC and sdrD in MRSA isolates were similar to those in methicillin-sensitive S. aureus (MSSA) isolates. The prevalence of co-existence of sdrC and sdrE among MRSA isolates was significantly higher than that among MSSA isolates (p<0.05). All ST1, ST5, ST7, and ST25 isolates were positive for sdrD. While all ST121 and ST398 isolates were negative for sdrD. All ST59 and ST88 isolates were positive for sdrE. All ST1 isolates were concomitantly positive for sdrC and sdrD. Concomitant carriage of sdrC, sdrD, and sdrE was found among all ST5, 75.0% (9/12) of ST1, 69.2% (9/13) of ST6, 78.6% (11/14) of ST25, and 90.9% (20/22) of ST88 isolates. sdrD was linked to CC5, CC7 and CC88 isolates, especially CC88 isolates. There was a strong association between the presence of sdrE and CC59, CC88, and CC5 isolates. A significant correlation between concomitant carriage of sdrC, sdrD, and sdrE and CC88 isolates was found. sdrC-positive, sdrD-positive and sdrE-negative gene profile was significantly associated with CC7 clone. There was an association between sdrC-positive, sdrD-negative, and sdrE-positive gene profile and CC59 isolates. A correlation between sdrC-positive, sdrD-negative, and sdrE-negative gene profile and CC121 clone was found. More CC59 isolates carried sdr

  5. Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential.

    Science.gov (United States)

    Decarpentrie, Fanny; Vernet, Nadège; Mahadevaiah, Shantha K; Longepied, Guy; Streichemberger, Eric; Aknin-Seifer, Isabelle; Ojarikre, Obah A; Burgoyne, Paul S; Metzler-Guillemain, Catherine; Mitchell, Michael J

    2012-06-15

    Mammalian ZFY genes are located on the Y chromosome, and code putative transcription factors with 12-13 zinc fingers preceded by a large acidic (activating) domain. In mice, there are two genes, Zfy1 and Zfy2, which are expressed mainly in the testis. Their transcription increases in germ cells as they enter meiosis, both are silenced by meiotic sex chromosome inactivation (MSCI) during pachytene, and Zfy2 is strongly reactivated later in spermatids. Recently, we have shown that mouse Zfy2, but not Zfy1, is involved in triggering the apoptotic elimination of specific types of sex chromosomally aberrant spermatocytes. In humans, there is a single widely transcribed ZFY gene, and there is no evidence for a specific role in the testis. Here, we characterize ZFY transcription during spermatogenesis in mice and humans. In mice, we define a variety of Zfy transcripts, among which is a Zfy2 transcript that predominates in spermatids, and a Zfy1 transcript, lacking an exon encoding approximately half of the acidic domain, which predominates prior to MSCI. In humans, we have identified a major testis-specific ZFY transcript that encodes a protein with the same short acidic domain. This represents the first evidence that ZFY has a conserved function during human spermatogenesis. We further show that, in contrast to the full acidic domain, the short domain does not activate transcription in yeast, and we hypothesize that this explains the functional difference observed between Zfy1 and Zfy2 during mouse meiosis.

  6. Erwinia carotovora DsbA mutants: evidence for a periplasmic-stress signal transduction system affecting transcription of genes encoding secreted proteins.

    Science.gov (United States)

    Vincent-Sealy, L V; Thomas, J D; Commander, P; Salmond, G P

    1999-08-01

    The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approximately two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene

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

    Science.gov (United States)

    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.

  8. DNA vaccination with a gene encoding Toxoplasma gondii Rhoptry Protein 17 induces partial protective immunity against lethal challenge in mice

    Directory of Open Access Journals (Sweden)

    Wang Hai-Long

    2016-01-01

    Full Text Available Toxoplasma gondii is an obligate intracellular apicomplexan parasite that affects humans and various vertebrate livestock and causes serious economic losses. To develop an effective vaccine against T. gondii infection, we constructed a DNA vaccine encoding the T. gondii rhoptry protein 17 (TgROP17 and evaluated its immune protective efficacy against acute T. gondii infection in mice. The DNA vaccine (p3×Flag-CMV-14-ROP17 was intramuscularly injected to BALB/c mice and the immune responses of the vaccinated mice were determined. Compared to control mice treated with empty vector or PBS, mice immunized with the ROP17 vaccine showed a relatively high level of specific anti-T. gondii antibodies, and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a specific lymphocyte proliferative response, a Th1-type cellular immune response with production of IFN-γ and interleukin-2, and increased number of CD8+ T cells. Immunization with the ROP17 DNA significantly prolonged the survival time (15.6 ± 5.4 days, P < 0.05 of mice after challenge infection with the virulent T. gondii RH strain (Type I, compared with the control groups which died within 8 days. Therefore, our data suggest that DNA vaccination with TgROP17 triggers significant humoral and cellular responses and induces effective protection in mice against acute T. gondii infection, indicating that TgROP17 is a promising vaccine candidate against acute toxoplasmosis.

  9. Daily expression pattern of protein-encoding genes and small noncoding RNAs in synechocystis sp. strain PCC 6803.

    Science.gov (United States)

    Beck, Christian; Hertel, Stefanie; Rediger, Anne; Lehmann, Robert; Wiegard, Anika; Kölsch, Adrian; Heilmann, Beate; Georg, Jens; Hess, Wolfgang R; Axmann, Ilka M

    2014-09-01

    Many organisms harbor circadian clocks with periods close to 24 h. These cellular clocks allow organisms to anticipate the environmental cycles of day and night by synchronizing circadian rhythms with the rising and setting of the sun. These rhythms originate from the oscillator components of circadian clocks and control global gene expression and various cellular processes. The oscillator of photosynthetic cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, linked to a complex regulatory network. Synechocystis sp. strain PCC 6803 possesses the standard cyanobacterial kaiABC gene cluster plus multiple kaiB and kaiC gene copies and antisense RNAs for almost every kai transcript. However, there is no clear evidence of circadian rhythms in Synechocystis sp. PCC 6803 under various experimental conditions. It is also still unknown if and to what extent the multiple kai gene copies and kai antisense RNAs affect circadian timing. Moreover, a large number of small noncoding RNAs whose accumulation dynamics over time have not yet been monitored are known for Synechocystis sp. PCC 6803. Here we performed a 48-h time series transcriptome analysis of Synechocystis sp. PCC 6803, taking into account periodic light-dark phases, continuous light, and continuous darkness. We found that expression of functionally related genes occurred in different phases of day and night. Moreover, we found day-peaking and night-peaking transcripts among the small RNAs; in particular, the amounts of kai antisense RNAs correlated or anticorrelated with those of their respective kai target mRNAs, pointing toward the regulatory relevance of these antisense RNAs. Surprisingly, we observed that the amounts of 16S and 23S rRNAs in this cyanobacterium fluctuated in light-dark periods, showing maximum accumulation in the dark phase. Importantly, the amounts of all transcripts, including small noncoding RNAs, did not show any rhythm under continuous light or darkness, indicating the absence

  10. Hypoxic regulation of the expression of genes encoded estrogen related proteins in U87 glioma cells: eff ect of IRE1 inhibition.

    Science.gov (United States)

    Do, Minchenko; Oo, Riabovol; Oo, Ratushna; Oh, Minchenko

    2017-01-01

    The aim of the present study was to examine the effect of inhibition of endoplasmic reticulum stress signaling, mediated by IRE1 (inositol requiring enzyme 1), which is a central mediator of the unfolded protein response on the expression of genes encoded estrogen related proteins (NRIP1/RIP140, TRIM16/EBBP, ESRRA/NR3B1, FAM162A/E2IG5, PGRMC2/PMBP, and SLC39A6/LIV-1) and their hypoxic regulation in U87 glioma cells for evaluation of their possible significance in the control of glioma cells proliferation. The expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells, transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia, was studied by a quantitative polymerase chain reaction. Inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 signaling enzyme function up-regulates the expression of EBBP, E2IG5, PGRMC2, and SLC39A6 genes is in U87 glioma cells in comparison with the control glioma cells, with more significant changes for E2IG5 and PGRMC2 genes. At the same time, the expression of NRIP1 and ESRRA genes is strongly down-regulated in glioma cells upon inhibition of IRE1. We also showed that hypoxia increases the expression of E2IG5, PGRMC2, and EBBP genes and decreases NRIP1 and ESRRA genes expression in control glioma cells. Furthermore, the inhibition of IRE1 in U87 glioma cells decreases the eff ect of hypoxia on the expression of E2IG5 and PGRMC2 genes, eliminates hypoxic regulation of NRIP1 gene, and enhances the sensitivity of ESRRA gene to hypoxic condition. Furthermore, the expression of SLC39A6 gene is resistant to hypoxia in both the glioma cells with and without IRE1 signaling enzyme function. Results of this investigation demonstrate that inhibition of IRE1 signaling enzyme function affects the expression of NRIP1, EBBP, ESRRA, E2IG5, PGRMC2, and SLC39A6 genes in U87 glioma cells in gene specific manner and these changes

  11. Molecular cloning and characterization of a gene encoding the proline transporter protein in common bean (Phaseolus vulgaris L.

    Directory of Open Access Journals (Sweden)

    Jibao Chen

    2016-10-01

    Full Text Available As a typical compatible solute, proline is accumulated in plants under environmental stresses. Proline transporter (ProT plays an important role in proline distribution between plant organs. Using a candidate gene approach, we cloned a cDNA sequence for ProT from common bean (Phaseolus vulgaris L. and designated the gene PvProT. The deduced amino acid sequence of PvProT showed high similarity to Bet/ProT proteins from other leguminous plants, and the highest similarity was observed with mothbean (Vigna aconitifolia L. VuProT. Relative quantification of the mRNA level of PvProT using real-time PCR analysis showed that the PvProT transcript level was higher in leaves than in stems and roots of common bean plants subjected to drought and salt stress. Under 20% (w/w PEG-6000 treatment, drought-resistant plants expressed a higher level of PvProT transcripts than drought-sensitive plants. Although heterologous expression of PvProT in the Escherichia coli mutant mkh13 showed that PvProT exhibited uptake activities for proline and betaine, no betaine content was detected in the common bean. These findings suggest that PvProT plays an important role in the transportation of proline in common bean plants exposed to drought and salt stress.

  12. Molecular cloning and characterization of a gene encoding the proline transporter protein in common bean (Phaseolus vulgaris L.)

    Institute of Scientific and Technical Information of China (English)

    Jibao Chen; Jing Wu; Yunfeng Lu; Yuannan Cao; Hui Zeng; Zhaoyuan Zhang; Lanfen Wang; Shumin Wang

    2016-01-01

    As a typical compatible solute, proline is accumulated in plants under environmental stresses. Proline transporter (ProT) plays an important role in proline distribution between plant organs. Using a candidate gene approach, we cloned a cDNA sequence for ProT from common bean (Phaseolus vulgaris L.) and designated the gene PvProT. The deduced amino acid sequence of PvProT showed high similarity to Bet/ProT proteins from other leguminous plants, and the highest similarity was observed with mothbean (Vigna aconitifolia L.) VuProT. Relative quantification of the mRNA level of PvProT using real-time PCR analysis showed that the PvProT transcript level was higher in leaves than in stems and roots of common bean plants subjected to drought and salt stress. Under 20% (w/w) PEG-6000 treatment, drought-resistant plants expressed a higher level of PvProT transcripts than drought-sensitive plants. Although heterologous expression of PvProT in the Escherichia coli mutant mkh13 showed that PvProT exhibited uptake activities for proline and betaine, no betaine content was detected in the common bean. These findings suggest that PvProT plays an important role in the transportation of proline in common bean plants exposed to drought and salt stress.

  13. Aggregatibacter actinomycetemcomitans QseBC is activated by catecholamines and iron and regulates genes encoding proteins associated with anaerobic respiration and metabolism

    Science.gov (United States)

    Weigel, WA; Demuth, DR; Torres-Escobar, A; Juárez-Rodríguez, MD

    2015-01-01

    Aggregatibacter actinomycetemcomitans QseBC regulates its own expression and is essential for biofilm growth and virulence. However, the signal that activates the QseC sensor has not been identified and the qseBC regulon has not been defined. In this study, we show that QseC is activated by catecholamine hormones and iron but not by either component alone. Activation of QseC requires an EYRDD motif in the periplasmic domain of the sensor and site-specific mutations in EYRDD or the deletion of the periplasmic domain inhibits catecholamine/iron-dependent induction of the ygiW-qseBC operon. Catecholamine/iron-dependent induction of transcription also requires interaction of the QseB response regulator with its binding site in the ygiW-qseBC promoter. Whole genome microarrays were used to compare gene expression profiles of A. actinomycetemcomitans grown in a chemically defined medium with and without catecholamine and iron supplementation. Approximately 11.5% of the A. actinomycetemcomitans genome was differentially expressed by at least two-fold upon exposure to catecholamines and iron. The expression of ferritin was strongly induced, suggesting that intracellular iron storage capacity is increased upon QseBC activation. Consistent with this, genes encoding iron binding and transport proteins were down-regulated by QseBC. Strikingly, 57% of the QseBC up-regulated genes (56/99) encode proteins associated with anaerobic metabolism and respiration. Most of these up-regulated genes were recently reported to be induced during in vivo growth of A. actinomycetemcomitans. These results suggest that detection of catecholamines and iron by QseBC may alter the cellular metabolism of A. actinomycetemcomitans for increased fitness and growth in an anaerobic host environment. PMID:25923132

  14. The novel white spot syndrome virus-induced gene, PmERP15, encodes an ER stress-responsive protein in black tiger shrimp, Penaeus monodon.

    Science.gov (United States)

    Leu, Jiann-Horng; Liu, Kuan-Fu; Chen, Kuan-Yu; Chen, Shu-Hwa; Wang, Yu-Bin; Lin, Chung-Yen; Lo, Chu-Fang

    2015-04-01

    By microarray screening, we identified a white spot syndrome virus (WSSV)-strongly induced novel gene in gills of Penaeus monodon. The gene, PmERP15, encodes a putative transmembrane protein of 15 kDa, which only showed some degree of similarity (54-59%) to several unknown insect proteins, but had no hits to shrimp proteins. RT-PCR showed that PmERP15 was highly expressed in the hemocytes, heart and lymphoid organs, and that WSSV-induced strong expression of PmERP15 was evident in all tissues examined. Western blot analysis likewise showed that WSSV strongly up-regulated PmERP15 protein levels. In WSSV-infected hemocytes, immunofluorescence staining showed that PmERP15 protein was colocalized with an ER enzyme, protein disulfide isomerase, and in Sf9 insect cells, PmERP15-EGFP fusion protein colocalized with ER -Tracker™ Red dye as well. GRP78, an ER stress marker, was found to be up-regulated in WSSV-infected P. monodon, and both PmERP15 and GRP78 were up-regulated in shrimp injected with ER stress inducers tunicamycin and dithiothreitol. Silencing experiments showed that although PmERP15 dsRNA-injected shrimp succumbed to WSSV infection more rapidly, the WSSV copy number had no significant changes. These results suggest that PmERP15 is an ER stress-induced, ER resident protein, and its induction in WSSV-infected shrimp is caused by the ER stress triggered by WSSV infection. Furthermore, although PmERP15 has no role in WSSV multiplication, its presence is essential for the survival of WSSV-infected shrimp.

  15. CHIR99021 promotes self-renewal of mouse embryonic stem cells by modulation of protein-encoding gene and long intergenic non-coding RNA expression

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yongyan [College of Veterinary Medicine, Northwest A and F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); Ai, Zhiying [Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); College of Life Sciences, Northwest A and F University, Yangling 712100, Shaanxi (China); Yao, Kezhen [College of Veterinary Medicine, Northwest A and F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); Cao, Lixia; Du, Juan; Shi, Xiaoyan [Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); College of Life Sciences, Northwest A and F University, Yangling 712100, Shaanxi (China); Guo, Zekun, E-mail: gzk@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A and F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); Zhang, Yong, E-mail: zhylab@hotmail.com [College of Veterinary Medicine, Northwest A and F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China)

    2013-10-15

    Embryonic stem cells (ESCs) can proliferate indefinitely in vitro and differentiate into cells of all three germ layers. These unique properties make them exceptionally valuable for drug discovery and regenerative medicine. However, the practical application of ESCs is limited because it is difficult to derive and culture ESCs. It has been demonstrated that CHIR99021 (CHIR) promotes self-renewal and enhances the derivation efficiency of mouse (m)ESCs. However, the downstream targets of CHIR are not fully understood. In this study, we identified CHIR-regulated genes in mESCs using microarray analysis. Our microarray data demonstrated that CHIR not only influenced the Wnt/β-catenin pathway by stabilizing β-catenin, but also modulated several other pluripotency-related signaling pathways such as TGF-β, Notch and MAPK signaling pathways. More detailed analysis demonstrated that CHIR inhibited Nodal signaling, while activating bone morphogenetic protein signaling in mESCs. In addition, we found that pluripotency-maintaining transcription factors were up-regulated by CHIR, while several developmental-related genes were down-regulated. Furthermore, we found that CHIR altered the expression of epigenetic regulatory genes and long intergenic non-coding RNAs. Quantitative real-time PCR results were consistent with microarray data, suggesting that CHIR alters the expression pattern of protein-encoding genes (especially transcription factors), epigenetic regulatory genes and non-coding RNAs to establish a relatively stable pluripotency-maintaining network. - Highlights: • Combined use of CHIR with LIF promotes self-renewal of J1 mESCs. • CHIR-regulated genes are involved in multiple pathways. • CHIR inhibits Nodal signaling and promotes Bmp4 expression to activate BMP signaling. • Expression of epigenetic regulatory genes and lincRNAs is altered by CHIR.

  16. Molecular characterization and evolution of a gene family encoding male-specific reproductive proteins in the African malaria vector Anopheles gambiae

    Directory of Open Access Journals (Sweden)

    Sharakhov Igor V

    2011-10-01

    Full Text Available Abstract Background During copulation, the major Afro-tropical malaria vector Anopheles gambiae s.s. transfers male accessory gland (MAG proteins to females as a solid mass (i.e. the "mating plug". These proteins are postulated to function as important modulators of female post-mating responses. To understand the role of selective forces underlying the evolution of these proteins in the A. gambiae complex, we carried out an evolutionary analysis of gene sequence and expression divergence on a pair of paralog genes called AgAcp34A-1 and AgAcp34A-2. These encode MAG-specific proteins which, based on homology with Drosophila, have been hypothesized to play a role in sperm viability and function. Results Genetic analysis of 6 species of the A. gambiae complex revealed the existence of a third paralog (68-78% of identity, that we named AgAcp34A-3. FISH assays showed that this gene maps in the same division (34A of chromosome-3R as the other two paralogs. In particular, immuno-fluorescence assays targeting the C-terminals of AgAcp34A-2 and AgAcp34A-3 revealed that these two proteins are localized in the posterior part of the MAG and concentrated at the apical portion of the mating plug. When transferred to females, this part of the plug lies in proximity to the duct connecting the spermatheca to the uterus, suggesting a potential role for these proteins in regulating sperm motility. AgAcp34A-3 is more polymorphic than the other two paralogs, possibly because of relaxation of purifying selection. Since both unequal crossing-over and gene conversion likely homogenized the members of this gene family, the interpretation of the evolutionary patterns is not straightforward. Although several haplotypes of the three paralogs are shared by most A. gambiae s.l. species, some fixed species-specific replacements (mainly placed in the N- and C-terminal portions of the secreted peptides were also observed, suggesting some lineage-specific adaptation. Conclusions

  17. Rj4, a Gene Controlling Nodulation Specificity in Soybeans, Encodes a Thaumatin-Like Protein But Not the One Previously Reported.

    Science.gov (United States)

    Tang, Fang; Yang, Shengming; Liu, Jinge; Zhu, Hongyan

    2016-01-01

    Rj4 is a dominant gene in soybeans (Glycine max) that restricts nodulation by many strains of Bradyrhizobium elkanii. The soybean-B. elkanii symbiosis has a low nitrogen-fixation efficiency, but B. elkanii strains are highly competitive for nodulation; thus, cultivars harboring an Rj4 allele are considered favorable. Cloning the Rj4 gene is the first step in understanding the molecular basis of Rj4-mediated nodulation restriction and facilitates the development of molecular tools for genetic improvement of nitrogen fixation in soybeans. We finely mapped the Rj4 locus within a small genomic region on soybean chromosome 1, and validated one of the candidate genes as Rj4 using both complementation tests and CRISPR/Cas9-based gene knockout experiments. We demonstrated that Rj4 encodes a thaumatin-like protein, for which a corresponding allele is not present in the surveyed rj4 genotypes, including the reference genome Williams 82. Our conclusion disagrees with the previous report that Rj4 is the Glyma.01G165800 gene (previously annotated as Glyma01g37060). Instead, we provide convincing evidence that Rj4 is Glyma.01g165800-D, a duplicated and unique version of Glyma.01g165800, that has evolved the ability to control symbiotic specificity. © 2016 American Society of Plant Biologists. All Rights Reserved.

  18. Rj4, a Gene Controlling Nodulation Specificity in Soybeans, Encodes a Thaumatin-Like Protein But Not the One Previously Reported1

    Science.gov (United States)

    Tang, Fang; Yang, Shengming; Liu, Jinge

    2016-01-01

    Rj4 is a dominant gene in soybeans (Glycine max) that restricts nodulation by many strains of Bradyrhizobium elkanii. The soybean-B. elkanii symbiosis has a low nitrogen-fixation efficiency, but B. elkanii strains are highly competitive for nodulation; thus, cultivars harboring an Rj4 allele are considered favorable. Cloning the Rj4 gene is the first step in understanding the molecular basis of Rj4-mediated nodulation restriction and facilitates the development of molecular tools for genetic improvement of nitrogen fixation in soybeans. We finely mapped the Rj4 locus within a small genomic region on soybean chromosome 1, and validated one of the candidate genes as Rj4 using both complementation tests and CRISPR/Cas9-based gene knockout experiments. We demonstrated that Rj4 encodes a thaumatin-like protein, for which a corresponding allele is not present in the surveyed rj4 genotypes, including the reference genome Williams 82. Our conclusion disagrees with the previous report that Rj4 is the Glyma.01G165800 gene (previously annotated as Glyma01g37060). Instead, we provide convincing evidence that Rj4 is Glyma.01g165800-D, a duplicated and unique version of Glyma.01g165800, that has evolved the ability to control symbiotic specificity. PMID:26582727

  19. Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto-Kakizaki rats.

    Science.gov (United States)

    Salem, K A; Adrian, T E; Qureshi, M A; Parekh, K; Oz, M; Howarth, F C

    2012-12-01

    There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0

  20. The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

    Science.gov (United States)

    Warner, T S; Sinclair, D A; Fitzpatrick, K A; Singh, M; Devlin, R H; Honda, B M

    1998-04-01

    Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

  1. The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.

    OpenAIRE

    Varnum, B C; Ma, Q F; T. H. Chi; Fletcher, B.; Herschman, H.R.

    1991-01-01

    The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately...

  2. The Maize Imprinted Gene Floury3 Encodes a PLATZ Protein Required for tRNA and 5S rRNA Transcription Through Interaction with RNA Polymerase III.

    Science.gov (United States)

    Li, Qi; Wang, Jiechen; Ye, Jianwei; Zheng, Xixi; Xiang, Xiaoli; Li, Changsheng; Fu, Miaomiao; Wang, Qiong; Zhang, Zhi-Yong; Wu, Yongrui

    2017-09-05

    Maize (Zea mays) floury3 (fl3) is a classic semi-dominant negative mutant that exhibits severe defects in the endosperm but fl3 plants otherwise appear normal. We cloned the fl3 gene and determined that it encodes a PLATZ (plant AT-rich sequence- and zinc-binding) protein. The mutation in fl3 resulted in an Asn to His replacement in the conserved PLATZ domain, creating a dominant allele. Fl3 is specifically expressed in starchy endosperm cells and regulated by genomic imprinting, which leads to the suppressed expression of fl3 when transmitted through the male, perhaps as a consequence the semi-dominant behavior. Yeast two-hybrid screening and bimolecular luciferase complementation (BiLC) experiments revealed that FL3 interacts with the RNA polymerase III subunit 53 (RPC53) and transcription factor class C 1 (TFC1), two critical factors of the RNA polymerase III (RNAPIII) transcription complex. In the fl3 endosperm, the levels of many tRNAs and 5S rRNA that are transcribed by RNAPIII are significantly reduced, suggesting that the incorrectly folded fl3 protein may impair the function of RNAPIII. The transcriptome is dramatically altered in fl3 mutants, in which the down-regulated genes are primarily enriched in pathways related to translation, ribosome, misfolded protein responses and nutrient reservoir activity. Collectively, these changes may lead to defects in endosperm development and storage reserve filling in fl3 seeds. © 2017 American Society of Plant Biologists. All rights reserved.

  3. Effect of the deletion of qmoABC and the promoter distal gene encoding a hypothetical protein on sulfate-reduction in Desulfovibrio vulgaris Hildenborough

    Energy Technology Data Exchange (ETDEWEB)

    Zane, Grant M.; Yen, Huei-chi Bill; Wall, Judy D.

    2010-03-18

    The pathway of electrons required for the reduction of sulfate in sulfate-reducing bacteria (SRB) is not yet fully characterized. In order to determine the role of a transmembrane protein complex suggested to be involved in this process, a deletion of Desulfovibrio vulgaris Hildenborough was created by marker exchange mutagenesis that eliminated four genes putatively encoding the QmoABC complex and a hypothetical protein (DVU0851). The Qmo complex (quinone-interacting membrane-bound oxidoreductase) is proposed to be responsible for transporting electrons to the dissimilatory adenosine-5?phosphosulfate (APS) reductase in SRB. In support of the predicted role of this complex, the deletion mutant was unable to grow using sulfate as its sole electron acceptor with a range of electron donors. To explore a possible role for the hypothetical protein in sulfate reduction, a second mutant was constructed that had lost only the gene that codes for DVU0851. The second constructed mutant grew with sulfate as the sole electron acceptor; however, there was a lag that was not present with the wild-type or complemented strain. Neither deletion strain was significantly impaired for growth with sulfite or thiosulfate as terminal electron acceptor. Complementation of the D(qmoABC-DVU0851) mutant with all four genes or only the qmoABC genes restored its ability to grow by sulfate respiration. These results confirmed the prediction that the Qmo complex is in the electron pathway for sulfate-reduction and revealed that no other transmembrane complex could compensate when Qmo was lacking.

  4. Molecular characterization and expression analysis of three homoeologous Ta14S genes encoding 14-3-3 proteins in wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    Xinguo Wang

    2016-06-01

    Full Text Available The purpose of this study was to characterize Ta14S homoeologs and assess their functions in wheat seed development. The genomic and cDNA sequences of three Ta14S homoeologous genes encoding 14-3-3 proteins were isolated. Sequence analysis revealed that the three homoeologs consisted of five exons and four introns and were very highly conserved in the coding regions and in exon/intron structure, whereas the cDNA sequences were variable in the 5′ and 3′-UTR. The three genes, designated as Ta14S-2A, Ta14S-2B and Ta14S-2D, were located in homoeologous group 2 chromosomes. The polypeptide chains of the three Ta14S genes were highly similar. These genes were most homologous to Hv14A from barley. Real-time quantitative PCR indicated that the three Ta14S genes were differentially expressed in different organs at different developmental stages and all exhibited greater expression in primary roots of 1-day-old germlings than in other tissues. Comparison of the expression patterns of the three homoeologous genes at different times after pollination also revealed that their expression was developmentally regulated. The transcription of Ta14S-2B was clearly higher during seed germination, whereas expressions of Ta14S-2A and Ta14S-2D were up-regulated at the beginning of seed imbibition (0–12 h, but declined thereafter. The results suggested that the three Ta14S homoeologous genes have regulatory roles in seed development and germination.

  5. Molecular characterization and expression analysis of three homoeologous Ta14S genes encoding 14-3-3 proteins in wheat(Triticum aestivum L.)

    Institute of Scientific and Technical Information of China (English)

    Xinguo Wang; Yanli Wang; Ruixia Xiao; Xin Chen; Jiangping Ren

    2016-01-01

    The purpose of this study was to characterize Ta14 S homoeologs and assess their functions in wheat seed development.The genomic and c DNA sequences of three Ta14 S homoeologous genes encoding 14-3-3 proteins were isolated.Sequence analysis revealed that the three homoeologs consisted of five exons and four introns and were very highly conserved in the coding regions and in exon/intron structure,whereas the c DNA sequences were variable in the 5′ and 3′-UTR.The three genes,designated as Ta14S-2A,Ta14S-2B and Ta14S-2D,were located in homoeologous group 2 chromosomes.The polypeptide chains of the three Ta14 S genes were highly similar.These genes were most homologous to Hv14 A from barley.Real-time quantitative PCR indicated that the three Ta14 S genes were differentially expressed in different organs at different developmental stages and all exhibited greater expression in primary roots of 1-day-old germlings than in other tissues.Comparison of the expression patterns of the three homoeologous genes at different times after pollination also revealed that their expression was developmentally regulated.The transcription of Ta14S-2B was clearly higher during seed germination,whereas expressions of Ta14S-2A and Ta14S-2D were up-regulated at the beginning of seed imbibition(0–12 h),but declined thereafter.The results suggested that the three Ta14 S homoeologous genes have regulatory roles in seed development and germination.

  6. PHH1, a novel gene from Arabidopsis thaliana that encodes a protein similar to plant blue-light photoreceptors and microbial photolyases.

    Science.gov (United States)

    Hoffman, P D; Batschauer, A; Hays, J B

    1996-11-27

    A cDNA from Arabidopsis thaliana similar to microbial photolyase genes, and designated AT-PHH1, was isolated using a photolyase-like cDNA from Sinapsis alba (SA-PHR1) as a probe. Multiple isolations yielded only PHH1 cDNAs, and a few blue-light-receptor CRY1 (HY4) cDNAs (also similar to microbial photolyase genes), suggesting the absence of any other highly similar Arabidopsis genes. The AT-PHH1 and SA-PHR1 cDNA sequences predict 89% identity at the protein level, except for an AT-PHH1 C-terminal extension (111 amino acids), also not seen in microbial photolyases. AT-PHH1 and CRY1 show less similarity (54% p4erein identity), including respective C-terminal extensions that are themselves mostly dissimilar. Analysis of fifteen AT-PHH1 genomic isolates reveals a single gene, with three introns in the coding sequence and one in the 5'-untranslated leader. Full-length AT-PHH1, and both AT-PHH1 and AT-PHH1 delta C-513 (truncated to be approximately the size of microbial photolyase genes) cDNAs, were overexpressed, respectively, in yeast and Escherichia coli mutants hypersensitive to ultraviolet light. The absence of significant effects on resistance suggests either that any putative AT-PHH1 DNA repair activity requires cofactors/chromophores not present in yeast or E. coli, or that AT-PHH1 encodes a blue-light/ultraviolet-A receptor rather than a DNA repair protein.

  7. Effect of the deletion of genes encoding proteins of the extracellular virion form of vaccinia virus on vaccine immunogenicity and protective effectiveness in the mouse model.

    Directory of Open Access Journals (Sweden)

    Clement A Meseda

    Full Text Available Antibodies to both infectious forms of vaccinia virus, the mature virion (MV and the enveloped virion (EV, as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.

  8. capA, a cspA-like gene that encodes a cold acclimation protein in the psychrotrophic bacterium Arthrobacter globiformis SI55.

    Science.gov (United States)

    Berger, F; Normand, P; Potier, P

    1997-09-01

    By use of Arthrobacter globiformis SI55, a psychrotrophic bacterium capable of growth between -5 and +32 degrees C, we cloned and sequenced capA, a gene homologous to cspA encoding the major cold shock protein in Escherichia coli. The deduced protein sequence has a high level of identity with the sequences of other CspA-related proteins from various sources, and no particular residue or domain that could be specific to cold-adapted microorganisms emerged. We show that CapA was produced very rapidly following cold shock, but unlike its mesophilic counterparts, it was still expressed during prolonged growth at low temperature. Its synthesis is regulated at the translational level, and we showed that growth resumption following a temperature downshift correlated with CapA expression. Transient inhibitions in protein synthesis during the first stages of the cold shock response severely impaired the subsequent acclimation of A. globiformis SI55 to low temperature and delayed CapA expression. The cold shock response in A. globiformis SI55 is an adaptative process in which CapA may play a crucial role. We suggest that low-temperature acclimation is conditioned mainly by the ability of cells to restore an active translational machinery after cold shock in a process that may be different from that present in mesophiles.

  9. Comparative analysis and molecular characterization of a gene BANF1 encoded a DNA-binding protein during mitosis from the Giant Panda and Black Bear.

    Science.gov (United States)

    Zeng, Yichun; Hou, Yi-Ling; Ding, Xiang; Hou, Wan-Ru; Li, Jian

    2014-01-01

    Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.

  10. Gravistimulation changes expression of genes encoding putative carrier proteins of auxin polar transport in etiolated pea epicotyls

    Science.gov (United States)

    Hoshino, T.; Hitotsubashi, R.; Miyamoto, K.; Tanimoto, E.; Ueda, J.

    STS-95 space experiment has showed that auxin polar transport in etiolated epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings is controlled by gravistimulation. In Arabidopsis thaliana auxin polar transport has considered to be regulated by efflux and influx carrier proteins in plasma membranes, AtPIN1 and AtAUX1, respectively. In order to know how gravistimuli control auxin polar transport in etiolated pea epicotyls at molecular levels, strenuous efforts have been made, resulting in successful isolation of full-length cDNAs of a putative auxin efflux and influx carriers, PsPIN2 and PsAUX1, respectively. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 (accession no. AY222857, Chawla and DeMason, 2003) and AtPINs, and also among PsAUX1, AtAUX1 and their related genes. Phylogenetic analyses based on the deduced amino acid sequences revealed that PsPIN2 belonged to a subclade including AtPIN3, AtPIN4 relating to lateral transport of auxin, while PsPIN1 belonged to the same clade as AtPIN1 relating to auxin polar transport. In the present study, we examined the effects of gravistimuli on the expression of PsPINs and PsAUX1 in etiolated pea seedlings by northern blot analysis. Expression of PsPIN1, PsPIN2 and PsAUX1 in hook region of 3.5-d-old etiolated pea seedlings grown under simulated microgravity conditions on a 3-D clinostat increased as compared with that of the seedlings grown under 1 g conditions. On the other hand, that of PsPIN1 and PsAUX1 in the 1st internode region under simulated microgravity conditions on a 3-D clinostat also increased, while that of PsPIN2 was affected little. These results suggest that expression of PsPIN1, PsPIN2 and PsAUX1 regulating polar/lateral transport of auxin is substantially under the control of gravity. A possible role of PsPINs and PsAUX1 of auxin polar transport in etiolated pea seedlings will also be discussed.

  11. Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate

    Directory of Open Access Journals (Sweden)

    Erma Sulistyaningsih

    2015-10-01

    Full Text Available Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recombinant GRA1-encoding gene was thendigesting using EcoRI restriction endonuclease and sequencing. The result showed that the recombinant GRA1-encoding gene consisted of DNA sequences encoding all signal peptide and mature peptide of GRA1 protein.Alignment of recombinant GRA1 sequence to gene encoding GRA1 protein of Toxoplasma gondii RH isolate showed100% homologous.Keywords: GRA1 protein, Toxoplasma gondii, tachyzoite, cloning, cDNA

  12. CsAGP1, a gibberellin-responsive gene from cucumber hypocotyls, encodes a classical arabinogalactan protein and is involved in stem elongation.

    Science.gov (United States)

    Park, Me Hea; Suzuki, Yoshihito; Chono, Makiko; Knox, J Paul; Yamaguchi, Isomaro

    2003-03-01

    Fluorescence differential display was used to isolate the gibberellin (GA)-responsive gene, CsAGP1, from cucumber (Cucumis sativus) hypocotyls. A sequence analysis of CsAGP1 indicated that the gene putatively encodes a "classical" arabinogalactan protein (AGP) in cucumber. Transgenic tobacco (Nicotiana tabacum) plants overexpressing CsAGP1 under the control of the cauliflower mosaic virus 35S promoter produced a Y(betaGlc)(3)-reactive proteoglycan in addition to AGPs present in wild-type tobacco plants. Immuno-dot blotting of the product, using anti-AGP antibodies, showed that the CsAGP1 protein had the AGP epitopes common to AGP families. The transcription level of CsAGP1 in cucumber hypocotyls increased in response not only to GA but also to indole-3-acetic acid. Although CsAGP1 is expressed in most vegetative tissues of cucumber, including the shoot apices and roots, the GA treatment resulted in an increase in the mRNA level of CsAGP1 only in the upper part of the hypocotyls. Y(betaGlc)(3), which selectively binds AGPs, inhibited the hormone-promoted elongation of cucumber seedling hypocotyls. Transgenic plants ectopically expressing CsAGP1 showed a taller stature and earlier flowering than the wild-type plants. These observations suggest that CsAGP1 is involved in stem elongation.

  13. Expression and Comparative Analysis of Genes Encoding Outer Membrane Proteins LipL21, LipL32 and OmpL1 in Epidemic Leptospires

    Institute of Scientific and Technical Information of China (English)

    Xiang-Yan ZHANG; Xiao-Kui GUO; Yang YU; Ping HE; Yi-Xuan ZHANG; Bao-Yu HU; Yang YANG; Yi-Xin NIE; Xiu-Gao JIANG; Guo-Ping ZHAO

    2005-01-01

    Leptospiral outer membrane proteins (OMPs) are highly conserved in different species, and play an essential role in the development of new immunoprotection and serodiagnosis strategies. The genes encoding LipL21, LipL32 and OmpL1 were cloned from the complete genome sequence of Leptospira interrogans serovar lai strain Lai and expressed in vitro. Sequence comparison analysis revealed that the three genes were highly conserved among distinct epidemic leptospires, including three major epidemic species Leptospira interrogans, Leptospira borgpetersenii and Leptospira weilii, in China. Immunoblot analysis was further performed to scrutinize 15 epidemic Leptospira reference strains using the antisera of the recombinant OMPs. Both immunoblot assay and reverse transcription-polymerase chain reaction demonstrated that these three OMPs were conservatively expressed in pathogenic L. interrogans strains and other pathogenic leptospires.Additionally, the use of these recombinant OMPs as antigens in enzyme-linked immunosorbent assay (ELISA)for serodiagnosis of leptospirosis was evaluated. The recombinant LipL32 and OmpL1 proteins showed a high degree of ELISA reactivity with sera from patients infected with L. interrogans strain Lai and other pathogenic leptospires. These results may contribute to the identification of candidates for broad-range vaccines and immunodiagnostic antigens in further research.

  14. Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    Ouyang Danming; Hu Yongxuan; Li Mulan; Zeng Xiaojun; He Zhixiong; Yuan Caijia

    2008-01-01

    Objective: To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosoma japonicum,(SjAslp) and transfer it into mammalian cells to express the objective protein. Methods: By polymerase chain reaction (PCR) technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK+/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1(+). After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Resnlts: The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion: SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.

  15. Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

    Science.gov (United States)

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

    2013-11-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons.

  16. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  17. In Silico Analysis of the Genes Encoding Proteins that Are Involved in the Biosynthesis of the RMS/MAX/D Pathway Revealed New Roles of Strigolactones in Plants

    Directory of Open Access Journals (Sweden)

    Marek Marzec

    2015-03-01

    Full Text Available Strigolactones were described as a new group of phytohormones in 2008 and since then notable large number of their functions has been uncovered, including the regulation of plant growth and development, interactions with other organisms and a plant’s response to different abiotic stresses. In the last year, investigations of the strigolactone biosynthesis pathway in two model species, Arabidopsis thaliana and Oryza sativa, resulted in great progress in understanding the functions of four enzymes that are involved in this process. We performed in silico analyses, including the identification of the cis-regulatory elements in the promoters of genes encoding proteins of the strigolactone biosynthesis pathway and the identification of the miRNAs that are able to regulate their posttranscriptional level. We also searched the databases that contain the microarray data for the genes that were analyzed from both species in order to check their expression level under different growth conditions. The results that were obtained indicate that there are universal regulations of expression of all of the genes that are involved in the strigolactone biosynthesis in Arabidopsis and rice, but on the other hand each stage of strigolactone production may be additionally regulated independently. This work indicates the presence of crosstalk between strigolactones and almost all of the other phytohormones and suggests the role of strigolactones in the response to abiotic stresses, such as wounding, cold or flooding, as well as in the response to biotic stresses.

  18. Down-regulation in multiple human cancers of a novel gene, DMHC, from 17q25.1 that encodes an integral membrane protein.

    Science.gov (United States)

    Mikami, I; Harada, H; Nagai, H; Tsuneizumi, M; Nobe, Y; Koizumi, K; Sugano, S; Tanaka, S; Emi, M

    2001-04-01

    Frequent observations of allelic loss in chromosomal band 17q25.1 in a variety of human cancers have suggested that one or more tumor suppressor genes are present in that region. Moreover, a genetic locus for hereditary focal non-epidermolytic palmoplantar keratoderma, a condition associated with cancer of the esophagus (TOC; Tylosis with Oesophageal Cancer), lies in the same region. We screened cell lines derived from a variety of human cancers by reverse transcription-polymerase chain reaction (RT-PCR) to detect alterations in expression of genes within the region in question, by examining expressed sequence tags located there. These experiments identified an 1834-bp full-length cDNA encoding a novel, 441-amino acid integral membrane protein with seven putative transmembrane domains. This gene showed loss or extreme decrease of expression in 6 of 10 uterine cancer-cell lines, 2 of 11 hepatic cell carcinoma-cell lines, 2 of 7 lung cancer-cell lines, 1 of 6 gastric cancer-cell lines, and 1 of 10 breast cancer-cell lines. (We named it DMHC ("down-regulated in multiple human cancers").) Our results suggest that loss of expression of DMHC at 17q25.1 may play an important role in development of variety of human cancers.

  19. A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

    Science.gov (United States)

    Zuo, Dong-Yun; Yi, Shu-Yuan; Liu, Rong-Jing; Qu, Bo; Huang, Tao; He, Wei-Jie; Li, Cheng; Li, He-Ping; Liao, Yu-Cai

    2016-06-01

    Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification.

  20. Expansion of genes encoding piRNA-associated argonaute proteins in the pea aphid: diversification of expression profiles in different plastic morphs.

    Directory of Open Access Journals (Sweden)

    Hsiao-Ling Lu

    Full Text Available Piwi-interacting RNAs (piRNAs are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are "specialised" in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction.

  1. GhHyPRP4, a cotton gene encoding putative hybrid proline-rich protein, is preferentially expressed in leaves and involved in plant response to cold stress

    Institute of Scientific and Technical Information of China (English)

    Gengqing Huang; Siying Gong; Wenliang Xu; Peng Li; Dejing Zhang; Lixia Qin; Wen Li; Xuebao Li

    2011-01-01

    Plant hybrid proline-rich proteins (HyPRPs) usually consist of an N-terminal signal peptide, a central prolinerich domain, and a conserved eight-cysteine motif C-terminal domain. In this study, one gene (designated as GhHyPRP4) encoding putative HyPRP was isolated from cotton cDNA library. Northern blot and quantitative reverse transcriptase-polymerase chain reaction analyses revealed that GhHyPRP4 was preferentially expressed in leaves. Under cold stress, GhHyPRP4 expression was significantly up-regulated in leaves of cotton seedlings.Using the genome walking approach, a promoter fragment of GhHyPRP4 gene was isolated from cotton genome.GUS (β-glucuronidase) gene driven by GhHyPRP4 promoter was specifically expressed in leaves and cotyledons of the transgenic 4rabidopsis thaliana. Furthermore,GUS expression in leaves was remarkably induced by cold stress. Overexpression of GhHyPRP4 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival rate upon treatment under -20℃ for 60 h.These data suggested that GhHyPRP4 may be involved in plant response to cold stress during seedling development of cotton.

  2. The promoter of the barley aleurone-specific gene encoding a putative 7 kDa lipid transfer protein confers aleurone cell-specific expression in transgenic rice.

    Science.gov (United States)

    Kalla, R; Shimamoto, K; Potter, R; Nielsen, P S; Linnestad, C; Olsen, O A

    1994-12-01

    This paper describes the aleurone-specific gene Ltp2 from barley, which encodes a putative 7 kDa non-specific lipid transfer protein. As shown by Northern and in situ hybridization analyses, the Ltp2 transcript is present in barley aleurone cells shortly after the initiation of aleurone cell differentiation. The expression of Ltp2 increases until grain mid-maturity, but the mRNA is absent from mature grains. The Ltp2 transcript is undetectable in the embryo and vegetative tissues, confirming the aleurone specificity of the Ltp2 gene. The ability of the isolated 801 bp Ltp2 promoter to direct aleurone-specific expression in immature barley grains is demonstrated by particle bombardment experiments. In these experiments, the activity of the Ltp2 promoter is 5% of the activity of the strong constitutive Actin1 promoter from rice, as quantified by GUS activity measurements. In stably transformed rice plants containing the Ltp2 promoter-Gus construct, the specificity of the Ltp2 promoter is confirmed in vivo by the presence of GUS activity exclusively in the aleurone layer. This study demonstrates the conserved nature of the regulatory signals involved in aleurone-specific gene transcription in cereal grains.

  3. An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily.

    Science.gov (United States)

    Bourouis, M; Moore, P; Ruel, L; Grau, Y; Heitzler, P; Simpson, P

    1990-09-01

    The product(s) of the gene shaggy (sgg) is required for seemingly unrelated events during the development of Drosophila melanogaster. In embryos, maternal and zygotically derived sgg products are required initially to construct a normal syncytial blastoderm and later for normal segmentation. Furthermore, in mutant animals a process of intercellular communication that is required for the segregation of the neural and epidermal lineage during the formation of the central nervous system and the adult peripheral nervous system is disrupted. Here we describe a transcription unit of approximately 40 kb lying within the cloned chromosomal interval 3B1, and provide evidence that it encodes the sgg+ function. Of seven developmentally regulated transcripts that are partially generated by alternative splicing, two seem to be responsible for early sgg activity. Sequence analysis of corresponding cDNA(s) predicts a protein of 514 amino acids with a canonical catalytic domain found in serine/threonine specific protein kinases, linked to an unusual region rich in Gly, Ala and Ser. A search for homologies as well as a comparative study of the kinase catalytic domain with that of other proteins, revealed that the protein kinase domain of sgg is distantly related to the members of the CDC28/cdc2+ subfamily of protein kinases, all of which play cardinal roles in the regulation of the yeast and mammalian cell cycles. Ubiquitous expression of sgg transcripts was found during embryonic stages. A possible role of the sgg protein in a signal transduction pathway necessary for intercellular communication at different stages of development is discussed.

  4. Epigenetics of human myometrium: DNA methylation of genes encoding contraction-associated proteins in term and preterm labor.

    Science.gov (United States)

    Mitsuya, Kohzoh; Singh, Natasha; Sooranna, Suren R; Johnson, Mark R; Myatt, Leslie

    2014-05-01

    Preterm birth involves the interaction of societal and environmental factors potentially modulating the length of gestation via the epigenome. An established form of epigenetic regulation is DNA methylation where promoter hypermethylation is associated with gene repression. We hypothesized we would find differences in DNA methylation in the myometrium of women with preterm labor of different phenotypes versus normal term labor. Myometrial tissue was obtained at cesarean section at term with or without labor, preterm without labor, idiopathic preterm labor, and twin gestations with labor. Genomic DNA was isolated, and samples in each group were combined and analyzed on a NimbleGen 2.1M human DNA methylation array. Differences in methylation from -8 to +3 kb of transcription start sites of 22 contraction-associated genes were determined. Cytosine methylation was not present in CpG islands of any gene but was present outside of CpG islands in shores and shelves in 19 genes. No differential methylation was found across the tissue groups for six genes (PTGES3L, PTGER2, PTGER4, PTGFRN, ESR2, and GJA1). For 13 genes, differential methylation occurred in several patterns between tissue groups. We find a correlation between hypomethylation and increased mRNA expression of PTGES/mPGES-1, indicating potential functional relevance of methylation, but no such correlation for PTGS2/COX-2, suggesting other regulatory mechanisms for PTGS2 at labor. The majority of differential DNA methylation of myometrial contraction-associated genes with different labor phenotypes occurs outside of CpG islands in gene promoters, suggesting that the entirety of DNA methylation across the genome should be considered.

  5. Prognostic Significance of B-cell Differentiation Genes Encoding Proteins in Diffuse Large B-cell Lymphoma and Follicular Lymphoma Grade 3

    Science.gov (United States)

    Borovečki, Ana; Korać, Petra; Nola, Marin; Ivanković, Davor; Jakšić, Branimir; Dominis, Mara

    2008-01-01

    Aim To define prognostic significance of B-cell differentiation genes encoding proteins and BCL2 and BCL6 gene abnormalities in diffuse large B-cell lymphoma and follicular lymphoma grade 3 with >75% follicular growth pattern. Methods In 53 patients with diffuse large B-cell lymphoma and 20 patients with follicular lymphoma grade 3 with >75% follicular growth pattern the following was performed: 1) determination of protein expression of BCL6, CD10, MUM1/IRF4, CD138, and BCL2 by immunohistochemistry; 2) subclassification into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) groups according to the results of protein expression; 3) detection of t(14;18)(q32;q21)/IgH-BCL2 and BCL6 abnormalities by fluorescent in situ hybridization in diffuse large B-cell lymphoma and follicular lymphoma grade 3 with >75% follicular growth pattern as well as in GCB and ABC groups; and 4) assessment of the influence of the analyzed characteristics and clinical prognostic factors on overall survival. Results Isolated BCL6 expression was more frequently found in follicular lymphoma grade 3 with >75% follicular growth pattern than in diffuse large B-cell lymphoma (P = 0.030). There were no differences in BCL2 and BCL6 gene abnormalities between diffuse large B-cell lymphoma and follicular lymphoma grade 3 with >75% follicular growth pattern. Diffuse large B-cell lymphoma and follicular lymphoma grade 3 with >75% follicular growth pattern patients were equally distributed in GCB and ABC groups. t(14;18)(q32;q21) was more frequently recorded in GCB group, and t(14;18)(q32;q21) with BCL2 additional signals or only BCL2 and IgH additional signals in ABC group (P = 0.004). The GCB and ABC groups showed no difference in BCL6 gene abnormalities. There was no overall survival difference between the patients with diffuse large B-cell lymphoma or follicular lymphoma grade 3 with >75% follicular growth pattern, however, GCB group had longer overall survival than ABC group (P

  6. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  7. Quantification of allele-specific expression of a gene encoding strawberry polygalacturonase-inhibiting protein (PGIP) using Pyrosequencing((TM))

    NARCIS (Netherlands)

    Schaart, J.G.; Mehli, L.; Schouten, H.J.

    2005-01-01

    Recent studies indicate that allele-specific differences in gene expression are a common phenomenon. The extent to which differential allelic expression exists might be underestimated, due to the limited accuracy of the methods used so far. To demonstrate allele-specific expression, we investigated

  8. A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans.

    Science.gov (United States)

    Mink, M; Fogelgren, B; Olszewski, K; Maroy, P; Csiszar, K

    2001-06-01

    A novel human gene, SARM, encodes the orthologue of a Drosophila protein (CG7915) and contains a unique combination of the sterile alpha (SAM) and the HEAT/Armadillo motifs. The SARM gene was identified on chromosome 17q11, between markers D17S783 and D17S841 on BAC clone AC002094, which also included a HERV repeat and keratin-18-like, MAC30, TNFAIP1, HSPC017, and vitronectin genes in addition to three unknown genes. The mouse SARM gene was located on a mouse chromosome 11 BAC clone (AC002324). The SARM gene is 1.8 kb centromeric to the vitronectin gene, and the two genes share a promoter region that directs a high level of liver-specific expression of both the SARM and the vitronectin genes. In addition to the liver, the SARM gene was highly expressed in the kidney. A 0.4-kb antisense transcript was coordinately expressed with the SARM gene in the kidney and liver, while in the brain and malignant cell lines, it appeared independent of SARM gene transcription. The SARM gene encodes a protein of 690 amino acids. Based on amino acid sequence homology, we have identified a SAM motif within this derived protein. Structure modeling and protein folding recognition studies confirmed the presence of alpha-alpha right-handed superhelix-like folds consistent with the structure of the Armadillo and HEAT repeats of the beta-catenin and importin protein families. Both motifs are known to be involved in protein-protein interactions promoting the formation of diverse protein complexes. We have identified the same conserved SAM/Armadillo motif combination in the mouse, Drosophila, and Caenorhabditis elegans SARM proteins. Copyright 2001 Academic Press.

  9. Identification and Cloning of Resistance Gene Analogues (RGAs) Encoding NBS-LRR Proteins from Gossypium arboreum L.

    Institute of Scientific and Technical Information of China (English)

    AZHAR Muhammad Tehseen; BASHIR Afiab; BRIDDON Rob W; MANSOOR Shahid

    2008-01-01

    @@ Plants have developed a complicated defense mechanism during evolution to resist the harmful pathogens they encountered.The mechanism involves the interaction of the plant resistance (R) gene product with the component from the pathogen.This interaction further activates the signal transdue tion pathway,thus leading to defense responses.These defense responses include a hypersensitive response that results in localized cell death,and other general responses such as strengthening of the cell wall,formation of phytoalexins,etc.

  10. Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus.

    Science.gov (United States)

    Guthrie, Alan J; Quan, Melvyn; Lourens, Carina W; Audonnet, Jean-Christophe; Minke, Jules M; Yao, Jiansheng; He, Ling; Nordgren, Robert; Gardner, Ian A; Maclachlan, N James

    2009-07-16

    We describe the development and preliminary characterization of a recombinant canarypox virus vectored (ALVAC) vaccine for protective immunization of equids against African horse sickness virus (AHSV) infection. Horses (n=8) immunized with either of two concentrations of recombinant canarypox virus vector (ALVAC-AHSV) co-expressing synthetic genes encoding the outer capsid proteins (VP2 and VP5) of AHSV serotype 4 (AHSV-4) developed variable titres (horse immunized with a commercial recombinant canarypox virus vectored vaccine expressing the haemagglutinin genes of two equine influenza H3N8 viruses was seronegative to AHSV and following infection with virulent AHSV-4 developed pyrexia, thrombocytopenia and marked oedema of the supraorbital fossae typical of the "dikkop" or cardiac form of African horse sickness. AHSV was detected by virus isolation and quantitative reverse transcriptase polymerase chain reaction in the blood of the control horse from 8 days onwards after challenge infection whereas AHSV was not detected at any time in the blood of the ALVAC-AHSV vaccinated horses. The control horse seroconverted to AHSV by 2 weeks after challenge infection as determined by both virus neutralization and ELISA assays, whereas six of eight of the ALVAC-AHSV vaccinated horses did not seroconvert by either assay following challenge infection with virulent AHSV-4. These data confirm that the ALVAC-AHSV vaccine will be useful for the protective immunization of equids against African horse sickness, and avoids many of the problems inherent to live-attenuated AHSV vaccines.

  11. A protein kinase C-encoding gene, pkcA, is essential to the viability of the filamentous fungus Aspergillus nidulans.

    Science.gov (United States)

    Ichinomiya, Masayuki; Uchida, Hirotaka; Koshi, Yukako; Ohta, Akinori; Horiuchi, Hiroyuki

    2007-11-01

    A protein kinase C (PKC)-encoding gene (pkcA) was isolated from the filamentous fungus Aspergillus nidulans. Although we attempted to isolate pkcA deletion mutants, we obtained only heterokaryons that had both DeltapkcA and pkcA(+) nuclei. Conidia produced by the heterokaryon germinated. The germ tubes, however, lysed frequently and no colony formation was observed, indicating that the pkcA gene is essential to the viability of A. nidulans. We constructed conditional mutants (alcA(p)-pkcA mutants) that expressed pkcA under the control of the alcA promoter (alcA(p)). Under alcA(p)-repressing conditions, their colonies were smaller than those of the wild-type strains and their hyphae lysed frequently. These phenotypes were not remedied under moderate- or high-osmolarity conditions; the growth defect deteriorated further under the latter. Under alcA(p)-inducing conditions, the alcA(p)-pkcA mutants also showed growth-sensitivity to cell wall destabilizing agents. These results indicate that pkcA plays an important role in the maintenance of cell integrity.

  12. Identification of flgZ as a flagellar gene encoding a PilZ domain protein that regulates swimming motility and biofilm formation in Pseudomonas.

    Science.gov (United States)

    Martínez-Granero, Francisco; Navazo, Ana; Barahona, Emma; Redondo-Nieto, Miguel; González de Heredia, Elena; Baena, Irene; Martín-Martín, Irene; Rivilla, Rafael; Martín, Marta

    2014-01-01

    Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.

  13. Stabilization of secondary structure elements by specific combinations of hydrophilic and hydrophobic amino acid residues is more important for proteins encoded by GC-poor genes.

    Science.gov (United States)

    Khrustalev, Vladislav Victorovich; Barkovsky, Eugene Victorovich

    2012-12-01

    Stabilization of secondary structure elements by specific combinations of hydrophobic and hydrophilic amino acids has been studied by the way of analysis of pentapeptide fragments from twelve partial bacterial proteomes. PDB files describing structures of proteins from species with extremely high and low genomic GC-content, as well as with average G + C were included in the study. Amino acid residues in 78,009 pentapeptides from alpha helices, beta strands and coil regions were classified into hydrophobic and hydrophilic ones. The common propensity scale for 32 possible combinations of hydrophobic and hydrophilic amino acid residues in pentapeptide has been created: specific pentapeptides for helix, sheet and coil were described. The usage of pentapeptides preferably forming alpha helices is decreasing in alpha helices of partial bacterial proteomes with the increase of the average genomic GC-content in first and second codon positions. The usage of pentapeptides preferably forming beta strands is increasing in coil regions and in helices of partial bacterial proteomes with the growth of the average genomic GC-content in first and second codon positions. Due to these circumstances the probability of coil-sheet and helix-sheet transitions should be increased in proteins encoded by GC-rich genes making them prone to form amyloid in certain conditions. Possible causes of the described fact that importance of alpha helix and coil stabilization by specific combinations of hydrophobic and hydrophilic amino acids is growing with the decrease of genomic GC-content have been discussed.

  14. Identification of flgZ as a flagellar gene encoding a PilZ domain protein that regulates swimming motility and biofilm formation in Pseudomonas.

    Directory of Open Access Journals (Sweden)

    Francisco Martínez-Granero

    Full Text Available Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC and one phosphodiesterase (BifA implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.

  15. Identification of an E-box DNA binding protein, activated protein 4, and its function in regulating the expression of the gene encoding diapause hormone and pheromone biosynthesis-activating neuropeptide in Helicoverpa armigera.

    Science.gov (United States)

    Hu, C-H; Hong, B; Xu, W-H

    2010-04-01

    Activated protein 4 (AP-4), an E-box DNA-binding protein, was cloned from the cotton bollworm, Helicoverpa armigera (Har). The expression of Har-AP-4 mRNA and the protein that it encodes are significantly higher in nondiapause pupae than in diapause pupae. In vitro-translated Har-AP-4 can bind specifically to the E-box motif on the promoter of the diapause hormone and pheromone biosynthesis-activating neuropeptide (DH-PBAN). Har-AP-4, fused with the green fluorescent protein (GFP), is localized to the nucleus, and overexpression of Har-AP-4 can significantly activate the promoter of the DH-PBAN gene that is involved in nondiapause pupal development in H. armigera. These results suggest that Har-AP-4, which binds to the promoter of DH-PBAN, may play a role in regulating pupal development in H. armigera.

  16. An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death

    Science.gov (United States)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

  17. An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death

    Science.gov (United States)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

  18. Distinct expression, localization and function of two Rab7 proteins encoded by paralogous genes in a free-living model eukaryote.

    Science.gov (United States)

    Osińska, Magdalena; Wiejak, Jolanta; Wypych, Emilia; Bilski, Henryk; Bartosiewicz, Rafał; Wyroba, Elżbieta

    2011-01-01

    Rab7 GTPases are involved in membrane trafficking in the late endosomal/lysosomal pathway. In Paramecium octaurelia Rab7a and Rab7b are encoded by paralogous genes. Antipeptide antibodies generated against divergent C-termini recognize Rab7a of 22.5 kDa and Rab7b of 25 kDa, respectively. In 2D gel electrophoresis two immunoreactive spots were identified for Rab7b at pI about 6.34 and about 6.18 and only one spot for Rab7a of pI about 6.34 suggesting post-translational modification of Rab7b. Mass spectrometry revealed eight identical phosphorylated residues in the both proteins. ProQ Emerald staining and ConA overlay of immunoprecipitated Rab7b indicated its putative glycosylation that was further supported by a faster electrophoretic mobility of this protein upon deglycosylation. Such a post-translational modification and substitution of Ala(140) in Rab7a for Ser(140) in Rab7b may result in distinct targeting to the oral apparatus where Rab7b associates with the microtubular structures as revealed by STED confocal and electron microscopy. Rab7a was mapped to phagosomal compartment. Absolute qReal-Time PCR analysis revealed that expression of Rab7a was 2.6-fold higher than that of Rab7b. Upon latex internalization it was further 2-fold increased for Rab7a and only slightly for Rab7b. Post-transcriptional gene silencing of rab7a suppressed phagosome formation by 70 % and impaired their acidification. Ultrastructural analysis with double immunogold labeling revealed that this effect was due to the lack of V-ATPase recruitment to phagolysosomes. No significant phenotype changes were noticed in cells upon rab7b silencing. In conclusion, Rab7b acquired a new function, whereas Rab7a can be assigned to the phagolysosomal pathway.

  19. The LuWD40-1 gene encoding WD repeat protein regulates growth and pollen viability in flax (Linum Usitatissimum L.).

    Science.gov (United States)

    Kumar, Santosh; Jordan, Mark C; Datla, Raju; Cloutier, Sylvie

    2013-01-01

    As a crop, flax holds significant commercial value for its omega-3 rich oilseeds and stem fibres. Canada is the largest producer of linseed but there exists scope for significant yield improvements. Implementation of mechanisms such as male sterility can permit the development of hybrids to assist in achieving this goal. Temperature sensitive male sterility has been reported in flax but the leakiness of this system in field conditions limits the production of quality hybrid seeds. Here, we characterized a 2,588 bp transcript differentially expressed in male sterile lines of flax. The twelve intron gene predicted to encode a 368 amino acid protein has five WD40 repeats which, in silico, form a propeller structure with putative nucleic acid and histone binding capabilities. The LuWD40-1 protein localized to the nucleus and its expression increased during the transition and continued through the vegetative stages (seed, etiolated seedling, stem) while the transcript levels declined during reproductive development (ovary, anthers) and embryonic morphogenesis of male fertile plants. Knockout lines for LuWD40-1 in flax failed to develop shoots while overexpression lines showed delayed growth phenotype and were male sterile. The non-viable flowers failed to open and the pollen grains from these flowers were empty. Three independent transgenic lines overexpressing the LuWD40-1 gene had ∼80% non-viable pollen, reduced branching, delayed flowering and maturity compared to male fertile genotypes. The present study provides new insights into a male sterility mechanism present in flax.

  20. The LuWD40-1 gene encoding WD repeat protein regulates growth and pollen viability in flax (Linum Usitatissimum L..

    Directory of Open Access Journals (Sweden)

    Santosh Kumar

    Full Text Available As a crop, flax holds significant commercial value for its omega-3 rich oilseeds and stem fibres. Canada is the largest producer of linseed but there exists scope for significant yield improvements. Implementation of mechanisms such as male sterility can permit the development of hybrids to assist in achieving this goal. Temperature sensitive male sterility has been reported in flax but the leakiness of this system in field conditions limits the production of quality hybrid seeds. Here, we characterized a 2,588 bp transcript differentially expressed in male sterile lines of flax. The twelve intron gene predicted to encode a 368 amino acid protein has five WD40 repeats which, in silico, form a propeller structure with putative nucleic acid and histone binding capabilities. The LuWD40-1 protein localized to the nucleus and its expression increased during the transition and continued through the vegetative stages (seed, etiolated seedling, stem while the transcript levels declined during reproductive development (ovary, anthers and embryonic morphogenesis of male fertile plants. Knockout lines for LuWD40-1 in flax failed to develop shoots while overexpression lines showed delayed growth phenotype and were male sterile. The non-viable flowers failed to open and the pollen grains from these flowers were empty. Three independent transgenic lines overexpressing the LuWD40-1 gene had ∼80% non-viable pollen, reduced branching, delayed flowering and maturity compared to male fertile genotypes. The present study provides new insights into a male sterility mechanism present in flax.

  1. Tuberculate fruit gene Tu encodes a C2 H2 zinc finger protein that is required for the warty fruit phenotype in cucumber (Cucumis sativus L.).

    Science.gov (United States)

    Yang, Xuqin; Zhang, Weiwei; He, Huanle; Nie, Jingtao; Bie, Beibei; Zhao, Junlong; Ren, Guoliang; Li, Yue; Zhang, Dabing; Pan, Junsong; Cai, Run

    2014-06-01

    Cucumber fruits that have tubercules and spines (trichomes) are known to possess a warty (Wty) phenotype. In this study, the tuberculate fruit gene Tu was identified by map-based cloning, and was found to encode a transcription factor (TF) with a single C2 H2 zinc finger domain. Tu was identified in all 38 Wty lines examined, and was completely absent from all 56 non-warty (nWty) lines. Cucumber plants transgenic for Tu (TCP) revealed that Tu was required for the Wty fruit phenotype. Subcellular localization showed that the fusion protein GFP-Tu was localized mainly to the nucleus. Based on analyses of semi-quantitative and quantitative reverse transcription polymerase chain reaction (RT-PCR), and mRNA in situ hybridization, we found that Tu was expressed specifically in fruit spine cells during development of fruit tubercules. Moreover, cytokinin (CTK) content measurements and cytological observations in Wty and nWty fruits revealed that the Wty fruit phenotype correlated with high endogenous CTK concentrations. As a result of further analyses on the transcriptomic profile of the nWty fruit epidermis and TCP fruit warts, expression of CTK-associated genes, and hormone content in nWty fruit epidermis, Wty fruit warts and epidermis, and TCP fruit warts and epidermis, we found that Tu probably promoted CTK biosynthesis in fruit warts. Here we show that Tu could not be expressed in the glabrous and tubercule-free mutant line gl that contained Tu, this result that futher confirmed the epistatic effect of the trichome (spine) gene Gl over Tu. Taken together, these data led us to propose a genetic pathway for the Wty fruit trait that could guide future mechanistic studies.

  2. The Bradyrhizobium japonicum fegA gene encodes an iron-regulated outer membrane protein with similarity to hydroxamate-type siderophore receptors.

    Science.gov (United States)

    LeVier, K; Guerinot, M L

    1996-12-01

    Iron is important in the symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, yet little is known about rhizobial iron acquisition strategies. Analysis of outer membrane proteins (OMPs) from B. japonicum 61A152 identified three iron-regulated OMPs in the size range of several known receptors for Fe(III)-scavenging siderophores. One of the iron-regulated proteins, FegA, was purified and microsequenced, and a reverse genetics approach was used to clone a fegA-containing DNA fragment. Sequencing of this fragment revealed a single open reading frame of 750 amino acids. A putative N-terminal signal sequence of 14 amino acids which would result in a mature protein of 736 amino acids with a molecular mass of 80,851 Da was predicted. FegA shares significant amino acid similarity with several Fe(III)-siderophore receptors from gram-negative bacteria and has greater than 50% amino acid similarity and 33% amino acid identity with two [corrected] bacterial receptors for hydroxamate-type Fe(III)-siderophores. A dendrogram describing total inferred sequence similarity among 36 TonB-dependent OMPs was constructed; FegA grouped with Fe(III)-hydroxamate receptors. The transcriptional start site of fegA was mapped by primer extension analysis, and a putative Fur-binding site was found in the promoter. Primer extension and RNA slot blot analysis demonstrated that fegA was expressed only in cells grown under iron-limiting conditions. This is the first report of the cloning of a gene encoding a putative Fe(III)-siderophore receptor from nitrogen-fixing rhizobia.

  3. The Ancestral Gene for Transcribed, Low-Copy Repeats in the Prader-Willi/Angleman Region Encodes a Large Protein Implicated in Protein Trafficking that is Deficient in Mice with Neuromuscular and

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Y.

    1999-01-01

    Transcribed, low-copy repeat elements are associated with the breakpoint regions of common deletions in Prader-Willi and Angelman syndromes. We report here the identification of the ancestral gene ( HERC2 ) and a family of duplicated, truncated copies that comprise these low-copy repeats. This gene encodes a highly conserved giant protein, HERC2, that is distantly related to p532 (HERC1), a guanine nucleotide exchange factor (GEF) implicated in vesicular trafficking. The mouse genome contains a single Herc2 locus, located in the jdf2 (juvenile development and fertility-2) interval of chromosome 7C. We have identified single nucleotide splice junction mutations in Herc2 in three independent N-ethyl-N-nitrosourea-induced jdf2 mutant alleles, each leading to exon skipping with premature termination of translation and/or deletion of conserved amino acids. Therefore, mutations in Herc2 lead to the neuromuscular secretory vesicle and sperm acrosome defects, other developmental abnormalities and juvenile lethality of jdf2 mice. Combined, these findings suggest that HERC2 is an important gene encoding a GEF involved in protein trafficking and degradation pathways in the cell.

  4. The Ep152R ORF of African Swine Fever Virus strain Georgia encodes for an essential gene that interacts with host protein BAG6

    Science.gov (United States)

    African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The viral genome encodes for more than 150 genes, and only a select few have been studied in some detail. Here we rep...

  5. A Puf RNA-binding protein encoding gene PlM90 regulates the sexual and asexual life stages of the litchi downy blight pathogen Peronophythora litchii.

    Science.gov (United States)

    Jiang, Liqun; Ye, Wenwu; Situ, Junjian; Chen, Yubin; Yang, Xinyu; Kong, Guanghui; Liu, Yaya; Tinashe, Runyanga J; Xi, Pinggen; Wang, Yuanchao; Jiang, Zide

    2017-01-01

    Sexual and asexual reproduction are two key processes in the pathogenic cycle of many filamentous pathogens. However in Peronophythora litchii, the causal pathogen for the litchi downy blight disease, critical regulator(s) of sexual or asexual differentiation has not been elucidated. In this study, we cloned a gene named PlM90 from P. litchii, which encodes a putative Puf RNA-binding protein. We found that PlM90 was highly expressed during asexual development, and much higher than that during sexual development, while relatively lower during cyst germination and plant infection. By polyethylene glycol (PEG)-mediated protoplast transformation, we generated three PlM90-silenced transformants and found a severely impaired ability in sexual spore production and a delay in stages of zoospore release and encystment. However, the pathogenicity of P. litchii was not affected by PlM90-silencing. Therefore we conclude that PlM90 specifically regulates the sexual and asexual differentiation of P. litchii.

  6. Autoantibody germ-line gene segment encodes V{sub H} and V{sub L} regions of a human anti-streptococcal monoclonal antibody recognizing streptococcal M protein and human cardiac myosin epitopes

    Energy Technology Data Exchange (ETDEWEB)

    Quinn, A.; Cunningham, M.W. [Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Adderson, E.E. [Univ. of Utah, Salt Lake City, UT (United States)] [and others

    1995-04-15

    Cross-reactivity of anti-streptococcal Abs with human cardiac myosin may result in sequelae following group A streptococcal infections. Molecular mimicry between group A streptococcal M protein and cardiac myosin may be the basis for the immunologic cross-reactivity. In this study, a cross-reactive human anti-streptococcal/antimyosin mAb (10.2.3) was characterized, and the myosin epitopes were recognized by the Ab identified. mAb 10.2.3 reacted with four peptides from the light meromyosin (LMM) tail fragment of human cardiac myosin, including LMM-10 (1411-1428), LMM-23 (1580-1597), LMM-27 (1632-1649), and LMM-30 (1671-1687). Only LMM-30 inhibited binding of mAb 10.2.3 to streptococcal M protein and human cardiac myosin. Human mAb 10.2.3 labeled cytoskeletal structures within rat heart cells in indirect immunofluorescence, and reacted with group A streptococci expressing various M protein serotypes, PepM5, and recombinant M protein. The nucleotide sequence of gene segments encoding the Ig heavy and light chain V region of mAb 10.2.3 was determined. The light chain V segment was encoded by a VK1 gene segment that was 98.5% identical with germ-line gene humig{sub K}Vi5. The V segment of the heavy chain was encoded by a V{sub H}3a gene segment that differed from the V{sub H}26 germ-line gene by a single base change. V{sub H}26 is expressed preferentially in early development and encodes autoantibodies with anti-DNA and rheumatoid factor specificities. Anti-streptococcal mAb 10.2.3 is an autoantibody encoded by V{sub H} and V{sub L} genes, with little or no somatic mutation. 63 refs., 11 figs.

  7. The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of "upstream" Planar Cell Polarity proteins.

    Science.gov (United States)

    Wang, Ying; Yan, Jie; Lee, Haeryun; Lu, Qiuheng; Adler, Paul N

    2014-10-01

    The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.

  8. Differential Translation Tunes Uneven Production of Operon-Encoded Proteins

    Directory of Open Access Journals (Sweden)

    Tessa E.F. Quax

    2013-09-01

    Full Text Available Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.

  9. Water deficit modulates gene expression in growing zones of soybean seedlings. Analysis of differentially expressed cDNAs, a new beta-tubulin gene, and expression of genes encoding cell wall proteins.

    Science.gov (United States)

    Creelman, R A; Mullet, J E

    1991-10-01

    Transfer of soybean seedlings to low-water-potential vermiculite (psi w = -0.3 MPa) results in a reversible decrease in hypocotyl growth and modulation of several polysomal mRNAs (Plant Physiol 92: 205-214). We report here the isolation of two cDNA clones (pGE16 and pGE95) which correspond to genes whose mRNA levels are increased, and one cDNA clone (pGE23) which corresponds to a gene whose mRNA level is decreased in the hypocotyl zone of cell elongation by water deficit. In well-watered seedlings mRNAs hybridizing to pGE16 and pGE95 are most abundant in mature regions of the seedling, but in water-deficient seedlings mRNA levels are reduced in mature regions and enhanced in elongating regions. RNA corresponding to soybean proline-rich protein 1 (sbPRP1) shows a similar tissue distribution and response to water deficit. In contrast, in well-watered seedlings, the gene corresponding to pGE23 was highly expressed in the hypocotyl and root growing zones. Transfer of seedlings to low-water-potential vermiculite caused a rapid decrease in mRNA hybridizing to pGE23. Sequence analysis revealed that pGE23 has high homology with beta-tubulin. Water deficit also reduced the level of mRNA hybridizing to JCW1, an auxin-modulated gene, although with different kinetics. Furthermore, mRNA encoding actin, glycine-rich proteins (GRPs), and hydroxyproline-rich glycoproteins (HRGPs) were down-regulated in the hypocotyl zone of elongation of seedlings exposed to water deficit. No effect of water deficit was observed on the expression of chalcone synthase. Decreased expression of beta-tubulin, actin, JCW1, HRGP and GRP and increased expression of sbPRP1, pGE95 and pGE16 in the hypocotyl zone of cell elongation could participate in the reversible growth inhibition observed in water-deficient soybean seedlings.

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

    Directory of Open Access Journals (Sweden)

    Hui-Yeng Y Yap

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

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

    Science.gov (United States)

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

    2015-01-01

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

  12. Identification and use of genes encoding amatoxin and phallotoxin

    Energy Technology Data Exchange (ETDEWEB)

    Hallen, Heather E.; Walton, Jonathan D.; Luo, Hong; Scott-Craig, John S.

    2016-12-13

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptide toxins and toxin production in mushrooms. In particular, the present invention relates to using genes and proteins from Amanita species encoding Amanita peptides, specifically relating to amatoxins and phallotoxins. In a preferred embodiment, the present invention also relates to methods for detecting Amanita peptide toxin genes for identifying Amanita peptide-producing mushrooms and for diagnosing suspected cases of mushroom poisoning. Further, the present inventions relate to providing kits for diagnosing and monitoring suspected cases of mushroom poisoning in patients.

  13. Two distinct genotypes of prtF2, encoding a fibronectin binding protein, and evolution of the gene family in Streptococcus pyogenes.

    Science.gov (United States)

    Ramachandran, V; McArthur, J D; Behm, C E; Gutzeit, C; Dowton, M; Fagan, P K; Towers, R; Currie, B; Sriprakash, K S; Walker, M J

    2004-11-01

    The group A Streptococcus (GAS) is an important pathogen that is responsible for a wide range of human diseases. Fibronectin binding proteins (FBPs) play an important role in promoting GAS adherence and invasion of host cells. The prtF2 gene encodes an FBP and is present in approximately 60% of GAS strains. In the present study we examined 51 prtF2-positive GAS strains isolated from the Northern Territory of Australia, and here we describe two genotypes of prtF2 which are mutually exclusive. The two genotypes have been identified previously as pfbp and fbaB. We show that these genotypes map to the same chromosomal location within the highly recombinatorial fibronectin-collagen-T antigen (FCT) locus, indicating that they arose from a common ancestor, and in this study these genotypes were designated the pfbp type and the fbaB type. Phylogenetic analysis of seven pfbp types, 14 fbaB types, and 11 prtF2-negative GAS strains by pulsed-field gel electrophoresis (PFGE) produced 32 distinct PFGE patterns. Interpretation of evolution based on the PFGE dendrogram by parsimony suggested that the pfbp type had a recent origin compared to the fbaB type. A comparison of multiple DNA sequences of the pfbp and fbaB types revealed a mosaic pattern for the amino-terminal region of the pfbp types. The fbaB type is generally conserved at the amino terminus but varies in the number of fibronectin binding repeats in the carboxy terminus. Our data also suggest that there is a possible association of the pfbp genotype with sof (84.2%), while the fbaB genotype was found in a majority of the GAS strains negative for sof (90.6%), indicating that these two prtF2 subtypes may be under different selective pressures.

  14. Polynucleotides encoding TRF1 binding proteins

    Science.gov (United States)

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  15. The Escherichia coli MntR Miniregulon Includes Genes Encoding a Small Protein and an Efflux Pump Required for Manganese Homeostasis ▿†

    OpenAIRE

    Waters, Lauren S.; Sandoval, Melissa; Storz, Gisela

    2011-01-01

    Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA...

  16. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

    NARCIS (Netherlands)

    Punt, P.J.; Gemeren, I.A. van; Drint-Kuijvenhoven, J.; Hessing, J.G.M.; Muijlwijk van - Harteveld, G.M.; Beijersbergen, A.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    1998-01-01

    The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bip

  17. Structure of the gene encoding columbid annexin Icp35.

    Science.gov (United States)

    Hitti, Y S; Horseman, N D

    1991-07-22

    The cp35 gene, encoding an annexin I (AnxI) cropsac 35-kDa protein (cp35) from the pigeon, consists of 13 exons and twelve introns. The borders of exons 2-13 were mapped by comparison with the known cDNA sequence. A 5-kb sequence containing exons 1, 2, and 3, and 1.4 kb of 5'-flanking DNA, is presented. The transcription start point was mapped by S1 nuclease protection. The region of the cp35 mRNA sequence, which we had previously shown to be profoundly different from mammalian anxI, is located in the first half of exon 3. Whereas human anxI is known to be single copy, Southern analysis of pigeon genomic DNA and genomic clones demonstrated multiple anxI genes in the pigeon, diverging significantly in their 5'-termini. Pigeon vimentin, on the other hand, is encoded by a single-copy gene as it is in other birds and mammals. These experiments have demonstrated that the cp35 mRNA is transcribed from its individual gene and is not a product of alternative processing of the pigeon homolog of mammalian anxI. We speculate that the diversification of anxI genes in Columbid birds allowed the recruitment of one of these genes (cp35) for unique regulation by prolactin in the absence of post-translational regulation via residues encoded by exons 2 and 3.

  18. Cloning and Characterization of a Novel cDNA Encoding Late Embryogenesis-Abundant Protein 5 Like (LEA-5) Gene from Cara Cara Navel Orange Fruit(Citrus sinensis Osbeck)

    Institute of Scientific and Technical Information of China (English)

    TAO Neng-guo; YE Jun-li; XU Juan; DENG Xiu-xin

    2006-01-01

    LEA5 gene was postulated related with both stress and hormone responses. In an attempt to find genes exclusively expressed during fruit ripening of Cara Cara navel orange, a novel cDNA clone encoding late embryogenesis-abundant protein 5 like gene (CitLEA5-1) was obtained. It was 582 bp in length, containing 97 deduced amino acids. Compared with the stress-induced LEA5 from leaves of Citrus sinensis, CitLEA5-1 had a shorter 3' untranslated region (UTR). Semiquantitative RT-PCR analysis revealed that CitLEA5-1 was transcriptional regulated during fruit ripening of Cara Cara navel orange.

  19. Chromosomal localization of the human gene encoding c-myc promoter-binding protein (MPB1) to chromosome 1p35-pter

    Energy Technology Data Exchange (ETDEWEB)

    White, R.A.; Dowler, L.L. [Univ. of Missouri, Kansas City, MO (United States); Adkison, L.R. [Mercer Univ. School of Medicine, Macon, GA (United States); Ray, R.B. [St. Louis Univ. Health Sciences Center, St. Louis, MO (United States)

    1997-02-01

    We report the mapping of the human gene MPB1 (c-myc promoter binding protein), a recently identified gene regulatory protein. MPB1 binds to the c-myc P2 promoter and exerts a negative regulatory role on c-myc transcription. Since exogenous expression from transfection of the MPB1 gene suppresses the tumorigenic property of breast cancer cells, there was interest in determining the chromosomal location of this gene. The human MPB1 gene was assigned to human chromosome 1p35-pter using Southern blot analyses of genomic DNAs from rodent-human somatic hybrid cell lines. A specific human genomic fragment was observed only in the somatic cell lines containing human chromosome 1 or the p35-pter region of the chromosome. 10 refs., 2 figs.

  20. Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments

    Energy Technology Data Exchange (ETDEWEB)

    Cotmore, S.F.; McKie, V.C.; Anderson, L.J.; Astell, C.R.; Tattersall, P.

    1986-11-01

    Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights for 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus.

  1. Characterization of the MSMEG_2631 gene (mmp) encoding a multidrug and toxic compound extrusion (MATE) family protein in Mycobacterium smegmatis and exploration of its polyspecific nature using biolog phenotype microarray.

    Science.gov (United States)

    Mishra, Mukti Nath; Daniels, Lacy

    2013-04-01

    In Mycobacterium, multidrug efflux pumps can be associated with intrinsic drug resistance. Comparison of putative mycobacterial transport genes revealed a single annotated open reading frame (ORF) for a multidrug and toxic compound extrusion (MATE) family efflux pump in all sequenced mycobacteria except Mycobacterium leprae. Since MATE efflux pumps function as multidrug efflux pumps by conferring resistance to structurally diverse antibiotics and DNA-damaging chemicals, we studied this gene (MSMEG_2631) in M. smegmatis mc(2)155 and determined that it encodes a MATE efflux system that contributes to intrinsic resistance of Mycobacterium. We propose that the MSMEG_2631 gene be named mmp, for mycobacterial MATE protein. Biolog Phenotype MicroArray data indicated that mmp deletion increased susceptibility for phleomycin, bleomycin, capreomycin, amikacin, kanamycin, cetylpyridinium chloride, and several sulfa drugs. MSMEG_2619 (efpA) and MSMEG_3563 mask the effect of mmp deletion due to overlapping efflux capabilities. We present evidence that mmp is a part of an MSMEG_2626-2628-2629-2630-2631 operon regulated by a strong constitutive promoter, initiated from a single transcription start site. All together, our results show that M. smegmatis constitutively encodes an Na(+)-dependent MATE multidrug efflux pump from mmp in an operon with putative genes encoding proteins for apparently unrelated functions.

  2. The Schizosaccharomyces pombe map1 gene encodes an SRF/MCM1-related protein required for P-cell specific gene expression

    DEFF Research Database (Denmark)

    Nielsen, O; Friis, T; Kjaerulff, S

    1996-01-01

    Cells of Schizosaccharomyces pombe undergo mating and meiosis when starved for a nitrogen source. In this process a P and and M cell first mate to generate a diploid zygote, which subsequently enters meiosis and sporulates. The P mating type is controlled by the mat1-Pc gene at the mating type lo...

  3. An approach to the production of soluble protein from a fungal gene encoding an aggregation-prone xylanase in Escherichia coli.

    Science.gov (United States)

    Le, Yilin; Peng, Jingjing; Wu, Huawei; Sun, Jianzhong; Shao, Weilan

    2011-04-08

    The development of new procedures and protocols that allow researchers to obtain recombinant proteins is of fundamental importance in the biotechnology field. A strategy was explored to overcome inclusion-body formation observed when expressing an aggregation-prone fungal xylanase in Escherichia coli. pHsh is an expression plasmid that uses a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (σ(32)). A derivative of pHsh was constructed by fusing a signal peptide to xynA2 gene to facilitate export of the recombinant protein to the periplasm. The xylanase was produced in a soluble form. Three factors were essential to achieving such soluble expression of the xylanase: 1) the target gene was under the control of the Hsh promoter, 2) the gene product was exported into the periplasm, and 3) gene expression was induced by a temperature upshift. For the first time we report the expression of periplasmic proteins under the control of an Hsh promoter regulated by σ(32). One unique feature of this approach was that over 200 copies of the Hsh promoter in an E. coli cell significantly increased the concentration of σ(32). The growth inhibition of the recombinant cells corresponded to an increase in the levels of soluble periplasmic protein. Therefore, an alternative protocol was designed to induce gene expression from pHsh-ex to obtain high levels of active soluble enzymes.

  4. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    Energy Technology Data Exchange (ETDEWEB)

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. (National Institutes of Health, Bethesda, MD (USA))

    1990-08-01

    We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

  5. Immunochemical method for detection of antibody against HTLV-III core protein based upon recombinant HTLV-III gag gene encoded protein

    Energy Technology Data Exchange (ETDEWEB)

    Chang, N.T.; Ghrayeb, J.

    1989-02-28

    A method is described of detecting antibody against HTLV-III core protein in a biological fluid, comprising the steps of: a. providing an antigen immunoadsorbent comprising a solid phase to which is attached a HTLV-III core antigen which is a chimeric antigen comprising an amino acid sequence beginning at amino acid number 1 through 99, and extending to amino acid number 228, the chimeric antigen being immunoreactive with antibody against HTLV-III core protein; b. incubating the immunoadsorbent with a sample of the biological fluid to be tested under conditions which allow antibody in the sample to complex with the antigen immunoadsorbent; c. separating the immmunoadsorbent from the sample; and d. determining antibody bound to the iuumoadsorbent as an indication of antibody against HTLV-III core protein in the sample.

  6. windbeutel, a gene required for dorsoventral patterning in Drosophila, encodes a protein that has homologies to vertebrate proteins of the endoplasmic reticulum

    OpenAIRE

    Konsolaki, Mary; Schüpbach, Trudi

    1998-01-01

    The formation of the dorsoventral axis of the Drosophila embryo depends on cell–cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The gene windbeutel functions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilizati...

  7. Identification of two Arabidopsis genes encoding a peroxisomal oxidoreductase-like protein and an acyl-CoA synthetase-like protein that are required for responses to pro-auxins.

    Science.gov (United States)

    Wiszniewski, Andrew A G; Zhou, Wenxu; Smith, Steven M; Bussell, John D

    2009-03-01

    Indole-3-butyric acid (IBA) and 2,4-dichlorophenoxybutyric acid (2,4-DB) are metabolised by peroxisomal beta-oxidation to active auxins that inhibit root growth. We screened Arabidopsis mutants for resistance to IBA and 2,4-DB and identified two new 2,4-DB resistant mutants. The mutant genes encode a putative oxidoreductase (SDRa) and a putative acyl-activating enzyme (AAE18). Both proteins are localised to peroxisomes. SDRa is coexpressed with core beta-oxidation genes, but germination, seedling growth and the fatty acid profile of sdra seedlings are indistinguishable from wild type. The sdra mutant is also resistant to IBA, but aae18 is not. AAE18 is the first example of a gene required for response to 2,4-DB but not IBA. The closest relative of AAE18 is AAE17. AAE17 is predicted to be peroxisomal, but an aae17 aae18 double mutant responded similarly to aae18 for all assays. We propose that AAE18 is capable of activating 2,4-DB but IBA activating enzymes remain to be discovered. We present an updated model for peroxisomal pro-auxin metabolism in Arabidopsis that includes SDRa and AAE18.

  8. Genes optimized by evolution for accurate and fast translation encode in Archaea and Bacteria a broad and characteristic spectrum of protein functions

    Directory of Open Access Journals (Sweden)

    Merkl Rainer

    2010-11-01

    Full Text Available Abstract Background In many microbial genomes, a strong preference for a small number of codons can be observed in genes whose products are needed by the cell in large quantities. This codon usage bias (CUB improves translational accuracy and speed and is one of several factors optimizing cell growth. Whereas CUB and the overrepresentation of individual proteins have been studied in detail, it is still unclear which high-level metabolic categories are subject to translational optimization in different habitats. Results In a systematic study of 388 microbial species, we have identified for each genome a specific subset of genes characterized by a marked CUB, which we named the effectome. As expected, gene products related to protein synthesis are abundant in both archaeal and bacterial effectomes. In addition, enzymes contributing to energy production and gene products involved in protein folding and stabilization are overrepresented. The comparison of genomes from eleven habitats shows that the environment has only a minor effect on the composition of the effectomes. As a paradigmatic example, we detailed the effectome content of 37 bacterial genomes that are most likely exposed to strongest selective pressure towards translational optimization. These effectomes accommodate a broad range of protein functions like enzymes related to glycolysis/gluconeogenesis and the TCA cycle, ATP synthases, aminoacyl-tRNA synthetases, chaperones, proteases that degrade misfolded proteins, protectants against oxidative damage, as well as cold shock and outer membrane proteins. Conclusions We made clear that effectomes consist of specific subsets of the proteome being involved in several cellular functions. As expected, some functions are related to cell growth and affect speed and quality of protein synthesis. Additionally, the effectomes contain enzymes of central metabolic pathways and cellular functions sustaining microbial life under stress situations. These

  9. Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

    Indian Academy of Sciences (India)

    Girdhar K Pandey; Amita Pandey; Vanga Siva Reddy; Renu Deswal; Alok Bhattacharya; Kailash C Upadhyaya; Sudhir K Sopory

    2007-03-01

    Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants.

  10. The Arabidopsis gene YS1 encoding a DYW protein is required for editing of rpoB transcripts and the rapid development of chloroplasts during early growth.

    Science.gov (United States)

    Zhou, Wenbin; Cheng, Yuxiang; Yap, Aaron; Chateigner-Boutin, Anne-Laure; Delannoy, Etienne; Hammani, Kamel; Small, Ian; Huang, Jirong

    2009-04-01

    Virescence, a phenotype in which leaves green more slowly than usual, is recognized to play a role in protection from photo-oxidative damage before healthy chloroplasts are developed. The elucidation of the molecular mechanisms underlying virescence will provide insights into how the development of chloroplasts is controlled. In this study, we find that knockout alleles of Yellow Seedlings 1 (YS1) in Arabidopsis lead to a virescent phenotype, which disappears by 3 weeks after germination. The ys1 mutation resulted in marked decreases in photosynthetic capacity and photosynthetic pigment complexes, and disturbed ultrastructure of thylakoid membranes in 8-day-old seedlings. However, cotyledons of ys1 seedlings pre-treated in the dark for 5 days turn green almost as fast as the wild type in light, revealing that the developmental defects in ys1 are limited to the first few days after germination. Inspection of all known plastid RNA editing and splicing events revealed that YS1 is absolutely required for editing of site 25992 in rpoB transcripts encoding the beta subunit of the plastid-encoded RNA polymerase (PEP). YS1 is a nuclear-encoded chloroplast-localized pentatricopeptide repeat protein differing from previously described editing factors in that it has a C-terminal DYW motif. A defect in PEP activity is consistent with the changes in plastid transcript patterns observed in ys1 seedlings. We conclude that the activity of PEP containing RpoB translated from unedited transcripts is insufficient to support rapid chloroplast differentiation. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

  11. Chronic intermittent hypoxia increases encoding pigment epithelium-derived factor gene expression, although not that of the protein itself, in the temporal cortex of rats,

    Directory of Open Access Journals (Sweden)

    Guilherme Silva Julian

    2015-02-01

    Full Text Available Objective: Obstructive sleep apnea syndrome is mainly characterized by intermittent hypoxia (IH during sleep, being associated with several complications. Exposure to IH is the most widely used animal model of sleep apnea, short-term IH exposure resulting in cognitive and neuronal impairment. Pigment epithelium-derived factor (PEDF is a hypoxia-sensitive factor acting as a neurotrophic, neuroprotective, and antiangiogenic agent. Our study analyzed performance on learning and cognitive tasks, as well as PEDF gene expression and PEDF protein expression in specific brain structures, in rats exposed to long-term IH. Methods: Male Wistar rats were exposed to IH (oxygen concentrations of 21-5% for 6 weeks-the chronic IH (CIH group-or normoxia for 6 weeks-the control group. After CIH exposure, a group of rats were allowed to recover under normoxic conditions for 2 weeks (the CIH+N group. All rats underwent the Morris water maze test for learning and memory, PEDF gene expression and PEDF protein expression in the hippocampus, frontal cortex, and temporal cortex being subsequently assessed. Results: The CIH and CIH+N groups showed increased PEDF gene expression in the temporal cortex, PEDF protein expression remaining unaltered. PEDF gene expression and PEDF protein expression remained unaltered in the frontal cortex and hippocampus. Long-term exposure to IH did not affect cognitive function. Conclusions: Long-term exposure to IH selectively increases PEDF gene expression at the transcriptional level, although only in the temporal cortex. This increase is probably a protective mechanism against IH-induced injury.

  12. Early host cell reactivation of an oxidatively damaged adenovirus-encoded reporter gene requires the Cockayne syndrome proteins CSA and CSB.

    Science.gov (United States)

    Leach, Derrik M; Rainbow, Andrew J

    2011-03-01

    Reduced host cell reactivation (HCR) of a reporter gene containing 8-oxoguanine (8-oxoG) lesions in Cockayne syndrome (CS) fibroblasts has previously been attributed to increased 8-oxoG-mediated inhibition of transcription resulting from a deficiency in repair. This interpretation has been challenged by a report suggesting reduced expression from an 8-oxoG containing reporter gene occurs in all cells by a mechanism involving gene inactivation by 8-oxoG DNA glycosylase and this inactivation is strongly enhanced in the absence of the CS group B (CSB) protein. The observation of reduced gene expression in the absence of CSB protein led to speculation that decreased HCR in CS cells results from enhanced gene inactivation rather than reduced gene reactivation. Using an adenovirus-based β-galactosidase (β-gal) reporter gene assay, we have examined the effect of methylene blue plus visible light (MB + VL)-induced 8-oxoG lesions on the time course of gene expression in normal and CSA and CSB mutant human SV40-transformed fibroblasts, repair proficient and CSB mutant Chinese hamster ovary (CHO) cells and normal mouse embryo fibroblasts. We demonstrate that MB + VL treatment of the reporter leads to reduced expression of the damaged β-gal reporter relative to control at early time points following infection in all cells, consistent with in vivo inhibition of RNA polII-mediated transcription. In addition, we have demonstrated HCR of reporter gene expression occurs in all cell types examined. A significant reduction in the rate of gene reactivation in human SV40-transformed cells lacking functional CSA or CSB compared to normal cells was found. Similarly, a significant reduction in the rate of reactivation in CHO cells lacking functional CSB (CHO-UV61) was observed compared to the wild-type parental counterpart (CHO-AA8). The data presented demonstrate that expression of an oxidatively damaged reporter gene is reactivated over time and that CSA and CSB are required for

  13. Identification of β-haemolysin-encoding genes in Streptococcus anginosus.

    Science.gov (United States)

    Asam, D; Mauerer, S; Walheim, E; Spellerberg, B

    2013-08-01

    Streptococcus anginosus is an emerging pathogen, but little is known about its virulence factors. To detect the genes responsible for β-haemolysis we performed genomic mutagenesis of the β-haemolytic S. anginosus type strain ATCC 12395 using the vector pGhost9:ISS1. Integration site analysis of 15 non-haemolytic mutants identified a gene cluster with high homology to the genes of the streptolysin S (SLS) encoding sag gene cluster of S. pyogenes. The gene cluster harbours 10 open reading frames displaying significant similarities to the S. pyogenes genes sagA-sagI, with the identities on protein level ranging from 38 to 87%. Complementation assays of S. anginosus sagB and sagD integration mutants with the respective genes confirmed their importance for β-haemolysin production and suggest the presence of post-translational modifications in S. anginosus SLS similar to SLS of S. pyogenes. Characterization of the S. anginosus haemolysin in comparison to the S. pyogenes SLS showed that the haemolysin is surface bound, but in contrast to S. pyogenes neither fetal calf serum nor RNA was able to stabilize the haemolysin of S. anginosus in culture supernatants. Inhibition of β-haemolysis by polyethylene glycol of different sizes was carried out, giving no evidence of a pore-forming haemolytic mechanism. Analysis of a whole genome shotgun sequence of Streptococcus constellatus, a closely related streptococcal species that belongs to the S. anginosus group, revealed a similar sag gene cluster. Employing a genomic mutagenesis strategy we were able to determine an SLS encoding gene cluster in S. anginosus and demonstrate its importance for β-haemolysin production in S. anginosus.

  14. Properties of virion transactivator proteins encoded by primate cytomegaloviruses

    Directory of Open Access Journals (Sweden)

    Barry Peter A

    2009-05-01

    Full Text Available Abstract Background Human cytomegalovirus (HCMV is a betaherpesvirus that causes severe disease in situations where the immune system is immature or compromised. HCMV immediate early (IE gene expression is stimulated by the virion phosphoprotein pp71, encoded by open reading frame (ORF UL82, and this transactivation activity is important for the efficient initiation of viral replication. It is currently recognized that pp71 acts to overcome cellular intrinsic defences that otherwise block viral IE gene expression, and that interactions of pp71 with the cell proteins Daxx and ATRX are important for this function. A further property of pp71 is the ability to enable prolonged gene expression from quiescent herpes simplex virus type 1 (HSV-1 genomes. Non-human primate cytomegaloviruses encode homologs of pp71, but there is currently no published information that addresses their effects on gene expression and modes of action. Results The UL82 homolog encoded by simian cytomegalovirus (SCMV, strain Colburn, was identified and cloned. This ORF, named S82, was cloned into an HSV-1 vector, as were those from baboon, rhesus monkey and chimpanzee cytomegaloviruses. The use of an HSV-1 vector enabled expression of the UL82 homologs in a range of cell types, and permitted investigation of their abilities to direct prolonged gene expression from quiescent genomes. The results show that all UL82 homologs activate gene expression, and that neither host cell type nor promoter target sequence has major effects on these activities. Surprisingly, the UL82 proteins specified by non-human primate cytomegaloviruses, unlike pp71, did not direct long term expression from quiescent HSV-1 genomes. In addition, significant differences were observed in the intranuclear localization of the UL82 homologs, and in their effects on Daxx. Strikingly, S82 mediated the release of Daxx from nuclear domain 10 substructures much more rapidly than pp71 or the other proteins tested. All

  15. Recombinant vectors construction for cellobiohydrolase encoding gene constitutive expression

    Directory of Open Access Journals (Sweden)

    Leontina GURGU

    2012-12-01

    Full Text Available Cellobiohydrolases (EC 3.2.1.91 are important exo enzymes involved in cellulose hydrolysis alongside endoglucanases (EC 3.2.1.4 and β-glucosidases (EC 3.2.1.21. Heterologous cellobiohydrolase gene expression under constitutive promoter control using Saccharomyces cerevisiae as host system is of great importance for a successful SSF process. From this point of view, the main objective of the work was to use Yeplac181 expression vector as a recipient for cellobiohdrolase - cbhB encoding gene expression under the control of the actin promoter, in Saccharomyces cerevisiae. Two hybridvectors, YEplac-Actp and YEplac-Actp-CbhB, were generated usingEscherichia coli XLI Blue for the cloning experiments. Constitutive cbhB gene expression was checked by proteine gel electrophoresis (SDS-PAGE after insertion of these constructs into Saccharomyces cerevisiae.

  16. Characterization of a novel mouse gene encoding an SYCP3-like protein that relocalizes from the XY body to the nucleolus during prophase of male meiosis I.

    Science.gov (United States)

    Tsutsumi, Makiko; Kogo, Hiroshi; Kowa-Sugiyama, Hiroe; Inagaki, Hidehito; Ohye, Tamae; Kurahashi, Hiroki

    2011-07-01

    Xlr6 is a novel but uncharacterized X-linked gene that is upregulated in meiotic prophase I during mouse spermatogenesis. Xlr6 belongs to the Xlr gene family, which includes a component of the axial/lateral element of the synaptonemal complex, Sycp3, and its transcripts are abundant in the fetal ovary and adult testis. Immunostaining and Western blot analysis demonstrate a diffuse localization pattern for this protein in the nucleus and an association with chromatin during the leptotene and zygotene stages. In males, XLR6 accumulates at the XY body of early pachytene to midpachytene spermatocytes, although the Xlr6 gene is subjected to meiotic sex chromosome inactivation. During the late pachytene and diplotene stages, the XLR6 protein relocalizes from the XY body to the nucleolus and, eventually, disappears by diakinesis. In females, XLR6 disappears at the pachytene stage, whereas it accumulates at the unpaired chromosomes occasionally observed in wild-type female mice. Although the amino acid sequence of XLR6 has a high similarity with SYCP3, its distinct localization pattern and dynamism suggest a unique chromatin modification function that leads to the transcriptional repression of ribosomal DNA in addition to sex chromosome genes.

  17. clk1, a serine/threonine protein kinase-encoding gene, is involved in pathogenicity of Colletotrichum lindemuthianum on common bean.

    Science.gov (United States)

    Dufresne, M; Bailey, J A; Dron, M; Langin, T

    1998-02-01

    A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.

  18. The milkweed pod1 Gene Encodes a KANADI Protein That Is Required for Abaxial/Adaxial Patterning in Maize Leaves[W

    Science.gov (United States)

    Candela, Héctor; Johnston, Robyn; Gerhold, Abigail; Foster, Toshi; Hake, Sarah

    2008-01-01

    Leaf primordia initiate from the shoot apical meristem with inherent polarity; the adaxial side faces the meristem, while the abaxial side faces away from the meristem. Adaxial/abaxial polarity is thought to be necessary for laminar growth of leaves, as mutants lacking either adaxial or abaxial cell types often develop radially symmetric lateral organs. The milkweed pod1 (mwp1) mutant of maize (Zea mays) has adaxialized sectors in the sheath, the proximal part of the leaf. Ectopic leaf flaps develop where adaxial and abaxial cell types juxtapose. Ectopic expression of the HD-ZIPIII gene rolled leaf1 (rld1) correlates with the adaxialized regions. Cloning of mwp1 showed that it encodes a KANADI transcription factor. Double mutants of mwp1-R with a microRNA-resistant allele of rld1, Rld1-N1990, show a synergistic phenotype with polarity defects in sheath and blade and a failure to differentiate vascular and photosynthetic cell types in the adaxialized sectors. The sectored phenotype and timing of the defect suggest that mwp1 is required late in leaf development to maintain abaxial cell fate. The phenotype of mwp1; Rld1 double mutants shows that both genes are also required early in leaf development to delineate leaf margins as well as to initiate vascular and photosynthetic tissues. PMID:18757553

  19. Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.

    Science.gov (United States)

    Tiner, Bethany L; Sha, Jian; Kirtley, Michelle L; Erova, Tatiana E; Popov, Vsevolod L; Baze, Wallace B; van Lier, Christina J; Ponnusamy, Duraisamy; Andersson, Jourdan A; Motin, Vladimir L; Chauhan, Sadhana; Chopra, Ashok K

    2015-04-01

    Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a

  20. Regulation of the rplY gene encoding 5S rRNA binding protein L25 in Escherichia coli and related bacteria.

    Science.gov (United States)

    Aseev, Leonid V; Bylinkina, Natalia S; Boni, Irina V

    2015-05-01

    Ribosomal protein (r-protein) L25 is one of the three r-proteins (L25, L5, L18) that interact with 5S rRNA in eubacteria. Specific binding of L25 with a certain domain of 5S r-RNA, a so-called loop E, has been studied in detail, but information about regulation of L25 synthesis has remained totally lacking. In contrast to the rplE (L5) and rplR (L18) genes that belong to the polycistronic spc-operon and are regulated at the translation level by r-protein S8, the rplY (L25) gene forms an independent transcription unit. The main goal of this work was to study the regulation of the rplY expression in vivo. We show that the rplY promoter is down-regulated by ppGpp and its cofactor DksA in response to amino acid starvation. At the level of translation, the rplY expression is subjected to the negative feedback control. The 5'-untranslated region of the rplY mRNA comprises specific sequence/structure features, including an atypical SD-like sequence, which are highly conserved in a subset of gamma-proteobacterial families. Despite the lack of a canonical SD element, the rplY'-'lacZ single-copy reporter showed unusually high translation efficiency. Expression of the rplY gene in trans decreased the translation yield, indicating the mechanism of autogenous repression. Site-directed mutagenesis of the rplY 5' UTR revealed an important role of the conserved elements in the translation control. Thus, the rplY expression regulation represents one more example of regulatory pathways that control ribosome biogenesis in Escherichia coli and related bacteria. © 2015 Aseev et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  1. Two Genes Encoding Uracil Phosphoribosyltransferase Are Present in Bacillus subtilis

    DEFF Research Database (Denmark)

    Martinussen, Jan; Glaser, Philippe; Andersen, Paal S.

    1995-01-01

    Uracil phosphoribosyltransferase (UPRTase) catalyzes the key reaction in the salvage of uracil in many microorganisms. Surprisingly, two genes encoding UPRTase activity were cloned from Bacillus subtilis by complementation of an Escherichia coli mutant. The genes were sequenced, and the putative...

  2. PFP1, a gene encoding an Epc-N domain-containing protein, is essential for pathogenicity of the barley pathogen Rhynchosporium commune.

    Science.gov (United States)

    Siersleben, Sylvia; Penselin, Daniel; Wenzel, Claudia; Albert, Sylvie; Knogge, Wolfgang

    2014-08-01

    Scald caused by Rhynchosporium commune is an important foliar disease of barley. Insertion mutagenesis of R. commune generated a nonpathogenic fungal mutant which carries the inserted plasmid in the upstream region of a gene named PFP1. The characteristic feature of the gene product is an Epc-N domain. This motif is also found in homologous proteins shown to be components of histone acetyltransferase (HAT) complexes of fungi and animals. Therefore, PFP1 is suggested to be the subunit of a HAT complex in R. commune with an essential role in the epigenetic control of fungal pathogenicity. Targeted PFP1 disruption also yielded nonpathogenic mutants which showed wild-type-like growth ex planta, except for the occurrence of hyphal swellings. Complementation of the deletion mutants with the wild-type gene reestablished pathogenicity and suppressed the hyphal swellings. However, despite wild-type-level PFP1 expression, the complementation mutants did not reach wild-type-level virulence. This indicates that the function of the protein complex and, thus, fungal virulence are influenced by a position-affected long-range control of PFP1 expression. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. The nucleotide sequence of a CpG island demonstrates the presence of the first exon of the gene encoding the human lysosomal membrane protein lamp2 and assigns the gene to Xq24.

    Science.gov (United States)

    Manoni, M; Tribioli, C; Lazzari, B; DeBellis, G; Patrosso, C; Pergolizzi, R; Pellegrini, M; Maestrini, E; Rivella, S; Vezzoni, P

    1991-03-01

    An EagI-EcoRI clone of human genomic DNA, p2-7, mapped to Xq24 has been sequenced. This analysis has confirmed the presence of a CpG island and has identified the first exon of the human LAMP2 gene, encoding a glycoprotein of the lysosomal membrane. Since the p2-7 clone corresponds to single-copy DNA, we can assign the human LAMP2 gene to Xq24.

  4. Tracing the origin and evolution of plant TIR-encoding genes.

    Science.gov (United States)

    Sun, Xiaoqin; Pang, Hui; Li, Mimi; Chen, Jianqun; Hang, Yueyu

    2014-08-10

    Toll-interleukin-1 receptor (TIR)-encoding proteins represent one of the most important families of disease resistance genes in plants. Studies that have explored the functional details of these genes tended to focus on only a few limited groups; the origin and evolutionary history of these genes were therefore unclear. In this study, focusing on the four principal groups of TIR-encoding genes, we conducted an extensive genome-wide survey of 32 fully sequenced plant genomes and Expressed Sequence Tags (ESTs) from the gymnosperm Pinus taeda and explored the origins and evolution of these genes. Through the identification of the TIR-encoding genes, the analysis of chromosome positions, the identification and analysis of conserved motifs, and sequence alignment and phylogenetic reconstruction, our results showed that the genes of the TIR-X family (TXs) had an earlier origin and a wider distribution than the genes from the other three groups. TIR-encoding genes experienced large-scale gene duplications during evolution. A skeleton motif pattern of the TIR domain was present in all spermatophytes, and the genes with this skeleton pattern exhibited a conserved and independent evolutionary history in all spermatophytes, including monocots, that followed their gymnosperm origin. This study used comparative genomics to explore the origin and evolutionary history of the four main groups of TIR-encoding genes. Additionally, we unraveled the mechanism behind the uneven distribution of TIR-encoding genes in dicots and monocots.

  5. Knock-out of SO1377 gene, which encodes the member of a conserved hypothetical bacterial protein family COG2268, results in alteration of iron metabolism, increased spontaneous mutation and hydrogen peroxide sensitivity in Shewanella oneidensis MR-1

    Directory of Open Access Journals (Sweden)

    Klingeman Dawn M

    2006-04-01

    Full Text Available Abstract Background Shewanella oneidensis MR-1 is a facultative, gram-negative bacterium capable of coupling the oxidation of organic carbon to a wide range of electron acceptors such as oxygen, nitrate and metals, and has potential for bioremediation of heavy metal contaminated sites. The complete 5-Mb genome of S. oneidensis MR-1 was sequenced and standard sequence-comparison methods revealed approximately 42% of the MR-1 genome encodes proteins of unknown function. Defining the functions of hypothetical proteins is a great challenge and may need a systems approach. In this study, by using integrated approaches including whole genomic microarray and proteomics, we examined knockout effects of the gene encoding SO1377 (gi24372955, a member of the conserved, hypothetical, bacterial protein family COG2268 (Clusters of Orthologous Group in bacterium Shewanella oneidensis MR-1, under various physiological conditions. Results Compared with the wild-type strain, growth assays showed that the deletion mutant had a decreased growth rate when cultured aerobically, but not affected under anaerobic conditions. Whole-genome expression (RNA and protein profiles revealed numerous gene and protein expression changes relative to the wild-type control, including some involved in iron metabolism, oxidative damage protection and respiratory electron transfer, e. g. complex IV of the respiration chain. Although total intracellular iron levels remained unchanged, whole-cell electron paramagnetic resonance (EPR demonstrated that the level of free iron in mutant cells was 3 times less than that of the wild-type strain. Siderophore excretion in the mutant also decreased in iron-depleted medium. The mutant was more sensitive to hydrogen peroxide and gave rise to 100 times more colonies resistant to gentamicin or kanamycin. Conclusion Our results showed that the knock-out of SO1377 gene had pleiotropic effects and suggested that SO1377 may play a role in iron

  6. pTAR-encoded proteins in plasmid partitioning.

    Science.gov (United States)

    Kalnin, K; Stegalkina, S; Yarmolinsky, M

    2000-04-01

    Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of Agrobacterium spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465-478, 1987). However, resequencing of the region revealed two small downstream genes, parB and orf-84, of which only parB was found to be essential for partitioning in A. tumefaciens. Purified ParA exhibited a weak ATPase activity that was modestly increased by nonspecific DNA. ParB bound in vitro to repeated sequences present in a region, parS, that possesses centromere and operator functions and within which we identified the primary transcription start site by primer extension. In certain respects the Par proteins behave normally in the foreign host Escherichia coli. In E. coli, as in A. tumefaciens, ParB repressed the partition operon; ParA, inactive alone, augmented this repression. Functional similarities between the partition system of pTAR and those of other plasmids and bacteria are prominent, despite differences in size, organization, and amino acid sequence.

  7. The gusBC genes of Escherichia coli encode a glucuronide transport system

    NARCIS (Netherlands)

    Liang, WJ; Wilson, KJ; Xie, H; Knol, J; Suzuki, S; Rutherford, NG; Henderson, PJF; Jefferson, RA

    2005-01-01

    Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of beta-glucuronides with synthetic [C-14] phenyl-l-thio-beta-D-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the

  8. Male-specific lethal 2, a dosage compensation gene of Drosophila, undergoes sex-specific regulation and encodes a protein with a RING finger and a metallothionein-like cysteine cluster.

    Science.gov (United States)

    Zhou, S; Yang, Y; Scott, M J; Pannuti, A; Fehr, K C; Eisen, A; Koonin, E V; Fouts, D L; Wrightsman, R; Manning, J E

    1995-06-15

    In Drosophila the equalization of X-linked gene products between males and females, i.e. dosage compensation, is the result of a 2-fold hypertranscription of most of these genes in males. At least four regulatory genes are required for this process. Three of these genes, maleless (mle), male-specific lethal 1 (msl-1) and male-specific lethal 3 (msl-3), have been cloned and their products have been shown to interact and to bind to numerous sites on the X chromosome of males, but not of females. Although binding to the X chromosome is negatively correlated with the function of the master regulatory gene Sex lethal (Sxl), the mechanisms that restrict this binding to males and to the X chromosome are not yet understood. We have cloned the last of the known autosomal genes involved in dosage compensation, male-specific lethal 2 (msl-2), and characterized its product. The encoded protein (MSL-2) consists of 769 amino acid residues and has a RING finger (C3HC4 zinc finger) and a metallothionein-like domain with eight conserved and two non-conserved cysteines. In addition, it contains a positively and a negatively charged amino acid residue cluster and a coiled coil domain that may be involved in protein-protein interactions. Males produce a msl-2 transcript that is shorter than in females, due to differential splicing of an intron of 132 bases in the untranslated leader. Using an antiserum against MSL-2 we have shown that the protein is expressed at a detectable level only in males, where it is physically associated with the X chromosome. Our observations suggest that MSL-2 may be the target of the master regulatory gene Sxl and provide the basic elements of a working hypothesis on the function of MSL-2 in mediating the 2-fold increase in transcription that is characteristic of dosage compensation.

  9. Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes

    DEFF Research Database (Denmark)

    Honoré, B; Rasmussen, H H; Vorum, H;

    1995-01-01

    and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term...... treatment with 4 beta-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4 beta-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H', and F were chromosome-mapped to 5q35...

  10. Expression of the rgMT gene, encoding for a rice metallothionein-like protein in Saccharomyces cerevisiae and Arabidopsis thaliana

    Indian Academy of Sciences (India)

    Shumei Jin; Dan Sun; Ji Wang; Ying Li; Xinwang Wang; Shenkui Liu

    2014-12-01

    Metallothioneins (MTs) are cysteine-rich proteins of low molecular weight with many attributed functions, such as providing protection against metal toxicity, being involved in regulation of metal ions uptake that can impact plant physiology and providing protection against oxidative stress. However, the precise function of the metallothionein-like proteins such as the one coded for rgMT gene isolated from rice (Oryza sativa L.) is not completely understood. The whole genome analysis of rice (O. sativa) showed that the rgMT gene is homologue to the Os11g47809 on chromosome 11 of O. sativa sp. japonica genome. This study used the rgMT coding sequence to create transgenic lines to investigate the subcellular localization of the protein, as well as the impact of gene expression in yeast (Saccharomyces cerevisiae) and Arabidopsis thaliana under heavy metal ion, salt and oxidative stresses. The results indicate that the rgMT gene was expressed in the cytoplasm of transgenic cells. Yeast cells transgenic for rgMT showed vigorous growth compared to the nontransgenic controls when exposed to 7mM CuCl2, 10 mM FeCl2, 1 M NaCl, 24 mM NaHCO3 and 3.2 mM H2O2, but there was no significant difference for other stresses tested. Similarly, Arabidopsis transgenic for rgMT displayed significantly improved seed germination rates over that of the control when the seeds were stressed with 100 M CuCl2 or 1 mM H2O2. Increased biomass was observed in the presence of 100 M CuCl2, 220 M FeCl2, 3 mM Na2CO3, 5 mM NaHCO3 or 1 mM H2O2. These results indicate that the expression of the rice rgMT gene in transgenic yeast and Arabidopsis is implicated in improving their tolerance for certain salt and peroxide stressors.

  11. Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain.

    Science.gov (United States)

    Ito, H; Fujitani, K; Usui, K; Shimizu-Nishikawa, K; Tanaka, S; Yamamoto, D

    1996-09-03

    We have isolated a new Drosophila mutant, satori (sat), the males of which do not court or copulate with female flies. The sat mutation comaps with fruitless (fru) at 91B and does not rescue the bisexual phenotype of fru, indicating that sat is allelic to fru (fru(sat)). The fru(sat) adult males lack a male-specific muscle, the muscle of Lawrence, as do adult males with other fru alleles. Molecular cloning and analyses of the genomic and complementary DNAs indicated that transcription of the fru locus yields several different transcripts. The sequence of fru cDNA clones revealed a long open reading frame that potentially encodes a putative transcription regulator with a BTB domain and two zinc finger motifs. In the 5' noncoding region, three putative transformer binding sites were identified in the female transcript but not in male transcripts. The fru gene is expressed in a population of brain cells, including those in the antennal lobe, that have been suggested to be involved in determination of male sexual orientation. We suggest that fru functions downstream of tra in the sex-determination cascade in some neural cells and that inappropriate sexual development of these cells in the fru mutants results in altered sexual orientation of the fly.

  12. A novel human gene spindlin1,encoding a protein localized in the cell nucleus and inducing NIH3T3 cell's transformation

    Institute of Scientific and Technical Information of China (English)

    GAO Yanhong; QIN Lipeng; ZHANG Peng; CHEN Lin; YUAN Hongfeng; BAI Cixian; YAN Fang; YUE Wen; PEI Xuetao

    2004-01-01

    A novel human gene, spindlin1, recently cloned in our laboratory, is highly expressed in the tissue of ovary cancer. To study its biological function, a vector expressing green fluorescent-spindlin1 fusion protein was constructed and transfected into COS-7 and NIH3T3 cells by lipofectamine methods. The results showed that the fusion protein pEGFP-N1-spindlin1 was localized in the nucleus of COS-7 and NIH3T3 cells. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the parental NIH3T3 cells displayed a complete morphological change, improved the cell growth and increased the percentage of cells in G2/M phase (12.6% vs control cells at 3.4%). Furthermore, overexpressed spindlin1 cells formed colonies in soft agar, more motile in migration assay in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may be a prooncogene which is associated with tumorigenesis.

  13. CERKL, a retinal disease gene, encodes an mRNA-binding protein that localizes in compact and untranslated mRNPs associated with microtubules.

    Directory of Open Access Journals (Sweden)

    Alihamze Fathinajafabadi

    Full Text Available The function of CERKL (CERamide Kinase Like, a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases.

  14. Specific amplification of gene encoding N-terminal region of catalase-peroxidase protein (KatG-N) for diagnosis of disseminated MAC disease in HIV patients.

    Science.gov (United States)

    Latawa, Romica; Singh, Krishna Kumar; Wanchu, Ajay; Sethi, Sunil; Sharma, Kusum; Sharma, Aman; Laal, Suman; Verma, Indu

    2014-10-01

    Disseminated Mycobacterium avium-intracellulare complex (MAC) infection is considered as severe complication of advanced HIV/AIDS disease. Currently available various laboratory investigations have not only limited ability to discriminate between MAC infection and tuberculosis but are also laborious and time consuming. The aim of this study was, therefore, to design a molecular-based strategy for specific detection of MAC and its differentiation from Mycobacterium tuberculosis (M. tb) isolated from the blood specimens of HIV patients. A simple PCR was developed based on the amplification of 120-bp katG-N gene corresponding to the first 40 amino acids of N-terminal catalase-peroxidase (KatG) protein of Mycobacterium avium that shows only ~13% sequence homology by clustal W alignment to N-terminal region of M. tb KatG protein. This assay allowed the accurate and rapid detection of MAC bacteremia, distinguishing it from M. tb in a single PCR reaction without any need for sequencing or hybridization protocol to be performed thereafter. This study produced enough evidence that a significant proportion of Indian HIV patients have disseminated MAC bacteremia, suggesting the utility of M. avium katG-N gene PCR for early detection of MAC disease in HIV patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. MBNL142 and MBNL143 gene isoforms, overexpressed in DM1-patient muscle, encode for nuclear proteins interacting with Src family kinases.

    Science.gov (United States)

    Botta, A; Malena, A; Tibaldi, E; Rocchi, L; Loro, E; Pena, E; Cenci, L; Ambrosi, E; Bellocchi, M C; Pagano, M A; Novelli, G; Rossi, G; Monaco, H L; Gianazza, E; Pantic, B; Romeo, V; Marin, O; Brunati, A M; Vergani, L

    2013-08-15

    Myotonic dystrophy type-1 (DM1) is the most prevalent form of muscular dystrophy in adults. This disorder is an RNA-dominant disease, caused by expansion of a CTG repeat in the DMPK gene that leads to a misregulation in the alternative splicing of pre-mRNAs. The longer muscleblind-like-1 (MBNL1) transcripts containing exon 5 and the respective protein isoforms (MBNL142-43) were found to be overexpressed in DM1 muscle and localized exclusively in the nuclei. In vitro assays showed that MBNL142-43 bind the Src-homology 3 domain of Src family kinases (SFKs) via their proline-rich motifs, enhancing the SFK activity. Notably, this association was also confirmed in DM1 muscle and myotubes. The recovery, mediated by an siRNA target to Ex5-MBNL142-43, succeeded in reducing the nuclear localization of both Lyn and MBNL142-43 proteins and in decreasing the level of tyrosine phosphorylated proteins. Our results suggest an additional molecular mechanism in the DM1 pathogenesis, based on an altered phosphotyrosine signalling pathway.

  16. Expression analysis of genes encoding mitogen-activated protein kinases in maize provides a key link between abiotic stress signaling and plant reproduction.

    Science.gov (United States)

    Sun, Wei; Chen, Hao; Wang, Juan; Sun, Hong Wei; Yang, Shu Ke; Sang, Ya Lin; Lu, Xing Bo; Xu, Xiao Hui

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) play important roles in stress responses and development in plants. Maize (Zea mays), an important cereal crop, is a model plant species for molecular studies. In the last decade, several MAPKs have been identified in maize; however, their functions have not been studied extensively. Genome-wide identification and expression analysis of maize MAPK genes could provide valuable information for understanding their functions. In this study, 20 non-redundant maize MAPK genes (ZmMPKs) were identified via a genome-wide survey. Phylogenetic analysis of MAPKs from maize, rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), poplar (Populus trichocarpa), and tomato (Solanum lycopersicum) classified them into four major classes. ZmMPKs in the same class had similar domains, motifs, and genomic structures. Gene duplication investigations suggested that segmental duplications made a large contribution to the expansion of ZmMPKs. A number of cis-acting elements related to plant development and response to stress and hormones were identified in the promoter regions of ZmMPKs. Furthermore, transcript profile analysis in eight tissues and organs at various developmental stages demonstrated that most ZmMPKs were preferentially expressed in reproductive tissues and organs. The transcript abundance of most ZmMPKs changed significantly under salt, drought, cold, or abscisic acid (ABA) treatments, implying that they might participate in abiotic stress and ABA signaling. These expression analyses indicated that ZmMPKs might serve as linkers between abiotic stress signaling and plant reproduction. Our data will deepen our understanding of the complexity of the maize MAPK gene family and provide new clues to investigate their functions.

  17. Accelerated evolution of fetuin family proteins in Protobothrops flavoviridis (habu snake) serum and the discovery of an L1-like genomic element in the intronic sequence of a fetuin-encoding gene.

    Science.gov (United States)

    Tanaka, Yasuyoshi; Oyama, Sachiko; Hori, Shin-ichi; Ushio, Koya; Shioi, Narumi; Terada, Shigeyuki; Deshimaru, Masanobu

    2013-01-01

    Habu serum factor (HSF) and HSF-like protein (HLP) are fetuin family proteins isolated from Protobothrops flavoviridis (habu snake) serum with different physiological activities. A comparison of their cDNAs and intronic sequences revealed that nucleotide substitutions were primarily in protein-coding regions, and the substitution patterns indicated accelerated evolution of these proteins. Genomic DNA fragment analysis, including intron 1, revealed a 6.6-kb insertion homologous to the full-length mammalian LINE1 (L1) retrotransposable element (PfL1) only in the HLP gene. This segment retains an open reading frame (ORF) that encodes a reverse transcriptase (RT)-like protein (PfRT). We further found that a large number of homologous segments have dispersed in the habu snake genome, although we could not determine the enzymatic activities of their products. Moreover, an analysis of habu snake liver RNA indicated active transcription of the PfRT genes, suggesting that high levels of RT activity in this snake have driven the evolution of unique phenotypes of venom enzymes and serum inhibitors of them.

  18. Plant Proteins Are Smaller Because They Are Encoded by Fewer Exons than Animal Proteins

    Directory of Open Access Journals (Sweden)

    Obed Ramírez-Sánchez

    2016-12-01

    Full Text Available Protein size is an important biochemical feature since longer proteins can harbor more domains and therefore can display more biological functionalities than shorter proteins. We found remarkable differences in protein length, exon structure, and domain count among different phylogenetic lineages. While eukaryotic proteins have an average size of 472 amino acid residues (aa, average protein sizes in plant genomes are smaller than those of animals and fungi. Proteins unique to plants are ∼81 aa shorter than plant proteins conserved among other eukaryotic lineages. The smaller average size of plant proteins could neither be explained by endosymbiosis nor subcellular compartmentation nor exon size, but rather due to exon number. Metazoan proteins are encoded on average by ∼10 exons of small size [∼176 nucleotides (nt]. Streptophyta have on average only ∼5.7 exons of medium size (∼230 nt. Multicellular species code for large proteins by increasing the exon number, while most unicellular organisms employ rather larger exons (>400 nt. Among subcellular compartments, membrane proteins are the largest (∼520 aa, whereas the smallest proteins correspond to the gene ontology group of ribosome (∼240 aa. Plant genes are encoded by half the number of exons and also contain fewer domains than animal proteins on average. Interestingly, endosymbiotic proteins that migrated to the plant nucleus became larger than their cyanobacterial orthologs. We thus conclude that plants have proteins larger than bacteria but smaller than animals or fungi. Compared to the average of eukaryotic species, plants have ∼34% more but ∼20% smaller proteins. This suggests that photosynthetic organisms are unique and deserve therefore special attention with regard to the evolutionary forces acting on their genomes and proteomes.

  19. Ectopic overexpression of castor bean LEAFY COTYLEDON2 (LEC2) in Arabidopsis triggers the expression of genes that encode regulators of seed maturation and oil body proteins in vegetative tissues.

    Science.gov (United States)

    Kim, Hyun Uk; Jung, Su-Jin; Lee, Kyeong-Ryeol; Kim, Eun Ha; Lee, Sang-Min; Roh, Kyung Hee; Kim, Jong-Bum

    2013-01-01

    The LEAFY COTYLEDON2 (LEC2) gene plays critically important regulatory roles during both early and late embryonic development. Here, we report the identification of the LEC2 gene from the castor bean plant (Ricinus communis), and characterize the effects of its overexpression on gene regulation and lipid metabolism in transgenic Arabidopsis plants. LEC2 exists as a single-copy gene in castor bean, is expressed predominantly in embryos, and encodes a protein with a conserved B3 domain, but different N- and C-terminal domains to those found in LEC2 from Arabidopsis. Ectopic overexpression of LEC2 from castor bean under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis plants induces the accumulation of transcripts that encodes five major transcription factors (the LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON1-LIKE (L1L), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE 3 (ABI3) transcripts for seed maturation, and WRINKELED1 (WRI1) transcripts for fatty acid biosynthesis), as well as OLEOSIN transcripts for the formation of oil bodies in vegetative tissues. Transgenic Arabidopsis plants that express the LEC2 gene from castor bean show a range of dose-dependent morphological phenotypes and effects on the expression of LEC2-regulated genes during seedling establishment and vegetative growth. Expression of castor bean LEC2 in Arabidopsis increased the expression of fatty acid elongase 1 (FAE1) and induced the accumulation of triacylglycerols, especially those containing the seed-specific fatty acid, eicosenoic acid (20:1(Δ11)), in vegetative tissues.

  20. Ectopic overexpression of castor bean LEAFY COTYLEDON2 (LEC2 in Arabidopsis triggers the expression of genes that encode regulators of seed maturation and oil body proteins in vegetative tissues

    Directory of Open Access Journals (Sweden)

    Hyun Uk Kim

    2014-01-01

    Full Text Available The LEAFY COTYLEDON2 (LEC2 gene plays critically important regulatory roles during both early and late embryonic development. Here, we report the identification of the LEC2 gene from the castor bean plant (Ricinus communis, and characterize the effects of its overexpression on gene regulation and lipid metabolism in transgenic Arabidopsis plants. LEC2 exists as a single-copy gene in castor bean, is expressed predominantly in embryos, and encodes a protein with a conserved B3 domain, but different N- and C-terminal domains to those found in LEC2 from Arabidopsis. Ectopic overexpression of LEC2 from castor bean under the control of the cauliflower mosaic virus (CaMV 35S promoter in Arabidopsis plants induces the accumulation of transcripts that encodes five major transcription factors (the LEAFY COTYLEDON1 (LEC1, LEAFY COTYLEDON1-LIKE (L1L, FUSCA3 (FUS3, and ABSCISIC ACID INSENSITIVE 3 (ABI3 transcripts for seed maturation, and WRINKELED1 (WRI1 transcripts for fatty acid biosynthesis, as well as OLEOSIN transcripts for the formation of oil bodies in vegetative tissues. Transgenic Arabidopsis plants that express the LEC2 gene from castor bean show a range of dose-dependent morphological phenotypes and effects on the expression of LEC2-regulated genes during seedling establishment and vegetative growth. Expression of castor bean LEC2 in Arabidopsis increased the expression of fatty acid elongase 1 (FAE1 and induced the accumulation of triacylglycerols, especially those containing the seed-specific fatty acid, eicosenoic acid (20:1Δ11, in vegetative tissues.

  1. Tlys, a newly identified Sulfolobus spindle-shaped virus 1 transcript expressed in the lysogenic state, encodes a DNA-binding protein interacting at the promoters of the early genes

    DEFF Research Database (Denmark)

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta

    2013-01-01

    the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and Tind) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon-helix-helix DNA......-binding motif. DNA-binding assays demonstrated that the recombinant F55, purified from Escherichia coli, is indeed a putative transcription factor able to recognize site specifically target sequences in the promoters of the early induced T5, T6, and Tind transcripts, as well as of its own promoter. Binding...... sites of F55 are included within a tandem-repeated sequence overlapping the transcription start sites and/or the B recognition element of the pertinent genes. The strongest binding was observed with the promoters of T5 and T6, and an apparent cooperativity in binding was observed with the Tind promoter...

  2. Therapeutic efficacy of a tuberculosis DNA vaccine encoding heat shock protein 65 of Mycobacterium tuberculosis and the human interleukin 2 fusion gene.

    Science.gov (United States)

    Changhong, Shi; Hai, Zhang; Limei, Wang; Jiaze, An; Li, Xi; Tingfen, Zhang; Zhikai, Xu; Yong, Zhao

    2009-01-01

    Use of therapeutic DNA vaccines is a promising strategy against tuberculosis (TB), however, their immunogenicity still needs to be improved. In this study, a plasmid DNA vaccine expressing heat shock protein 65 (HSP65) and the human interleukin 2 (IL-2) fusion gene was constructed. Immune responses induced by the vaccine in the mice and protection against Mycobacterium tuberculosis (MTB) were investigated, along with the therapeutic effect of the DNA vaccine on tuberculosis in mice. Administration of the HSP65-IL-2-DNA vaccine enhanced Th1-type cellular responses by producing greater amounts of interferon-gamma (IFN-gamma) and IL-2 with a higher titer of antigen-specific anti-Hsp65 IgG2a. Compared with the Bacille Calmette-Guérin (BCG) vaccine, the DNA vaccine was able to evoke both CD4 and CD8 T-cell responses, with an especially high percentage of CD8 T-cells. The DNA vaccine was also able to induce high antigen-specific cytotoxicity activity against target cells. When the mice were challenged with virulent MTB H37Rv, a dramatic decrease in the numbers of MTB colony forming units in the spleen and lungs was observed in the mice immunized with HSP65-IL-2-DNA (P<0.05). Meanwhile, the bacterial numbers in TB infected mice treated with the DNA vaccine were also significantly reduced. The protective and therapeutic effects of the HSP65-IL-2-DNA vaccine in the spleen and lungs were superior to that of the HSP65-DNA vaccine (P<0.05). These results suggest that the DNA vaccine expression of IL-2 and the HSP65 fusion gene enhances the immunogenicity and protective as well as therapeutic effects of the HSP65-DNA vaccine against TB in mice by improving the Th1-type response.

  3. DNA-SEQUENCE DETERMINATION AND FUNCTIONAL-CHARACTERIZATION OF THE OCT-PLASMID-ENCODED ALKJKL GENES OF PSEUDOMONAS-OLEOVORANS

    NARCIS (Netherlands)

    van Beilen, J.B.; EGGINK, G; ENEQUIST, H; Witholt, Bernard; Bos, R

    1992-01-01

    The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodalt

  4. How are exons encoding transmembrane sequences distributed in the exon-intron structure of genes?

    Science.gov (United States)

    Sawada, Ryusuke; Mitaku, Shigeki

    2011-01-01

    The exon-intron structure of eukaryotic genes raises a question about the distribution of transmembrane regions in membrane proteins. Were exons that encode transmembrane regions formed simply by inserting introns into preexisting genes or by some kind of exon shuffling? To answer this question, the exon-per-gene distribution was analyzed for all genes in 40 eukaryotic genomes with a particular focus on exons encoding transmembrane segments. In 21 higher multicellular eukaryotes, the percentage of multi-exon genes (those containing at least one intron) within all genes in a genome was high (>70%) and with a mean of 87%. When genes were grouped by the number of exons per gene in higher eukaryotes, good exponential distributions were obtained not only for all genes but also for the exons encoding transmembrane segments, leading to a constant ratio of membrane proteins independent of the exon-per-gene number. The positional distribution of transmembrane regions in single-pass membrane proteins showed that they are generally located in the amino or carboxyl terminal regions. This nonrandom distribution of transmembrane regions explains the constant ratio of membrane proteins to the exon-per-gene numbers because there are always two terminal (i.e., the amino and carboxyl) regions - independent of the length of sequences.

  5. The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

    DEFF Research Database (Denmark)

    Christensen, P U; Davey, William John; Nielsen, O;

    1997-01-01

    In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is onl...

  6. The 216-bp marB gene of the marRAB operon in Escherichia coli encodes a periplasmic protein which reduces the transcription rate of marA.

    Science.gov (United States)

    Vinué, Laura; McMurry, Laura M; Levy, Stuart B

    2013-08-01

    The marRAB operon is conserved in seven genera of enteric bacteria (Escherichia, Shigella, Klebsiella, Enterobacter, Salmonella, Cronobacter, and Citrobacter). MarA is a transcriptional regulator affecting many genes involved in resistance to stresses, and MarR is an autorepressor of the operon, but a role for the marB gene has been unclear. A recent work reported that deletion of marB causes resistance to certain stresses and increases the amount of marA transcript. We show here that the small (216 bp) marB gene encodes a protein, not an sRNA, because two different stop codons within the predicted open reading frame of marB prevented plasmid-borne marB from complementing ΔmarB::Kan. The ΔmarB::Kan mutation did not increase the stability of the marA transcript, suggesting that MarB does not destabilize the marA transcript but rather reduces its rate of transcription. Placing the putative signal sequence of MarB upstream of signal-sequence-less alkaline phosphatase guided the phosphatase to its normal periplasmic location. We conclude that MarB is a small periplasmic protein that represses the marRAB promoter by an indirect mechanism, possibly involving a signal to one of the cytoplasmic regulators of that promoter.

  7. Nucleic acid compositions and the encoding proteins

    Science.gov (United States)

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  8. The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pak1p, involved in peroxisome integrity

    NARCIS (Netherlands)

    Klei, Ida J. van der; Heide, Meis van der; Baerends, Richard J.S.; Rechinger, Karl-Björn; Nicolay, Klaas; Kiel, Jan A.K.W.; Veenhuis, Marten

    1998-01-01

    The Hansenula polymorpha per6-210 mutant is impaired in respect of growth on methanol (Mut–) and is characterized by aberrant peroxisome formation. The functionally complementing DNA fragment contains two open reading frames. The first encodes dihydroxyacetone kinase (DAK), a cytosolic enzyme essent

  9. Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles

    DEFF Research Database (Denmark)

    Hummel, R; Nørgaard, P; Andreasen, P H

    1992-01-01

    . In accordance with the predicted molecular mass of the TF-ag protein, antibodies raised against a cro-lacZ'-TF-ag fusion protein specifically recognized a 45,000 M(r) protein in Western blots of total T. thermophila protein. Immunoelectron microscopy demonstrated that the TF-ag is associated with membranes...

  10. Multiple genes encode the major surface glycoprotein of Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Powell, F; Edman, J C;

    1993-01-01

    this antigen is a good candidate for development as a vaccine to prevent or control P. carinii infection. We have cloned and sequenced seven related but unique genes encoding the major surface glycoprotein of rat P. carinii. Partial amino acid sequencing confirmed the identity of these genes. Based on Southern...... hydrophobic region at the carboxyl terminus. The presence of multiple related msg genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is a possible mechanism for evading host defenses. Further characterization of this family of genes should allow the development...

  11. Genes encoding pentatricopeptide repeat (PPR proteins are not conserved in location in plant genomes and may be subject to diversifying selection

    Directory of Open Access Journals (Sweden)

    Brown Gregory G

    2007-05-01

    Full Text Available Abstract Background The pentatricopeptide repeat (PPR is a degenerate 35 amino acid motif that occurs in multiple tandem copies in members of a recently recognized eukaryotic gene family. Most analyzed eukaryotic genomes contain only a small number of PPR genes, but in plants the family is greatly expanded. The factors that underlie the expansion of this gene family in plants are not as yet understood. Results We show that the location of PPR genes is highly variable in comparisons between orthologous, closely related, and otherwise co-linear chromosomal regions of the Brassica rapa or radish and Arabidopsis thaliana. This observation also pertains to paralogous duplicated segments of the genomes of Arabidopsis thaliana and Brassica rapa. In addition, we show that PPR genes that seem closely linearly aligned in these comparisons are not generally found to be closely related to one another at the nucleotide and amino acid sequence level. We observe a relatively high level of non-synonomous vs synonomous changes among a group tandemly repeated radish PPR genes, suggesting that these, and possibly other PPR genes, are subject to diversifying selection. We also show that a duplicated region of the Arabidopsis genome possesses a relatively high density of PPR genes showing high similarity to restorers of fertility of cytoplasmic male sterile (CMS systems of petunia, radish and rice. The PPR genes in these regions, together with the restorer genes, are more highly similar to one another, in sequence as well as in structure, than to other PPR genes, even within the same sub-family. Conclusion Our results suggest are consistent with a model in which at least some PPR genes undergo a "birth and death" process that involves transposition to unrelated chromosomal sites. PPR genes hold certain features in common with disease resistance genes (R genes, and their "nomadic" character suggests that their evolutionary expansion in plants may have involved novel

  12. Characterization of a Soil Metagenome-Derived Gene Encoding Wax Ester Synthase.

    Science.gov (United States)

    Kim, Nam Hee; Park, Ji-Hye; Chung, Eunsook; So, Hyun-Ah; Lee, Myung Hwan; Kim, Jin-Cheol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-02-01

    A soil metagenome contains the genomes of all microbes included in a soil sample, including those that cannot be cultured. In this study, soil metagenome libraries were searched for microbial genes exhibiting lipolytic activity and those involved in potential lipid metabolism that could yield valuable products in microorganisms. One of the subclones derived from the original fosmid clone, pELP120, was selected for further analysis. A subclone spanning a 3.3 kb DNA fragment was found to encode for lipase/esterase and contained an additional partial open reading frame encoding a wax ester synthase (WES) motif. Consequently, both pELP120 and the full length of the gene potentially encoding WES were sequenced. To determine if the wes gene encoded a functioning WES protein that produced wax esters, gas chromatography-mass spectroscopy was conducted using ethyl acetate extract from an Escherichia coli strain that expressed the wes gene and was grown with hexadecanol. The ethyl acetate extract from this E. coli strain did indeed produce wax ester compounds of various carbon-chain lengths. DNA sequence analysis of the full-length gene revealed that the gene cluster may be derived from a member of Proteobacteria, whereas the clone does not contain any clear phylogenetic markers. These results suggest that the wes gene discovered in this study encodes a functional protein in E. coli and produces wax esters through a heterologous expression system.

  13. Isolation and characterization of the gene encoding the starch debranching enzyme limit dextrinase from germinating barley

    DEFF Research Database (Denmark)

    Kristensen, Michael; Lok, Finn; Planchot, Véronique

    1999-01-01

    The gene encoding the starch debranching enzyme limit dextrinase, LD, from barley (Hordeum vulgare), was isolated from a genomic phage library using a barley cDNA clone as probe. The gene encodes a protein of 904 amino acid residues with a calculated molecular mass of 98.6 kDa. This is in agreement...... fragments coupled with matrix assisted laser desorption mass spectrometry. The sequenced peptide fragments cover 70% of the entire protein sequence, which shows 62% and 77% identity to that of starch debranching enzymes from spinach and rice and 37% identity to Klebsiella pullulanase. Sequence alignment...

  14. Characterization of the Lactococcus lactis Nisin A Operon Genes nisP, Encoding a Subtilisin-Like Serine Protease Involved in Precursor Processing, and nisR, Encoding a Regulatory Protein Involved in Nisin Biosynthesis

    NARCIS (Netherlands)

    Polman, Joyce; Beerthuyzen, Marke M.; Siezen, Roland J.; Kuipers, Oscar P.; Vos, Willem M. de

    1993-01-01

    Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NIZO R5 relies on the presence of the conjugative transposon Tn5276 in the chromosome. A 12-kb DNA fragment of Tn5276 including the nisA gene and about 10 kb of downstream DNA was cloned in L. lactis, resulting in the production of

  15. Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae.

    Directory of Open Access Journals (Sweden)

    Baolong Zhang

    Full Text Available Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.

  16. ABA-Mediated Inhibition of Germination Is Related to the Inhibition of Genes Encoding Cell-Wall Biosynthetic and Architecture:Modifying Enzymes and Structural Proteins in Medicago truncatula Embryo Axis

    Institute of Scientific and Technical Information of China (English)

    Christine Gimeno-Gilles; Eric Lelièvre; Laure Viau; Mustafa Malik-Ghulam; Claudie Ricoult; Andreas Niebel; Nathalie Leduc; Anis M. Limami

    2009-01-01

    Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid (ABA) but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.

  17. DNA binding sites recognised in vitro by a knotted class 1 homeodomain protein encoded by the hooded gene, k, in barley (Hordeum vulgare)

    DEFF Research Database (Denmark)

    Krusell, L; Rasmussen, I; Gausing, K

    1997-01-01

    of knotted1 from maize was isolated from barley seedlings and expressed as a maltose binding protein fusion in E. coli. The purified HvH21-fusion protein selected DNA fragments with 1-3 copies of the sequence TGAC. Gel shift experiments showed that the TGAC element was required for binding and the results...

  18. Characterization of two genes encoding metal tolerance proteins from Beta vulgaris subspecies maritima that confers manganese tolerance in yeast

    DEFF Research Database (Denmark)

    Erbasol, Isil; Bozdag, Gonensin Ozan; Koc, Ahmet

    2013-01-01

    Manganese (Mn) is an essential micronutrient in plants. However increased Mn levels are toxic to plant cells. Metal tolerance proteins (MTPs), member of cation diffusion facilitator protein (CDF) family, have important roles in metal homeostatis in different plant species and catalyse efflux of e...... in planta localization and function as the Arabidopsis Mn-CDF homolog AtMTP11 and this conservation shows the evolutionary importance of these vesicular proteins in heavy metal homeostatis among plant species.......Manganese (Mn) is an essential micronutrient in plants. However increased Mn levels are toxic to plant cells. Metal tolerance proteins (MTPs), member of cation diffusion facilitator protein (CDF) family, have important roles in metal homeostatis in different plant species and catalyse efflux...

  19. Functional analysis of plastid-encoded genes

    OpenAIRE

    Swiatek, Magdalena

    2002-01-01

    Plastid chromosomes from the variety of plant species contain several conserved open reading frames of unknown function, which most probably represent functional genes. The primary aim of this thesis was the analysis of the role of two such ORFs, designated ycfs or hypothetical chloroplast reading frames, namely ycf9 (ORF62) and ycf10 (ORF229, cemA). Both were analyzed in Nicotiana tabacum (tobacco) via their inactivation using biolistic plastid transformation. A new experiment...

  20. The gene encoding acyl-CoA-binding protein is subject to metabolic regulation by both sterol regulatory element-binding protein and peroxisome proliferator-activated receptor alpha in hepatocytes

    DEFF Research Database (Denmark)

    Sandberg, Maria B; Bloksgaard, Maria; Duran-Sandoval, Daniel

    2005-01-01

    gene in hepatocytes. Members of the SREBP family activate the rat ACBP gene through binding sites for SREBP and the auxiliary factors Sp1 and nuclear factor Y in the proximal promoter. In addition, we show that ACBP is a peroxisome proliferator-activated receptor (PPAR) alpha target gene in cultured...... observation that ACBP expression in rodent liver is down-regulated by fasting, and we show that insulin but not glucose is the inducer of ACBP expression in primary rat hepatocytes. In keeping with the regulation by insulin, we show that ACBP is a sterol regulatory element-binding protein 1c (SREBP-1c) target...... hepatocytes and is induced in the liver by fibrates in a PPARalpha-dependent manner. Thus, ACBP is a dual PPARalpha and SREBP-1c target gene in hepatocytes. Fasting leads to reduced activity of SREBP but increased activity of PPARalpha in hepatocytes, and in keeping with ACBP being a dual target gene, we show...

  1. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    Science.gov (United States)

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

  2. Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12.

    Science.gov (United States)

    Sung, Y C; Parsell, D; Anderson, P M; Fuchs, J A

    1987-06-01

    The gene in Escherichia coli for cyanase, designated cynS, was localized to a BglII restriction site approximately 1.7 kilobases from the lacA end of the lac operon. The gene was cloned into the pUC13 vector. Maxicell analysis of plasmid-encoded proteins confirmed that the BglII site is in the region encoding the structural gene for cyanase. Cyanase-deficient strains had increased sensitivity to cyanate and were not able to use cyanate as a nitrogen source.

  3. Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12.

    OpenAIRE

    Sung, Y C; Parsell, D; Anderson, P. M.; Fuchs, J A

    1987-01-01

    The gene in Escherichia coli for cyanase, designated cynS, was localized to a BglII restriction site approximately 1.7 kilobases from the lacA end of the lac operon. The gene was cloned into the pUC13 vector. Maxicell analysis of plasmid-encoded proteins confirmed that the BglII site is in the region encoding the structural gene for cyanase. Cyanase-deficient strains had increased sensitivity to cyanate and were not able to use cyanate as a nitrogen source.

  4. Cloning of two genes encoding Rab7 in Paramecium.

    Science.gov (United States)

    Surmacz, Liliana; Wiejak, Jolanta; Wyroba, Elzbieta

    2006-01-01

    Rab7 is a small GTPase that plays a crucial role in the regulation of transport from early to late endosomes and lysosomes, phagosome maturation and in lysosomal biogenesis in mammalian cells. It contains conserved and unique sequence elements that mediate its function. Two Rab7 genes, Rab7a (703 bp) and Rab7b (707 bp) were identified in the unicellular eukaryote Paramecium by PCR amplification. They contain three short introns of different lengths (28-32 bp) and sequence located at identical positions in both genes. The presence of two Rab7 genes in the Paramecium genome was confirmed by Southern hybridization analysis performed with six different restriction enzymes. Expression of both genes was assessed by Northern blot and RT-PCR. Two transcripts of 1.8 and 2.2 kb were identified by hybridization analysis. The cloned complementary DNAs, both of 618 nucleotides in length, encode polypeptides of 206 amino acids that are 97.6% identical and differ in their C-termini. The predicted protein sequences of Rab7a and Rab7b contain all characteristic domains essential for Rab function: the effector domain (YRATVGADF) and four GTP-binding consensus sequences (GDSGVGKT, WDTAGQ, NKLD, SAK) as well as the prenylation motif (-CC) at the C-terminus indispensable for Rab binding to the membrane. Similarity searches revealed 81.6-82.1% homology of Paramecium Rab7 isoforms to human Rab7 and a lack of an insert typical for the Kinetoplastida - the species that appeared earlier in evolution. Paramecium is the first free-living lower eukaryote in which homologues of Rab7 have been identified that exhibit features similar to those of mammalian Rab7.

  5. Molecular cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum, and their relation to increased resistance to two fungal pathogens

    Science.gov (United States)

    Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). Inhibiting by PGIPs directly reduces potential PG activity in specific plant pathogenic fungi, reducing their aggressiveness. Here, we isolated and functionally chara...

  6. Mutations in the gene encoding the PML nuclear body protein Sp110 are associated with immunodeficiency and hepatic veno-occlusive disease.

    OpenAIRE

    Roscioli, Tony; Simon T Cliffe; Bloch, Donald B.; Bell, Christopher G.; Mullan, Glenda; Taylor, Peter J; Sarris, Maria; Wang, Joanne; Donald, Jennifer A.; Kirk, Edwin P; Ziegler, John B.; Salzer, Ulrich; McDonald, George B.; Wong, Melanie; Lindeman, Robert

    2006-01-01

    We describe mutations in the PML nuclear body protein Sp110 in the syndrome veno-occlusive disease with immunodeficiency, an autosomal recessive disorder of severe hypogammaglobulinemia, combined T and B cell immunodeficiency, absent lymph node germinal centers, absent tissue plasma cells and hepatic veno-occlusive disease. This is the first report of the involvement of a nuclear body protein in a human primary immunodeficiency and of high-penetrance genetic mutations in hepatic veno-occlusiv...

  7. Bioinformatics analysis and detection of gelatinase encoded gene in Lysinibacillussphaericus

    Science.gov (United States)

    Repin, Rul Aisyah Mat; Mutalib, Sahilah Abdul; Shahimi, Safiyyah; Khalid, Rozida Mohd.; Ayob, Mohd. Khan; Bakar, Mohd. Faizal Abu; Isa, Mohd Noor Mat

    2016-11-01

    In this study, we performed bioinformatics analysis toward genome sequence of Lysinibacillussphaericus (L. sphaericus) to determine gene encoded for gelatinase. L. sphaericus was isolated from soil and gelatinase species-specific bacterium to porcine and bovine gelatin. This bacterium offers the possibility of enzymes production which is specific to both species of meat, respectively. The main focus of this research is to identify the gelatinase encoded gene within the bacteria of L. Sphaericus using bioinformatics analysis of partially sequence genome. From the research study, three candidate gene were identified which was, gelatinase candidate gene 1 (P1), NODE_71_length_93919_cov_158.931839_21 which containing 1563 base pair (bp) in size with 520 amino acids sequence; Secondly, gelatinase candidate gene 2 (P2), NODE_23_length_52851_cov_190.061386_17 which containing 1776 bp in size with 591 amino acids sequence; and Thirdly, gelatinase candidate gene 3 (P3), NODE_106_length_32943_cov_169.147919_8 containing 1701 bp in size with 566 amino acids sequence. Three pairs of oligonucleotide primers were designed and namely as, F1, R1, F2, R2, F3 and R3 were targeted short sequences of cDNA by PCR. The amplicons were reliably results in 1563 bp in size for candidate gene P1 and 1701 bp in size for candidate gene P3. Therefore, the results of bioinformatics analysis of L. Sphaericus resulting in gene encoded gelatinase were identified.

  8. Campylobacter jejuni gene cj0511 encodes a serine peptidase essential for colonisation

    Directory of Open Access Journals (Sweden)

    A.V. Karlyshev

    2014-01-01

    Full Text Available According to MEROPS peptidase database, Campylobacter species encode 64 predicted peptidases. However, proteolytic properties of only a few of these proteins have been confirmed experimentally. In this study we identified and characterised a Campylobacter jejuni gene cj0511 encoding a novel peptidase. The proteolytic activity associated with this enzyme was demonstrated in cell lysates. Moreover, enzymatic studies conducted with a purified protein confirmed a prediction of it being a serine peptidase. Furthermore, cj0511 mutant was found to be severely attenuated in chicken colonisation model, suggesting a role of the Cj0511 protein in infection.

  9. pTAR-Encoded Proteins in Plasmid Partitioning

    OpenAIRE

    Kalnin, Kirill; Stegalkina, Svetlana; Yarmolinsky, Michael

    2000-01-01

    Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of Agrobacterium spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465–478, 1987). However, resequencing of the region revealed two small downstream g...

  10. Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase

    Directory of Open Access Journals (Sweden)

    I MADE ARTIKA

    2006-03-01

    Full Text Available Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody.

  11. Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles

    DEFF Research Database (Denmark)

    Hummel, R; Nørgaard, P; Andreasen, P H

    1992-01-01

    of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking. Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded. The abundance...

  12. Cloning and characterization of a novel oocyte-specific gene encoding an F-Box protein in rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified a novel transcript which is represented by ESTs only from the oocyte library. The novel...

  13. Bacillus caldolyticus prs gene encoding phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-1-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  14. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ostlund, Cecilia [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Guan, Tinglu [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Figlewicz, Denise A. [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Hays, Arthur P. [Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Worman, Howard J. [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Gerace, Larry [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Schirmer, Eric C., E-mail: e.schirmer@ed.ac.uk [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR (United Kingdom)

    2009-11-13

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

  15. A common W556S mutation in the LDL receptor gene of Danish patients with familial hypercholesterolemia encodes a transport-defective protein

    DEFF Research Database (Denmark)

    Jensen, H K; Holst, H; Jensen, L G;

    1997-01-01

    -Trp-Thr-Asp in the epidermal growth factor homology region, was studied in transfected COS-7 cells expressing normal and mutant LDL receptor cDNAs. Results obtained by immunofluorescence flow cytometry and confocal microscopy, as well as by immunoprecipitation, were compatible with complete retention of the mutant protein...

  16. Molecular characterization and expression analyses of cDNAs encoding the thioredoxin-interacting protein and selenoprotein P genes and histological changes in Nile tilapia (Oreochromis niloticus) in response to silver nanoparticle exposure.

    Science.gov (United States)

    Thummabancha, Kubpaphas; Onparn, Nuttaphon; Srisapoome, Prapansak

    2016-02-15

    Herein, Nile tilapia thioredoxin-interacting protein (On-TXNIP) and selenoprotein P (On-SEPP) cDNAs were cloned and characterized. The full-length On-TXNIP cDNA contained 2 arrestin domains, 2 conserved cysteine residues that bind to thioredoxin to inhibit thioredoxin function, and 2 PPXY motifs, which negatively regulate the protein by stimulating binding to E3 ubiquitin ligase. The On-SEPP cDNA contained 17 selenocysteines (Sec) encoded by the TGA codon, which can be recognized as either a stop codon or a Sec codon. The On-SEPP cDNA also carried 2 typical SECIS elements located in the 3'UTR that are important for selenocysteine translation. Evolutionary analyses of both the On-TXNIP and On-SEPP genes revealed that these genes are closely related to the TXNIP and SEPP genes in zebrafish (Danio rerio), with amino acid similarities of 91.8% and 61.9%, respectively. A normal tissue distribution analysis indicated that the On-TXNIP and On-SEPP genes were ubiquitously expressed in all tissues examined, and the highest expression levels of these genes were observed in peripheral blood leukocytes (PBLs) and the trunk kidney, respectively. The expression levels of On-TXNIP and On-SEPP transcripts were acutely and chronically analyzed following the injection of fish with 1, 10 or 100mg/kg silver nanoparticles (Ag NPs). Significant up-regulation of On-TXNIP and On-SEPP transcripts was observed in the liver, spleen, and head kidney at the early phase of Ag NP exposure (hours 6 through 48). Down-regulation of On-SEPP transcripts was clearly observed in the liver at weeks 1 to 4. Histopathology analysis demonstrated that the fish livers exhibited a dramatic infiltration of Kupffer cells, elevated bi-nucleated cells, expanded sinusoidal blood congestion and severe necrosis in a dose-dependent manner. Based on these findings, coupling of the expression analysis of these two cellular stress response genes and histopathological observation of fish exposed to Ag NPs should be

  17. An intron in a ribosomal protein gene from Tetrahymena

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Andreasen, Per Hove; Dreisig, Hanne

    1986-01-01

    We have cloned and sequenced a single copy gene encoding a ribosomal protein from the ciliate Tetrahymena thermophila. The gene product was identified as ribosomal protein S25 by comparison of the migration in two-dimensional polyacrylamide gels of the protein synthesized by translation in vitro...... of hybrid-selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979-bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene...

  18. 牛ACOT11基因克隆及其编码蛋白生物信息学分析%Cloning and Encoding Protein Bioinformatics Analysis of Bovine ACOT11 Gene

    Institute of Scientific and Technical Information of China (English)

    胡晓维; 靳聪飞; 刘新峰; 郭宏

    2015-01-01

    本研究旨在克隆牛ACOT11基因,并对其基因结构和蛋白质结构进行生物学特性分析.以GenBank数据库中公布的人ACOT11基因序列作为探针,检索得到牛的EST序列,进一步设计引物,再利用RT-PCR方法得到cDNA片段序列填补牛EST序列间的空缺,获得牛ACOT11基因.序列分析表明,牛ACOT11基因跨越大约4.9 kb的DNA区域,牛与人ACOT11基因所转录的mRNA同源性为87%.克隆的牛ACOT11基因含有16个外显子和15个内含子,编码区长1 620 bp,编码539个氨基酸.蛋白质结构分析表明,该基因蛋白二级结构预测以α螺旋为主;其35个磷酸化位点分布于丝氨酸(Ser)、苏氨酸(Thr)和酪氨酸(Tyr)残基上;保守结构域预测显示,该基因包含一个START功能结构域;亚细胞定位推测表明,牛ACOT11蛋白分泌通路信号肽所占的比例很小,大部分分布于细胞核内,不属于分泌蛋白.%In this study, bovine ACOT11 was cloned, and encoding protein was analyzed by bioinformatics methods. The human ACOT11 gene sequence in GenBank was chosen as probe to BLASTand the expressed sequence tags (ESTs) of bovine ACOT11 gene were gotten. The bovine ACOT11 gene was amplified by sequencing assembling of ESTs and cDNA sequence, which was amplified by reverse transcription polymerase chain reaction (RT-PCR). Sequence analyses show that the DNA region of cattle ACOT11 gene spans approximately 4.9 kb and the homology of mRNA between cattle and human ACOT11 is 87%. The bovine ACOT11 gene contains 16 exons and 15 introns, whose coding region is 1 620 bp and 539 amino acids were encoded. The secondary structure prediction of ACOT11 protein is mainly based on alpha helix, and ACOT11 protein contains 35 phosphorylation sites and one START domain, the phosphorylation sites locate in serine (Ser), threonine (Thr) and tyrosine (Tys) residue, the subcellular localization of ACOT11 is predicted in the nucleus, which didn't belong to the secreted protein.

  19. Human germline antibody gene segments encode polyspecific antibodies.

    Science.gov (United States)

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  20. [Immunoglobulin genes encoding antibodies directed to oncodevelopmental carbohydrate antigens].

    Science.gov (United States)

    Zenita, K; Yago, K; Fujimoto, E; Kannagi, R

    1990-07-01

    We investigated the immunoglobulin genes which encode the variable region of the monoclonal antibodies directed to the onco-developmental carbohydrate antigens such SSEA-1, fucosyl SSEA-1, SSEA-3 and SSEA-4. The VH region of these antibodies was preferentially encoded by the gene members of the X24, VH7183 and Q52 families, the families which are known to be located at the 3'-end region of the murine germ line VH gene. This result is interesting particularly when considering that the members of the 3'-end VH families are known to be preferentially expressed in embryonic B lymphocytes by an intrinsic genetic program. The comparative study of the nucleic acid sequences of mRNAs encoding these antibodies and the sequences of the corresponding germ line VH genes disclosed that the sequences encoding the antibodies contain no mutation from the germ line VH genes, or contain only a few somatic mutations, which are thought to be insignificant for the reactivity of the antibodies to the nominal antigens. These results imply that some of the embryonic B lymphocytes that express the unmutated germ line VH genes of the 3'-end families can be reactive with embryonic carbohydrate antigens, albeit rearranged with appropriate D-JH gene segments, and coupled with proper light chains. The VH region of the syngenic monoclonal anti-idiotypic antibodies directed to these anti-carbohydrate antibodies were also encoded preferentially by the members of the 3'-end VH families. We propose here that a part of the virgin embryonic B lymphocytes, which express the antibody encoded by the gene members of the 3'-end VH families at the cell surface, will be stimulated by the embryonic carbohydrate antigens which are abundantly present in the internal milieu of the embryo. The clonally expanded B lymphocytes, in turn, will facilitate the proliferation of other populations of embryonic B lymphocytes expressing the corresponding anti-idiotypic antibodies, which are also encoded by the gene members

  1. The implications of alternative splicing in the ENCODE protein complement

    DEFF Research Database (Denmark)

    Tress, Michael L.; Martelli, Pier Luigi; Frankish, Adam;

    2007-01-01

    Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been...

  2. Polymorphisms in host genes encoding NOSII, C-reactive protein, and adhesion molecules thrombospondin and E-selectin are risk factors for Plasmodium falciparum malaria in India.

    Science.gov (United States)

    Kanchan, K; Pati, S S; Mohanty, S; Mishra, S K; Sharma, S K; Awasthi, S; Venkatesh, V; Habib, S

    2015-10-01

    Cytoadherence of Plasmodium falciparum-infected red blood cells (RBCs) in host microvasculature and complex regulation of the immune response are important contributors to the clinical outcome of disease. We tested the association of 23 single nucleotide polymorphisms (SNPs) and a microsatellite repeat in adhesion molecule genes THBS1 and ESEL, and immune regulatory molecule genes NOSII, CRP, and MBL2 with falciparum malaria in populations residing in a malaria-endemic and a non-endemic region of India. The THBS1 haplotype CCCCA (rs1478604, rs7170682, rs2664141, rs12912082, rs3743125) was a risk factor in the endemic region (relative risk = 3.78) and an ESEL SNP (rs5368, His468Tyr) associated with cerebral malaria (CM) [CM vs. non-cerebral malaria (NCM), odds ratio (OR) = 2.23, p = 0.03]. In the non-endemic region, an ESEL 3'UTR SNP (rs5359) associated with enhanced risk of disease (OR = 3.62, p = 1 × 10(-4)) and the CT genotype of the CRP promoter SNP (C/T/A) strongly associated with protection (severe vs. control, OR = 0.29, p = 6 × 10(-5)). Long repeat alleles of the NOSII promoter microsatellite (CCTTT)n exhibited strong association with protection and the NOSII ATG haplotype (rs3729508, rs2297520, rs9282801) was strongly protective against severe malaria in both regions (endemic, severe vs. control, OR = 0.05, p = 0.0001; non-endemic, severe vs. control, OR = 0.3, p = 1 × 10(-5)). Our results suggest differential contribution of variants of the investigated genes in determining the outcome of malaria in Indian populations.

  3. A gene encoding a protein with a proline-rich domain (MtPPRD1), revealed by suppressive subtractive hybridization (SSH), is specifically expressed in the Medicago truncatula embryo axis during germination.

    Science.gov (United States)

    Bouton, Sophie; Viau, Laure; Lelièvre, Eric; Limami, Anis M

    2005-03-01

    A gene MtPPRD1, encoding a protein of 132 amino acids containing a proline-rich domain (PRD), has been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of embryo axes harvested immediately before and after radicle emergence. Although at the protein level MtPPRD1 showed low homology with plant lipid transfer proteins (LTPs), it did exhibit the eight cysteine residues conserved in all plant LTPs, a characteristic signature that allows the formation of a hydrophobic cavity adapted for loading hydrophobic molecules. Expression studies of MtPPRD1 have been carried out by quantitative real time RT-PCR throughout germination and post-germination processes in control seeds and seeds in which germination was delayed by abscisic acid (ABA) or the glutamine synthetase inhibitor methionine sulphoximine (MSX) treatments. The results showed that MtPPRD1 expression is developmentally regulated, induced in the embryo axis immediately before radicle emergence, reaches its maximum expression and declines during the early post-germination phase. Organ specificity studies showed that, except for a low and probably constitutive expression in roots, MtPPRD1 is specifically expressed in the embryo axis. Based on both experimental and in silico studies several putative roles are proposed for MtPPRD1 in Medicago truncatula, this protein can intervene (i) as an LTP in membrane biogenesis and regulation of the intracellular fatty acid pool by binding and transferring fatty acids and phospholipids between membranes, (ii) in the control of a developmental process specific to late germination and to early phases of post-germination, and (iii) and/or pathogen defence.

  4. Genome-wide comparative analysis of NBS-encoding genes in four Gossypium species

    Science.gov (United States)

    Nucleotide binding site (NBS) genes encode a large family of disease resistance (R) proteins in plants. The availability of genomic data of the two diploid cotton species, Gossypium arboreum and Gossypium raimondii, and the two allotetraploid cotton species, Gossypium hirsutum (TM-1) and Gossypium ...

  5. DNA polymerase III accessory proteins. I. holA and holB encoding delta and delta'.

    Science.gov (United States)

    Dong, Z; Onrust, R; Skangalis, M; O'Donnell, M

    1993-06-05

    The genes encoding the delta and delta' subunits of the 10-subunit Escherichia coli replicase, DNA polymerase III holoenzyme, have been identified and sequenced. The holA gene encoding delta is located downstream of rlpB at 15.2 min and predicts a 38.7 kda protein. The holB gene encoding delta' is located at 24.3 min and predicts a 36.9-kDa protein. Hence the delta and delta' subunits are unrelated proteins encoded by separate genes. The genes have been used to express and purify delta and delta' in quantity. The predicted amino acid sequence of delta' is homologous to the sequences of the tau and gamma subunits revealing a large amount of structural redundancy within the holoenzyme.

  6. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

    Directory of Open Access Journals (Sweden)

    Evaristus Chibunna Mbanefo

    Full Text Available BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6 M and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation, and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

  7. Rapid identification of genes encoding DNA polymerases by function-based screening of metagenomic libraries derived from glacial ice.

    Science.gov (United States)

    Simon, Carola; Herath, Judith; Rockstroh, Stephanie; Daniel, Rolf

    2009-05-01

    Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.

  8. Rapid Identification of Genes Encoding DNA Polymerases by Function-Based Screening of Metagenomic Libraries Derived from Glacial Ice▿

    OpenAIRE

    2009-01-01

    Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.

  9. Characterization of genes encoding poly(A polymerases in plants: evidence for duplication and functional specialization.

    Directory of Open Access Journals (Sweden)

    Lisa R Meeks

    Full Text Available BACKGROUND: Poly(A polymerase is a key enzyme in the machinery that mediates mRNA 3' end formation in eukaryotes. In plants, poly(A polymerases are encoded by modest gene families. To better understand this multiplicity of genes, poly(A polymerase-encoding genes from several other plants, as well as from Selaginella, Physcomitrella, and Chlamydomonas, were studied. METHODOLOGY/PRINCIPAL FINDINGS: Using bioinformatics tools, poly(A polymerase-encoding genes were identified in the genomes of eight species in the plant lineage. Whereas Chlamydomonas reinhardtii was found to possess a single poly(A polymerase gene, other species possessed between two and six possible poly(A polymerase genes. With the exception of four intron-lacking genes, all of the plant poly(A polymerase genes (but not the C. reinhardtii gene possessed almost identical intron positions within the poly(A polymerase coding sequences, suggesting that all plant poly(A polymerase genes derive from a single ancestral gene. The four Arabidopsis poly(A polymerase genes were found to be essential, based on genetic analysis of T-DNA insertion mutants. GFP fusion proteins containing three of the four Arabidopsis poly(A polymerases localized to the nucleus, while one such fusion protein was localized in the cytoplasm. The fact that this latter protein is largely pollen-specific suggests that it has important roles in male gametogenesis. CONCLUSIONS/SIGNIFICANCE: Our results indicate that poly(A polymerase genes have expanded from a single ancestral gene by a series of duplication events during the evolution of higher plants, and that individual members have undergone sorts of functional specialization so as to render them essential for plant growth and development. Perhaps the most interesting of the plant poly(A polymerases is a novel cytoplasmic poly(A polymerase that is expressed in pollen in Arabidopsis; this is reminiscent of spermatocyte-specific cytoplasmic poly(A polymerases in

  10. Gene regulation in response to protein disulphide isomerase deficiency

    DEFF Research Database (Denmark)

    Nørgaard, Per; Tachibana, Christine; Bruun, Anette W

    2003-01-01

    We have examined the activities of promoters of a number of yeast genes encoding resident endoplasmic reticulum proteins, and found increased expression in a strain with severe protein disulphide isomerase deficiency. Serial deletion in the promoter of the MPD1 gene, which encodes a PDI1-homologue...... element. The sequence (GACACG) does not resemble the unfolded protein response element. It is present in the upstream regions of the MPD1, MPD2, KAR2, PDI1 and ERO1 genes....

  11. Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis.

    NARCIS (Netherlands)

    Hollander, A.I. den; Koenekoop, R.K.; Mohamed, M.D.; Arts, H.H.; Boldt, K.; Towns, K.V.; Sedmak, T.; Beer, M. de; Nagel-Wolfrum, K.; McKibbin, M.; Dharmaraj, S.; Lopez, I.; Ivings, L.; Williams, G.A.; Springell, K.; Woods, C.G.; Jafri, H.; Rashid, Y.; Strom, T.M.; Zwaag, B. van der; Gosens, I.; Kersten, F.F.J.; Wijk, E. van; Veltman, J.A.; Zonneveld, M.N.; Beersum, S.E.C. van; Maumenee, I.H.; Wolfrum, U.; Cheetham, M.E.; Ueffing, M.; Cremers, F.P.M.; Inglehearn, C.F.; Roepman, R.

    2007-01-01

    Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We dete

  12. The highly conserved serine threonine kinase StkP of Streptococcus pneumoniae contributes to penicillin susceptibility independently from genes encoding penicillin-binding proteins

    Directory of Open Access Journals (Sweden)

    Dias Ricardo

    2009-06-01

    Full Text Available Abstract Background The serine/threonine kinase StkP of Streptococcus pneumoniae is a major virulence factor in the mouse model of infection. StkP is a modular protein with a N-terminal kinase domain a C-terminal PASTA domain carrying the signature of penicillin-binding protein (PBP and prokaryotic serine threonine kinase. In laboratory cultures, one target of StkP is the phosphoglucosamine mutase GlmM involved in the first steps of peptidoglycan biosynthesis. In order to further elucidate the importance of StkP in S. pneumoniae, its role in resistance to β-lactams has been assessed by mutational analysis in laboratory cultures and its genetic conservation has been investigated in isolates from infected sites (virulent, asymptomatic carriers, susceptible and non-susceptible to β-lactams. Results Deletion replacement mutation in stkP conferred hypersensitivity to penicillin G and was epistatic on mutations in PBP2X, PBP2B and PBP1A from the resistant 9V clinical isolate URA1258. Genetic analysis of 55 clinical isolates identified 11 StkP alleles differing from the reference R6 allele. None relevant mutation in the kinase or the PASTA domains were found to account for susceptibility of the isolates. Rather the minimal inhibitory concentration (MIC values of the strains appeared to be determined by their PBP alleles. Conclusion The results of genetic dissection analysis in lab strain Cp1015 reveal that StkP is involved in the bacterial response to penicillin and is epistatic on mutations PBP 2B, 2X and 1A. However analysis of the clinical isolates did not allow us to find the StkP alleles putatively involved in determining the virulence or the resistance level of a given strain, suggesting a strong conservation of StkP in clinical isolates.

  13. Coat protein mutations in an attenuated Cucumber mosaic virus encoding mutant 2b protein that lacks RNA silencing suppressor activity induces chlorosis with photosynthesis gene repression and chloroplast abnormalities in infected tobacco plants.

    Science.gov (United States)

    Mochizuki, Tomofumi; Yamazaki, Ryota; Wada, Tomoya; Ohki, Satoshi T

    2014-05-01

    In tobacco plants, the Cucumber mosaic virus (CMV) pepo strain induces mosaic symptoms, including pale green chlorosis and malformed tissues. Here, we characterized the involvement of 2b protein and coat protein (CP) in the development of mosaic symptoms. A 2b mutant (R46C) that lacks viral suppressor of RNA silencing (VSR) activity showed an asymptomatic phenotype with low levels of virus accumulation. Tomato spotted wilt virus NSs protein did not complement the virulence of the R46C, although it did restore high-level virus accumulation. However, R46C mutants expressing mutated CP in which the amino acid P129 was mutated to A, E, C, Q, or S induced chlorosis that was associated with reduced expression of chloroplast and photosynthesis related genes (CPRGs) and abnormal chloroplasts with fewer thylakoid membranes. These results suggest that the CP of the CMV pepo strain acquires virulence by amino acid mutations, which causes CPRG repression and chloroplast abnormalities.

  14. The Maize Divergent spindle-1 (dv1) Gene Encodes a Kinesin-14A Motor Protein Required for Meiotic Spindle Pole Organization.

    Science.gov (United States)

    Higgins, David M; Nannas, Natalie J; Dawe, R Kelly

    2016-01-01

    The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged β-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation.

  15. The maize Divergent spindle-1 (dv1 gene encodes a kinesin-14A motor protein required for meiotic spindle pole organization

    Directory of Open Access Journals (Sweden)

    David M Higgins

    2016-08-01

    Full Text Available The classic maize mutant divergent spindle-1 (dv1 causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged β-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation.

  16. Activating E17K mutation in the gene encoding the protein kinase AKT1 in a subset of squamous cell carcinoma of the lung.

    Science.gov (United States)

    Malanga, Donatella; Scrima, Marianna; De Marco, Carmela; Fabiani, Fernanda; De Rosa, Nicla; De Gisi, Silvia; Malara, Natalia; Savino, Rocco; Rocco, Gaetano; Chiappetta, Gennaro; Franco, Renato; Tirino, Virginia; Pirozzi, Giuseppe; Viglietto, Giuseppe

    2008-03-01

    Somatic mutation (E17K) that constitutively activates the protein kinase AKT1 has been found in human cancer patients. We determined the role of the E17K mutation of AKT1 in lung cancer, through sequencing of AKT1 exon 4 in 105 resected, clinically annotated non-small cell lung cancer specimens. We detected a missense mutations G-->A transition at nucleotide 49 (that results in the E17K substitution) in two squamous cell carcinoma (2/36) but not in adenocarcinoma (0/53). The activity of the endogenous kinase carrying the E17K mutation immunoprecipitated by tumour tissue was significantly higher compared with the wild-type kinase immunoprecipitated by the adjacent normal tissue as determined both by in vitro kinase assay using a consensus peptide as substrate and by in vivo analysis of the phosphorylation status of AKT1 itself (pT308, pS473) or of known downstream substrates such as GSK3 (pS9/S22) and p27 (T198). Immunostaining or immunoblot analysis on membrane-enriched extracts indicated that the enhanced membrane localization exhibited by the endogenous E17K-AKT1 may account for the observed increased activity of mutant E17K kinase in comparison with the wild-type AKT1 from adjacent normal tissue. In conclusion, this is the first report of AKT1 mutation in lung cancer. Our data provide evidence that, although AKT1 mutations are apparently rare in lung cancer (1.9%), the oncogenic properties of E17K-AKT1 may contribute to the development of a fraction of lung carcinoma with squamous histotype (5.5%).

  17. On the Role of PDZ Domain-Encoding Genes in Drosophila Border Cell Migration

    OpenAIRE

    Aranjuez, George; Kudlaty, Elizabeth; Longworth, Michelle S; McDonald, Jocelyn A.

    2012-01-01

    Cells often move as collective groups during normal embryonic development and wound healing, although the mechanisms governing this type of migration are poorly understood. The Drosophila melanogaster border cells migrate as a cluster during late oogenesis and serve as a powerful in vivo genetic model for collective cell migration. To discover new genes that participate in border cell migration, 64 out of 66 genes that encode PDZ domain-containing proteins were systematically targeted by in v...

  18. Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis

    Directory of Open Access Journals (Sweden)

    St Clair Dina A

    2007-10-01

    alternative splicing was found for at least 12 genes, 11 of which encode TIR-NBS-LRR proteins. There was no obvious correlation between expression pattern, phylogenetic relationship or genomic location of the NBS-LRR-encoding and related genes studied. Conclusion Transcripts of many NBS-LRR-encoding and related genes were defined. Most were present at low levels and exhibited tissue-specific expression patterns. Expression data are consistent with most Arabidopsis NBS-LRR-encoding and related genes functioning in plant defense responses but do not preclude other biological roles.

  19. Cloning of Mouse Enamel Matrix Serine Proteinase Encoding Mature Protein

    Institute of Scientific and Technical Information of China (English)

    MU Ya-bing; SUN Hong-chen; ZHANG Ze-bing; OUYANG Jie

    2003-01-01

    Objective: To clone cDNA of enamel matrix serine proteinase (EMSP1) encoding mature protein from mouse dental germs. Methods: Total RNA was isolated from developing incisors and molars of 7 days mouse pups and reverse-transcribed into cDNA. Two pairs of specific primers was designed to obtain the desired gene by Touchdown PCR and Nested PCR. The segment was inserted into Vector pMD-18T, and recombined vectors was transformed into E.coli JM109.The positive clone was chose and analysed by restriction endonuclease mapping and DNA sequencing. Results:700 bp of cDNA of mouse EMSP1 was sueccessfully cloned from mouse tooth germs tissue. The sequence was consistent with that displayed in PubMed. Conclusion:The mouse EMSP1 cDNA encoding mature protein is obtained for further study.%目的:克隆小鼠牙胚组织中釉基质丝氨酸蛋白酶(EMSP1)成熟肽编码区基因.方法:提取出生后7 d昆明种小白鼠切牙、磨牙牙胚总RNA,逆转录为cDNA,设计两对特异性引物,采用Touchdown PCR 和嵌套PCR方法,扩增出小鼠EMSP1起始密码子至终止密码子基因片段.将目的基因连入载体pMD-18T,转化入大肠杆菌JM109,通过蓝白筛选,挑选阳性克隆培养扩增,纯化重组质粒进行限制性酶切和核苷酸序列分析鉴定.结果:限制性酶切图谱和核苷酸序列分析均表明所克隆cDNA为小鼠700 bp的EMSP1成熟肽基因编码.结论:成功地克隆了小鼠编码EMSP1成熟肽基因片段.

  20. Differential translation tunes uneven production of operon-encoded proteins

    NARCIS (Netherlands)

    Quax, T.E.F.; Wolf, Y.I.; Koehorst, J.J.; Wurtzel, O.; Oost, van der R.; Ran, W.; Blombach, F.; Makarova, K.S.; Brouns, S.J.J.; Forster, A.C.; Wagner, E.G.H.; Sorek, R.; Koonin, E.V.; Oost, van der J.

    2013-01-01

    Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in

  1. Designing genes for successful protein expression.

    Science.gov (United States)

    Welch, Mark; Villalobos, Alan; Gustafsson, Claes; Minshull, Jeremy

    2011-01-01

    DNA sequences are now far more readily available in silico than as physical DNA. De novo gene synthesis is an increasingly cost-effective method for building genetic constructs, and effectively removes the constraint of basing constructs on extant sequences. This allows scientists and engineers to experimentally test their hypotheses relating sequence to function. Molecular biologists, and now synthetic biologists, are characterizing and cataloging genetic elements with specific functions, aiming to combine them to perform complex functions. However, the most common purpose of synthetic genes is for the expression of an encoded protein. The huge number of different proteins makes it impossible to characterize and catalog each functional gene. Instead, it is necessary to abstract design principles from experimental data: data that can be generated by making predictions followed by synthesizing sequences to test those predictions. Because of the degeneracy of the genetic code, design of gene sequences to encode proteins is a high-dimensional problem, so there is no single simple formula to guarantee success. Nevertheless, there are several straightforward steps that can be taken to greatly increase the probability that a designed sequence will result in expression of the encoded protein. In this chapter, we discuss gene sequence parameters that are important for protein expression. We also describe algorithms for optimizing these parameters, and troubleshooting procedures that can be helpful when initial attempts fail. Finally, we show how many of these methods can be accomplished using the synthetic biology software tool Gene Designer.

  2. A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance

    Science.gov (United States)

    van Rooij, Eva; Quiat, Daniel; Johnson, Brett A.; Sutherland, Lillian B.; Qi, Xiaoxia; Richardson, James A.; Kelm, Robert J.; Olson, Eric N.

    2009-01-01

    Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related microRNAs (miRNAs) within their introns, which, in turn, control muscle myosin content, myofiber identity and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, co-expresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 are functionally redundant, and play a dominant role in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance. PMID:19922871

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

    Science.gov (United States)

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

    2015-11-01

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

  4. Bacteriophage-encoded shiga toxin gene in atypical bacterial host

    Directory of Open Access Journals (Sweden)

    Casas Veronica

    2011-07-01

    Full Text Available Abstract Background Contamination from fecal bacteria in recreational waters is a major health concern since bacteria capable of causing human disease can be found in animal feces. The Dog Beach area of Ocean Beach in San Diego, California is a beach prone to closures due to high levels of fecal indicator bacteria (FIB. A potential source of these FIB could be the canine feces left behind by owners who do not clean up after their pets. We tested this hypothesis by screening the DNA isolated from canine feces for the bacteriophage-encoded stx gene normally found in the virulent strains of the fecal bacterium Escherichia coli. Results Twenty canine fecal samples were collected, processed for total and bacterial fraction DNA, and screened by PCR for the stx gene. The stx gene was detected in the total and bacterial fraction DNA of one fecal sample. Bacterial isolates were then cultivated from the stx-positive fecal sample. Eighty nine of these canine fecal bacterial isolates were screened by PCR for the stx gene. The stx gene was detected in five of these isolates. Sequencing and phylogenetic analyses of 16S rRNA gene PCR products from the canine fecal bacterial isolates indicated that they were Enterococcus and not E. coli. Conclusions The bacteriophage-encoded stx gene was found in multiple species of bacteria cultivated from canine fecal samples gathered at the shoreline of the Dog Beach area of Ocean Beach in San Diego, California. The canine fecal bacteria carrying the stx gene were not the typical E. coli host and were instead identified through phylogenetic analyses as Enterococcus. This suggests a large degree of horizontal gene transfer of exotoxin genes in recreational waters.

  5. Structure and expression of nuclear genes encoding rubisco activase. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Zielinski, R.E.

    1994-06-01

    Rubisco activase (Rca) is a soluble chloroplast protein that catalyzes the activation of rubisco, the enzyme that initiates the photosynthetic carbon reduction cycle, to catalytic competency. Rca in barley consists of three polypeptides, one of 46- and two of 42-kDa, but the quaternary structure of the protein is not known. The authors have isolated and completely sequenced 8.8 kb of barley genomic DNA containing two, tandemly oriented activase genes (RcaA and RcaB) and three different cDNAs encoding the 42- and 46-kDa Rca polypeptide isoforms. Genomic Southern blot assays indicate that these sequences represent the entire Rca gene family in barley. Pre-mRNAs transcribed from the RcaA gene are alternatively spliced to give mRNAs encoding both 46- (RcaA1) and 42-kDa (RcaA2) Rca isoforms. The RcaB gene encodes a single polypeptide of 42 kDa. Primer extension and northern blot assays indicate that RcaB mRNA is expressed at a level that is 10- to 100-fold lower than RcaA mRNA. Analyses at the mRNA and protein level showed that Rca gene expression is coordinated by that of the rubisco subunits during barley leaf development.

  6. Proteins encoded near the adenovirus late messenger RNA leader segments

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, J.B.; Anderson, C.W.

    1983-01-01

    Small fragments of adenovirus 2 DNA cloned into the single-strand phage M13 were used to select adenoviral messenger RNAs transcribed from the R-strand between map positions 16 and 30. Cell-free translation of these mRNAs produced proteins of 13.5K, 13.6K, and 11.5K, respectively encoded between the first and second segments of the tripartite major late leader, within the ''i''-leader segment, and immediately preceding the third leader segment. Partial sequence analysis of the 13.6K protein is consistent with the hypothesis that it is encoded within the i-leader segment.

  7. Expression of a synthetic gene encoding human insulin-like growth factor I in cultured mouse fibroblasts.

    OpenAIRE

    Bayne, M L; Cascieri, M A; Kelder, B; Applebaum, J; Chicchi, G; Shapiro, J A; Pasleau, F.; Kopchick, J. J.

    1987-01-01

    A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fi...

  8. Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda.

    OpenAIRE

    Barik, S

    1993-01-01

    The predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage lambda exhibited a high degree of similarity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to PP1, PP2A, and PP2B groups. Cloning and expression of the orf221 gene in Escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase. The single-subunit recombinant enzyme was purified in soluble form and shown to po...

  9. Identification and characterization of the Vibrio anguillarum prtV gene encoding a new metalloprotease

    Institute of Scientific and Technical Information of China (English)

    莫照兰; 郭东升; 茅云翔; 叶旭红; 邹玉霞; 肖鹏; 郝斌

    2010-01-01

    We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar,lower protease activity against azocasein,and lower activity for four glycosidases.This prtV mutant strain also had increased activity for two esterases in its extracellular products,as analyzed by the API ZYM system.In additi...

  10. Resistance to β-Lactams in Neisseria ssp Due to Chromosomally Encoded Penicillin-Binding Proteins

    Directory of Open Access Journals (Sweden)

    André Zapun

    2016-09-01

    Full Text Available Neisseria meningitidis and Neisseria gonorrhoeae are human pathogens that cause a variety of life-threatening systemic and local infections, such as meningitis or gonorrhoea. The treatment of such infection is becoming more difficult due to antibiotic resistance. The focus of this review is on the mechanism of reduced susceptibility to penicillin and other β-lactams due to the modification of chromosomally encoded penicillin-binding proteins (PBP, in particular PBP2 encoded by the penA gene. The variety of penA alleles and resulting variant PBP2 enzymes is described and the important amino acid substitutions are presented and discussed in a structural context.

  11. Direct Pathogenic Effects of HERV-encoded Proteins

    DEFF Research Database (Denmark)

    Hansen, Dorte Tranberg; Møller-Larsen, Anné; Petersen, Thor;

    and the possible direct pathogenic effects of HERV-encoded Env proteins on the CNS. Methods: Construction and characterization of a panel of recombinant Env-proteins is initiated and their pathogenic potential will be investigated: Fusiogenic potential analyzed by flow cytometry and confocal microscopy. Analysis...... of Env-induced apoptosis/necrosis in CNS cells will be performed by both DNA fragmentation ELISA and qPCR. Furthermore, the cellular localization of HERV-antigens on cells from patients with MS will be determined by confocal microscopy. A flow cytometric/confocal method has been optimized...

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

    Science.gov (United States)

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

    2015-01-01

    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. PMID:26475106

  13. Identification and characterization of an oleate hydratase-encoding gene from Bifidobacterium breve

    Science.gov (United States)

    O'Connell, Kerry Joan; Motherway, Mary O'Connell; Hennessey, Alan A; Brodhun, Florian; Ross, R Paul; Feussner, Ivo; Stanton, Catherine; Fitzgerald, Gerald F; van Sinderen, Douwe

    2013-01-01

    Bifidobacteria are common commensals of the mammalian gastrointestinal tract. Previous studies have suggested that a bifidobacterial myosin cross reactive antigen (MCRA) protein plays a role in bacterial stress tolerance, while this protein has also been linked to the biosynthesis of conjugated linoleic acid (CLA) in bifidobacteria. In order to increase our understanding on the role of MCRA in bifidobacteria we created and analyzed an insertion mutant of the MCRA-encoding gene of B. breve NCFB 2258. Our results demonstrate that the MCRA protein of B. breve NCFB 2258 does not appear to play a role in CLA production, yet is an oleate hydratase, which contributes to bifidobacterial solvent stress protection. PMID:23851389

  14. Protein Collapse is Encoded in the Folded State Architecture

    CERN Document Server

    Samanta, Himadri S; Hinczewski, Michael; Hori, Naoto; Chakrabarti, Shaon; Thirumalai, D

    2016-01-01

    Natural protein sequences that self-assemble to form globular structures are compact with high packing densities in the folded states. It is known that proteins unfold upon addition of denaturants, adopting random coil structures. The dependence of the radii of gyration on protein size in the folded and unfolded states obeys the same scaling laws as synthetic polymers. Thus, one might surmise that the mechanism of collapse in proteins and polymers ought to be similar. However, because the number of amino acids in single domain proteins is not significantly greater than about two hundred, it has not been resolved if the unfolded states of proteins are compact under conditions that favor the folded states - a problem at the heart of how proteins fold. By adopting a theory used to derive polymer-scaling laws, we find that the propensity for the unfolded state of a protein to be compact is universal and is encoded in the contact map of the folded state. Remarkably, analysis of over 2000 proteins shows that protei...

  15. Genome-wide identification and mapping of NBS-encoding resistance genes in Solanum tuberosum group phureja.

    Directory of Open Access Journals (Sweden)

    Roberto Lozano

    Full Text Available 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.

  16. The human HNRPD locus maps to 4q21 and encodes a highly conserved protein.

    Science.gov (United States)

    Dempsey, L A; Li, M J; DePace, A; Bray-Ward, P; Maizels, N

    1998-05-01

    The hnRNP D protein interacts with nucleic acids both in vivo and in vitro. Like many other proteins that interact with RNA, it contains RBD (or "RRM") domains and arg-gly-gly (RGG) motifs. We have examined the organization and localization of the human and murine genes that encode the hnRNP D protein. Comparison of the predicted sequences of the hnRNP D proteins in human and mouse shows that they are 96.9% identical (98.9% similar). This very high level of conservation suggests a critical function for hnRNP D. Sequence analysis of the human HNRPD gene shows that the protein is encoded by eight exons and that two additional exons specify sequences in the 3' UTR. Use of two of the coding exons is determined by alternative splicing of the HNRPD mRNA. The human HNRPD gene maps to 4q21. The mouse Hnrpd gene maps to the F region of chromosome 3, which is syntenic with the human 4q21 region.

  17. Identification of protein features encoded by alternative exons using Exon Ontology.

    Science.gov (United States)

    Tranchevent, Léon-Charles; Aubé, Fabien; Dulaurier, Louis; Benoit-Pilven, Clara; Rey, Amandine; Poret, Arnaud; Chautard, Emilie; Mortada, Hussein; Desmet, François-Olivier; Chakrama, Fatima Zahra; Moreno-Garcia, Maira Alejandra; Goillot, Evelyne; Janczarski, Stéphane; Mortreux, Franck; Bourgeois, Cyril F; Auboeuf, Didier

    2017-06-01

    Transcriptomic genome-wide analyses demonstrate massive variation of alternative splicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing variation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named "Exon Ontology," based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encoded protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applying this strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splicing. We also show that Exon Ontology helps to unravel biological processes that depend on suites of coregulated alternative exons, as we uncovered a role of epithelial cell-enriched splicing factors in the AKT signaling pathway and of mesenchymal cell-enriched splicing factors in driving splicing events impacting on autophagy. Freely available on the web, Exon Ontology is the first computational resource that allows getting a quick insight into the protein features encoded by alternative exons and investigating whether coregulated exons contain the same biological information. © 2017 Tranchevent et al.; Published by Cold Spring Harbor Laboratory Press.

  18. Detection of Leishmania spp. based on the gene encoding HSP20

    OpenAIRE

    Montalvo, Ana M; Departamento de Parasitología, Instituto de Medicina Tropical Pedro Kourí. La Habana, Cuba.; Fraga, Jorge; Departamento de Parasitología, Instituto de Medicina Tropical Pedro Kourí. La Habana, Cuba.; Rodríguez, Omaira; Laboratorio de referencia e investigación en enfermedades tropicales de sanidad militar. Bogotá, Colombia.; Blanco, Orestes; Departamento de Parasitología, Instituto de Medicina Tropical Pedro Kourí. La Habana, Cuba.; Llanos-Cuentas, Alejandro; Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia. Lima, Perú.; García, Ana L.; Universidad de San Simón. Cochabamba, Bolivia.; Valencia, Braulio M; Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia. Lima, Perú.; Muskus, Carlos; Programa de Estudio y Control de Enfermedades Tropicales, Universidad de Antioquia. Medellín, Colombia.; Van der Auwera, Gert; Biomedical Sciences Department. Institute of Tropical Medicine of Antwerp. Amberes, Bélgica.; Requena, José M; Centro de Biología Molecular Severo Ochoa. Madrid, España.

    2014-01-01

    Objectives. Explore a new target for molecular diagnosis of Leishmania. Materials and methods. We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. Results. The anal...

  19. A gene encoding a new cold-active lipase from an Antarctic isolate of Penicillium expansum.

    Science.gov (United States)

    Mohammed, Suja; Te'o, Junior; Nevalainen, Helena

    2013-08-01

    Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12-C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5'- and 3'-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.

  20. Escherichia coli contains a protein that is homologous in function and N-terminal sequence to the protein encoded by the nifS gene of Azotobacter vinelandii and that can participate in the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase.

    Science.gov (United States)

    Flint, D H

    1996-07-05

    In this paper, I report the purification of a protein from Escherichia coli that is very similar in sequence, molecular weight, and the reactions it can catalyze to the protein encoded by the Azotobacter vinelandii nifS gene. This E. coli protein contains pyridoxal phosphate as a cofactor and catalyzes the removal of sulfur from cysteine to form alanine and S0. When dithiothreitol is present along with cysteine, the S0 formed is reduced to S2-. This protein has a reactive sulfhydryl group that is essential for activity. As isolated, this sulfhydryl group appears to be in a disulfide linkage with the sulfhydryl group from the phosphopantetheine moiety of the acyl carrier protein. The purified E. coli protein can mobilize the sulfur from cysteine and contribute it to the formation of a [4Fe-4S] cluster on the apoprotein of E. coli dihydroxy-acid dehydratase. A mechanism is proposed for the early stages of the synthesis of Fe-S clusters using this protein and sulfur in the S0 oxidation state.

  1. [Mutations in the gene encoding filaggrin cause ichthyosis vulgaris].

    Science.gov (United States)

    Prasad, Sumangali Chandra; Rasmussen, Kirsten; Bygum, Anette

    2011-02-14

    Ichthyosis vulgaris is a common genetic skin disorder with an estimated prevalence of 1:250 caused by mutations in the gene encoding filaggrin. This disorder manifests itself within the first year of life and is clinically characterized by dry, scaly skin, keratosis pilaris, palmar hyperlinearity and atopic manifestations. Patients with a severe phenotype are homozygous or compound heterozygous for the mutations, whereas heterozygous patients show mild disease, suggesting semidominant inheritance with incomplete penetrance. We present a patient with classic severe ichthyosis vulgaris, atopic eczema and two loss-of-function mutations.

  2. Screening and analysis of hepatocellular carcinomaassociated antigens and their encoding genes

    Institute of Scientific and Technical Information of China (English)

    SHI Yongyu; WANG Hongcheng; LI Yan; PANG Xuewen; SUN Wensheng; CHEN Weifeng

    2003-01-01

    Identification of hepatocellular carcinoma- associated tumor antigens is necessary and pivotal for specific immunotherapy in hepatocellular carcinoma (HCC) patients. In the present study, HCC cDNAs are constructed into ZAP cDNA expression library and screened by sera of patients with HCC. The positive clones are DNA sequenced and analyzed by bioinformatics. Thirty-one genes of hepatocellular carcinoma-associated tumor antigens are identified, of which 1 is unknown and 30 are known. The proteins encoded by these known genes can be classified into 8 categories: constitutive molecules of hepatocytes, RNA transcription and splicing-associated molecules, protein metabolism-associated molecules, energy synthesis-associated molecules, signal transduction molecules, cell adhesion molecules, immunosuppressive molecules, and proteins with unknown function. Among these genes, CAGE is a cancer-testis (CT) antigen. It is concluded that identification of hepatocellular carcinoma-associated tumor antigens provides potential targets for immunotherapy of HCC patients and facilitates explanation of carcinogenesis of HCC.

  3. Construction, cloning, and expression of synthetic genes encoding spider dragline silk.

    Science.gov (United States)

    Prince, J T; McGrath, K P; DiGirolamo, C M; Kaplan, D L

    1995-08-29

    Synthetic genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the synthetic spider silks have substantial beta-sheet structure.

  4. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    Science.gov (United States)

    Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

    2007-03-13

    The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

  5. Antifungal activity of a virally encoded gene in transgenic wheat.

    Science.gov (United States)

    Clausen, M; Kräuter, R; Schachermayr, G; Potrykus, I; Sautter, C

    2000-04-01

    The cDNA encoding the antifungal protein KP4 from Ustilago maydis-infecting virus was inserted behind the ubiquitin promoter of maize and genetically transferred to wheat varieties particularly susceptible to stinking smut (Tilletia tritici) disease. The transgene was integrated and inherited over several generations. Of seven transgenic lines, three showed antifungal activity against U. maydis. The antifungal activity correlated with the presence of the KP4 transgene. KP4-transgenic, soil-grown wheat plants exhibit increased endogenous resistance against stinking smut.

  6. PEDV ORF3 encodes an ion channel protein and regulates virus production.

    Science.gov (United States)

    Wang, Kai; Lu, Wei; Chen, Jianfei; Xie, Shiqi; Shi, Hongyan; Hsu, Haojen; Yu, Wenjing; Xu, Ke; Bian, Chao; Fischer, Wolfgang B; Schwarz, Wolfgang; Feng, Li; Sun, Bing

    2012-02-17

    Several studies suggest that the open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to viral infectivity and pathogenicity, but its function remains unknown. Here, we propose a structure model of the ORF3 protein consisting of four TM domains and forming a tetrameric assembly. ORF3 protein can be detected in PEDV-infected cells and it functions as an ion channel in both Xenopus laevis oocytes and yeast. Mutation analysis showed that Tyr170 in TM4 is important for potassium channel activity. Furthermore, viral production is reduced in infected Vero cells when ORF3 gene is silenced by siRNA. Interestingly, the ORF3 gene from an attenuated PEDV encodes a truncated protein with 49 nucleotide deletions, which lacks the ion channel activity.

  7. The pea gene NA encodes ent-kaurenoic acid oxidase.

    Science.gov (United States)

    Davidson, Sandra E; Elliott, Robert C; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2003-01-01

    The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.

  8. AIdentification of encoding proteins related to SARS-CoV

    Institute of Scientific and Technical Information of China (English)

    MEI Hu; SUN Lili; ZHOU Yuan; XIONG Qing; LI Zhiliang

    2004-01-01

    By sampling 100 encoding proteins from SARS-coronavirus (SARS-CoV, NC 004718) and other six coronaviruses and selecting 23 variables through stepwise multiple regression (SMR) from 172 variables, the multiple linear regression (MLR) model was established with good results of the quantitative modelling correlation coefficient R2 = 0.645 and the cross-validation correlation coefficient 0.375. After removing 4 outliers, the quantitative modelling and cross-validation correlation coefficients were R2 = 0.743 and R2CV=0.543, respectively.

  9. Analysis of the Genes Encoding the Histones of Microsporidia Nosema bombycis

    Directory of Open Access Journals (Sweden)

    Liu Yang

    2013-02-01

    Full Text Available Histone proteins are essential components of eukaryotic chromosomes, the objective of the study is to provide some new insights into its evolution through analysis of N. bombycis Histone genes at genomic level. In the study, genes encoding core Histone H2A, H2B, H3 and H4 from Nosema bombycis were analyzed by multiple sequence alignments. Analysis showed that: each type of the core Histone genes, sharing high similarity with each other in both coding and non-coding regions, has low copy number. Multiple sequence alignments showed N. bombycis core Histones diverge obviously, relative-rate test revealed Histone proteins have accelerated in the evolutionary rate of amino acid substitution. The distance between the stop codon and consensus poly (A signal is compacted, no conserved hair-pin element was found in 3'-untranslated regions of Histone mRNAs and overlapping gene transcription was observed in the downstream region of Histone variant H3_3, that implies there maybe have only single class of core Histone genes encoding replication-independent Histones in N. bombycis. Surveying the upstream of the coding region of all core Histone genes, there were no canonical TATA or CAAT boxes except that a common Histone motif (TTTCCCTCC was discovered. Moreover, no similar Histone motif mentioned above existed in Encephalitozoon cuniculi, the closely related organisms. That means that similar Histone motif maybe exists in microsporidian last common ancestor, N. bombycis retained Histone motif, while E. cuniculi have lost Histone motif after the differentiation from the common ancestor with the change of the host. Therefore the analysis of the genes encoding the Histones ofN. bombycis revealed that there maybe have two evolution directions in microsporidia, that is, genome extreme compact and mild compact, during the course of evolution. It contributes us to have the knowledge of that there have different genome size in microsporidia and provide useful

  10. Silencing of the major family of NBS-LRR-encoding genes in lettuce results in the loss of multiple resistance specificities

    NARCIS (Netherlands)

    Wroblewski, T.; Piskurewicz, U.; Finkers-Tomczak, A.M.; Ochoa, O.; Michelmore, R.

    2007-01-01

    The RGC2 gene cluster in lettuce (Lactuca sativa) is one of the largest known families of genes encoding nucleotide binding site¿leucine-rich repeat (NBS¿LRR) proteins. One of its members, RGC2B, encodes Dm3 which determines resistance to downy mildew caused by the oomycete Bremia lactucae carrying

  11. A corm-specific gene encodes tarin, a major globulin of taro (Colocasia esculenta L. Schott).

    Science.gov (United States)

    Bezerra, I C; Castro, L A; Neshich, G; de Almeida, E R; de Sá, M F; Mello, L V; Monte-Neshich, D C

    1995-04-01

    A gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (G1, ca. 28 kDa) was isolated from a lambda Charon 35 library, using a cDNA derived from a highly abundant corm-specific mRNA, as probe. The gene, named tar1, and the corresponding cDNA were characterized and compared. No introns were found. The major transcription start site was determined by primer extension analysis. The gene has an open reading frame (ORF) of 765 bp, and the deduced amino acid sequence indicated a precursor polypeptide of 255 residues that is post-translationally processed into two subunits of about 12.5 kDa each. The deduced protein is 45% homologous to curculin, a sweet-tasting protein found in the fruit pulp of Curculigo latifolia and 40% homologous to a mannose-binding lectin from Galanthus nivalis. Significant similarity was also found at the nucleic acid sequence level with genes encoding lectins from plant species of the Amaryllidaceae and Lilliaceae families.

  12. Interspecies transfer of the penicillin-binding protein 3-encoding gene ftsI between Haemophilus influenzae and Haemophilus haemolyticus can confer reduced susceptibility to β-lactam antimicrobial agents.

    Science.gov (United States)

    Søndergaard, Annette; Witherden, Elizabeth A; Nørskov-Lauritsen, Niels; Tristram, Stephen G

    2015-07-01

    Mutations in ftsI, encoding penicillin-binding protein 3, can cause decreased β-lactam susceptibility in Haemophilus influenzae. Sequencing of ftsI from clinical strains has indicated interspecies recombination of ftsI between H. influenzae and Haemophilus haemolyticus. This study documented apparently unrestricted homologous recombination of ftsI between H. influenzae and H. haemolyticus in vitro. Transfer of ftsI from resistant isolates conferred similar but not identical increases in the MICs of susceptible strains of H. influenzae and H. haemolyticus.

  13. [Cloning and structure of gene encoded alpha-latrocrustoxin from the Black widow spider venom].

    Science.gov (United States)

    Danilevich, V N; Luk'ianov, S A; Grishin, E V

    1999-07-01

    The primary structure of the crusta gene encoding alpha-latrocrustoxin (alpha-LCT), a high molecular mass neurotoxin specific to crustaceans, was determined in the black widow spider Latrodectus mactans tredicimguttatus genome. The total length of the sequenced DNA was 4693 bp. The structural part of the black widow spider chromosome gene encoding alpha-LCT does not contain introns. The sequenced DNA contains a single extended open reading frame (4185 bp) and encodes a protein precursor of alpha-LCT, comprising 1395 aa. We assume the Met residue at position -10 relative to the N-terminal residue of Glu1 of the mature toxin to be the first one in the protein precursor. The calculated molecular mass of the precursor (156147 Da) exceeds that of the mature toxin by approximately 30 kDa. These data are in agreement with the notion that over the course of maturation the protein precursor undergoes double processing--cleavage of a decapeptide from the N-terminal part and of a approximately 200-aa fragment from the C-terminal part. alpha-LCT displayed a number of imperfect ankyrin-like repeats and areas of structural homology with earlier studied latrotoxins; the highest homology degree (62%) was revealed with alpha-latroinsectotoxin (alpha-LIT).

  14. The mouse nac1 gene, encoding a cocaine-regulated Bric-a-brac Tramtrac Broad complex/Pox virus and Zinc finger protein, is regulated by AP1.

    Science.gov (United States)

    Mackler, S A; Homan, Y X; Korutla, L; Conti, A C; Blendy, J A

    2003-01-01

    NAC1 cDNA was identified as a novel transcript induced in the nucleus accumbens from rats chronically treated with cocaine. NAC1 is a member of the Bric-a-brac Tramtrac Broad complex/Pox virus and Zinc finger family of transcription factors and has been shown by overexpression studies to prevent the development of behavioral sensitization resulting from repeated cocaine treatment. This paper reports the cloning and characterization of the corresponding gene. The mouse Nac1 gene consist of six exons, with exon 2 containing an alternative splice donor, providing a molecular explanation of the splice variants observed in mouse and rat. Transcripts of Nac1 were ubiquitously detected in different mouse tissues with prominent expression in the brain. The mouse Nac1 gene was localized to chromosome 8, suggesting a highly plausible candidate gene to explain differences in cocaine-induced behaviors between C57BL6/J and DBA/2J mice that had previously been mapped to the area. In addition, a functional AP1 binding site has been identified in an intron 1 enhancer of the Nac1 gene that plays an essential role in the activation of the gene in differentiation of neuroblastoma cells. Co-transfection with c-jun and c-fos expression plasmids, which encode the two subunits of AP1, activated the wild type Nac1 intron 1 enhancer two-fold over basal, nearly at the level of NAC1 enhancer activity seen in differenti