Sample records for ii nptii gene

  1. Transfer and expression of the bacterial NPT-II gene in chick embryos using a Schmidt-Ruppin retrovirus vector.

    PubMed Central

    Hippenmeyer, P J; Krivi, G G; Highkin, M K

    1988-01-01

    In an effort to introduce foreign genes into chickens, the bacterial neomycin phosphotransferase (NPT-II) gene was cloned into an infectious avian retroviral vector derived from the Schmidt-Ruppin A strain of RSV. The NPT-II gene was stable in the vector during passage in vitro and infected cells were resistant to G418. Fertilized chicken embryos were inoculated with the recombinant virus on day 0 and screened on day 20 for the NPT-II gene in blood cell DNA. Approximately 12% of the embryos were positive for the NPT-II gene. Screening of DNA from the brain, muscle, liver and foot of the positive embryos indicated that the NPT-II gene copy number could vary in a single embryo. However, some embryos had nearly equal NPT-II copy number in each tissue examined. To determine the expression of the bacterial gene, tissue extracts from the positive embryos were assayed for NPT-II activity. The results indicated that NPT-II activity varied depending on the tissue, with activity being highest in muscle and foot regardless of NPT-II gene copy number. Images PMID:2842731

  2. Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.).

    PubMed

    Herzog, Katja; Flachowsky, Henryk; Deising, Holger B; Hanke, Magda-Viola

    2012-04-25

    Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene. PMID:22349025

  3. Small RNAs were involved in homozygous state-associated silencing of a marker gene (Neomycin phosphotransferase II: nptII) in transgenic tomato plants.

    PubMed

    Deng, Lei; Pan, Yu; Chen, Xuqing; Chen, Guoping; Hu, Zongli

    2013-07-01

    Homozygous state-associated co-suppression is not a very common phenomenon. In our experiments, two transgenic plants 3A29 and 1195A were constructed by being transformed with the constructs pBIN-353A and pBIN119A containing nptII gene as a marker respectively. The homozygous progeny from these two independent transgenic lines 3A29 and 1195A, displayed kanamycin-sensitivity and produced a short main root without any lateral roots as untransformed control (wild-type) seedlings when germinated on kanamycin media. For the seedlings derived from putative hemizygous plants, the percentage of the seedlings showing normal growth on kanamycin media was about 50% and lower than the expected percentage (75%). Southern analysis of the genomic DNA confirmed that the homozygous and hemizygous plants derived from the same lines contained the same multiple nptII transgenes, which were located on the same site of chromosome. Northern analysis suggested that the marker nptII gene was expressed in the primary and the hemizygous transformants, but it was silenced in the homozygous transgenic plants. Further Northern analysis indicated that antisense and sense small nptII-derived RNAs were present in the transgenic plants and the blotting signal of nptII-derived small RNA was much higher in the homozygous transgenic plants than that of hemizygous transgenic plants. Additionally, read-through transcripts from the TRAMP gene to the nptII gene were detected. These results suggest that the read-through transcripts may be involved in homozygous state-associated silencing of the nptII transgene in transgenic tomato plants and a certain threshold level of the nptII-derived small RNAs is required for the homozygous state-associated co-suppression of the nptII transgene. PMID:23612328

  4. Survival of plant seeds, their UV screens, and nptII DNA for 18 months outside the International Space Station.

    PubMed

    Tepfer, David; Zalar, Andreja; Leach, Sydney

    2012-05-01

    The plausibility that life was imported to Earth from elsewhere can be tested by subjecting life-forms to space travel. Ultraviolet light is the major liability in short-term exposures (Horneck et al., 2001 ), and plant seeds, tardigrades, and lichens-but not microorganisms and their spores-are candidates for long-term survival (Anikeeva et al., 1990 ; Sancho et al., 2007 ; Jönsson et al., 2008 ; de la Torre et al., 2010 ). In the present study, plant seeds germinated after 1.5 years of exposure to solar UV, solar and galactic cosmic radiation, temperature fluctuations, and space vacuum outside the International Space Station. Of the 2100 exposed wild-type Arabidopsis thaliana and Nicotiana tabacum (tobacco) seeds, 23% produced viable plants after return to Earth. Survival was lower in the Arabidopsis Wassilewskija ecotype and in mutants (tt4-8 and fah1-2) lacking UV screens. The highest survival occurred in tobacco (44%). Germination was delayed in seeds shielded from solar light, yet full survival was attained, which indicates that longer space travel would be possible for seeds embedded in an opaque matrix. We conclude that a naked, seed-like entity could have survived exposure to solar UV radiation during a hypothetical transfer from Mars to Earth. Chemical samples of seed flavonoid UV screens were degraded by UV, but their overall capacity to absorb UV was retained. Naked DNA encoding the nptII gene (kanamycin resistance) was also degraded by UV. A fragment, however, was detected by the polymerase chain reaction, and the gene survived in space when protected from UV. Even if seeds do not survive, components (e.g., their DNA) might survive transfer over cosmic distances. PMID:22680697

  5. Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics

    PubMed Central

    2012-01-01

    Background The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes ?-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. Results The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3?years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. Conclusions Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest. PMID:22794278

  6. Tobacco (Nicotiana tobaccum) nuclear transgenics with high copy number can express NPTII driven by the chloroplast psbA promoter.

    PubMed

    Ye, G N; Pang, S Z; Sanford, J C

    1996-03-01

    A chloroplast expression vector containing the NPTII gene under the control of apsbA promoter (psbA-NPTII) was constructed, and was biolistically delivered into both suspension cells and leaf strips of tobacco (Nicotiana tabaccum). Analyses of subsequently recovered kanamycin-resistant transgenic plants indicate that the psbA-NPTII gene was not located in the chloroplast, but was in the nucleus in very high copy number. This conclusion was based upon results from: (1) Southern hybridization analyses of chloroplast and nuclear DNAs using NPTII, chloroplast-marker, and nuclear-marker probes; (2) pulse-field gel electrophoresis; and (3) kanamycin screening of sexual progenies. This study suggests that the nuclear expression of the NPTII gene may have been associated with many copies of the psbA-NPTII construction. Very high copy number in the nucleus might either allow NPTII expression from the otherwise inadequate psbA promoter, or might increase the chance of recombining with upstream tobacco regulatory sequences. PMID:24178457

  7. Evaluation of an ELISA assay for rapid detection and quantification of neomycin phosphotransferase II in transgenic plants

    Microsoft Academic Search

    Roland J. Nagel; John M. Manners; Robert G. Birch

    1992-01-01

    Neomycin phosphotransferase II (neo) is a selectable marker gene used extensively in plant transformation experiments. Here we evaluate immunological detection\\u000a of its gene product (NPTII) as an alternative to widely used radioactive assays. We have taken a commercially available non-radioactive\\u000a NPTII Enzyme linked-Immunosorbant Assay (ELISA) kit, modified the protocol for application to plant tissues, and used it to\\u000a quantify levels

  8. Investigation on gene transfer from genetically modified corn (Zea mays L.) plants to soil bacteria

    Microsoft Academic Search

    B. L. Ma; Robert E. Blackshaw; Julie Roy; Tianpei He

    2011-01-01

    Knowledge about the prevalence and diversity of antibiotic resistance genes in soil bacteria communities is required to evaluate the possibility and ecological consequences of the transfer of these genes carried by genetically modified (GM) plants to soil bacteria. The neomycin phosphotransferase gene (nptII) conferring resistance to kanamycin and neomycin is one of the antibiotic resistance genes commonly present in GM

  9. Transformation of Brassica napus L. using Agrobacterium tumefaciens : developmentally regulated expression of a reintroduced napin gene

    Microsoft Academic Search

    S. E. Radke; B. M. Andrews; M. M. Moloney; M. L. Crouch; J. C. Kridl; V. C. Knauf

    1988-01-01

    Genetically transformed plants of Brassica napus L. (oilseed rape) were obtained from hypocotyl expiants using Agrobacterium tumefaciens vectors. Hypocotyl explants were inoculated with disarmed or oncogenic A. tumefaciens strains, EHA101 and A281, and then cultured on media containing kanamycin. The A. tumefaciens strains harbored a binary vector, which contained a neomycin phosphotransferase II (NPTII) gene driven by the 35S promoter

  10. Effect of over-expression of Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE ( LuPLR ) gene in transgenic Phyllanthus amarus

    Microsoft Academic Search

    Anindita Banerjee; Sharmila Chattopadhyay

    2010-01-01

    Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin\\u000a phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l?1, kan

  11. Agrobacterium -mediated transformation of cotton ( Gossypium hirsutum ) using a heterologous bean chitinase gene

    Microsoft Academic Search

    Masoud Tohidfar; Mojtaba Mohammadi; Behzad Ghareyazie

    2005-01-01

    Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l?1

  12. Monitoring field releases of genetically modified sugar beets for persistence of transgenic plant DNA and horizontal gene transfer

    Microsoft Academic Search

    Frank Gebhard; Kornelia Smalla

    1999-01-01

    Field releases of transgenic rizomania-resistant sugar beet (Beta vulgaris) plants were accompanied by a study of the persistence of DNA from transgenic sugar beet litter in soil and of horizontal gene transfer of plant DNA to bacteria. The transgenic sugar beets contained the marker genes nptII and bar under the control of the bidirectional TR1\\/2 promoter conferring kanamycin (Km) and

  13. No impact of transgenic nptII-leafy Pinus radiata (Pinales: Pinaceae) on Pseudocoremia suavis (Lepidoptera: Geometridae) or its endoparasitoid Meteorus pulchricornis (Hymenoptera: Braconidae).

    PubMed

    Burgess, E P J; Barraclough, E I; Kean, A M; Walter, C; Malone, L A

    2011-10-01

    To investigate the biosafety to insects of transgenic Pinus radiata D. Don containing the antibiotic resistance marker gene nptII and the reproductive control gene leafy, bioassays were conducted with an endemic lepidopteran pest of New Zealand plantation pine forests and a hymenopteran endoparasitoid. Larvae of the common forest looper, Pseudocoremia suavis (Butler), were fed from hatching on P. radiata needles from either one of two nptII-leafy transgenic clones, or an isogenic unmodified control line. For both unparasitized P. suavis and those parasitized by Meteorus pulchricornis (Wesmael), consuming transgenic versus control pine had no impact on larval growth rate or mass at any age, larval duration, survival, pupation or successful emergence as an adult. Total larval duration was 1 d (3%) longer in larvae fed nptII-2 than nptII-1, but this difference was considered trivial and neither differed from the control. In unparasitized P. suavis larvae, pine type consumed did not affect rate of pupation or adult emergence, pupal mass, or pupal duration. Pine type had no effect on the duration or survival of M. pulchricornis larval or pupal stages, mass of cocoons, stage at which they died, adult emergence, or fecundity. Parasitism by M. pulchricornis reduced P. suavis larval growth rate, increased the duration of the third larval stadium, and resulted in the death of all host larvae before pupation. The lack of impact of an exclusive diet of nptII-leafy transgenic pines on the life history of P. suavis and M. pulchricornis suggests that transgenic plantation pines expressing nptII are unlikely to affect insect populations in the field. PMID:22251744

  14. 40 CFR 174.521 - Neomycin phosphotransferase II; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...phosphotransferase II; exemption from the requirement of a tolerance. Residues of the neomycin phosphotransferase II (NPTII) enzyme are exempted from the requirement of a tolerance in all food commodities when used as a plant-incorporated protectant...

  15. 40 CFR 174.521 - Neomycin phosphotransferase II; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...phosphotransferase II; exemption from the requirement of a tolerance. Residues of the neomycin phosphotransferase II (NPTII) enzyme are exempted from the requirement of a tolerance in all food commodities when used as a plant-incorporated protectant...

  16. 40 CFR 174.521 - Neomycin phosphotransferase II; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...phosphotransferase II; exemption from the requirement of a tolerance. Residues of the neomycin phosphotransferase II (NPTII) enzyme are exempted from the requirement of a tolerance in all food commodities when used as a plant-incorporated protectant...

  17. ROR1/RPA2A, a Putative Replication Protein A2, Functions in Epigenetic Gene Silencing and in Regulation of Meristem Development in ArabidopsisW?

    PubMed Central

    Xia, Ran; Wang, Junguo; Liu, Chunyan; Wang, Yu; Wang, Youqun; Zhai, Jixian; Liu, Jun; Hong, Xuhui; Cao, Xiaofeng; Zhu, Jian-Kang; Gong, Zhizhong

    2006-01-01

    We screened for suppressors of repressor of silencing1 (ros1) using the silenced 35S promoter-neomycin phosphotransferase II (Pro35S:NPTII) gene as a marker and identified two allelic mutants, ror1-1 and ror1-2 (for suppressor of ros1). Map-based cloning revealed that ROR1 encodes a 31-kD protein similar to DNA replication protein A2 (RPA2A). Mutations in ROR1 reactivate the silenced Pro35S:NPTII gene but not RD29A promoter-luciferase in the ros1 mutant. DNA methylation in rDNA, centromeric DNA, and RD29A promoter regions is not affected by ror1. However, chromatin immunoprecipitation data suggest that histone H3 acetylation is increased and histone H3K9 dimethylation is decreased in the 35S promoter in the ror1 ros1 mutant compared with ros1. These results indicate that release of silenced Pro35S:NPTII by ror1 mutations is independent of DNA methylation. ROR1/RPA2A is strongly expressed in shoot and root meristems. Mutations in ROR1/RPA2A affect cell division in meristems but not final cell sizes. Our work suggests important roles of ROR1/RPA2A in epigenetic gene silencing and in the regulation of plant development. PMID:16326925

  18. [Transformation of wheat with insecticide gene of arrowhead proteinase inhibitor by pollen tube pathway and analysis of transgenic plants] [In Process Citation

    PubMed

    Mu; Liu; Zhou; Wen; Zhang; Wei

    1999-01-01

    Arrowhead Proteinase Inhibitor(API), one kind of pure natural material, was derived from storage organ of Sagittaria trifolia. It belongs to serine proteinase inhibitor, and can inhibit trypsin, chemotrypsin and kallikrein. Furthermore, API is toxical to some species of insects such as lepidotera, Coleoptera and Diptrea etc. By means of pollen tube pathway, plasmid pBIAH-A(B) containing insect-resistant genes of API-A, API-B and selective marker gene of NPT-II were transferred into three lines of local winter wheat--JD-1, 8866, 866554. Then, Kanamycin-resistant screening and PCR analysis of genetic transformed plants showed that three of Kmr green plants (two from 866554, one from JD-1) were PCR positive with the positive rate of 0.29%. When the fragment of API gene was used as probe to hybrid with genomic DNA of Kmr green plants separately, all of three PCR positive ones displayed a single strong hybridizing band. Such results demonstrated that foreign target gene had been integrated into wheat genome already. Simultaneously, PCR analysis and Southern hybridization were carried out among selfiedoffsprings of transformed positive plant of the line 899554-3, some of them were PCR and Southern blotting positive, indicating that foreign gene integrated into wheat genome could stably transmitted into next generation. In addition, the expression level of NPT-II gene was checked via ELISA in our study, all of three PCR and Southern blot positive plants could yield high level of NPT-II. This data provided a more powerful evidence for integration of insecticide gene into wheat genome. PMID:10876664

  19. Gene duplication and gene conversion in class II MHC genes of New Zealand robins (Petroicidae).

    PubMed

    Miller, Hilary C; Lambert, David M

    2004-06-01

    In contrast to mammals, the evolution of MHC genes in birds appears to be characterized by high rates of gene duplication and concerted evolution. To further our understanding of the evolution of passerine MHC genes, we have isolated class II B sequences from two species of New Zealand robins, the South Island robin (Petroica australis australis), and the endangered Chatham Island black robin (Petroica traversi). Using an RT-PCR based approach we isolated four transcribed class II B MHC sequences from the black robin, and eight sequences from the South Island robin. RFLP analysis indicated that all class II B loci were contained within a single linkage group. Analysis of 3'-untranslated region sequences enabled putative orthologous loci to be identified in the two species, and indicated that multiple rounds of gene duplication have occurred within the MHC of New Zealand robins. The orthologous relationships are not retained within the coding region of the gene, instead the sequences group within species. A number of putative gene conversion events were identified across the length of our sequences that may account for this. Exon 2 sequences are highly diverse and appear to have diverged under balancing selection. It is also possible that gene conversion involving short stretches of sequence within exon 2 adds to this diversity. Our study is the first report of putative orthologous MHC loci in passerines, and provides further evidence for the importance of gene duplication and gene conversion in the evolution of the passerine MHC. PMID:15138734

  20. MicroRNA genes are transcribed by RNA polymerase II

    Microsoft Academic Search

    Yoontae Lee; Minju Kim; Jinju Han; Kyu-Hyun Yeom; Sanghyuk Lee; Sung Hee Baek; V Narry Kim

    2004-01-01

    MicroRNAs (miRNAs) constitute a large family of noncod- ing RNAs that function as guide molecules in diverse gene silencing pathways. Current efforts are focused on the regulatory function of miRNAs, while little is known about how these unusual genes themselves are regulated. Here we present the first direct evidence that miRNA genes are transcribed by RNA polymerase II (pol II).

  1. Testing Groups of Genes Part II: Scoring Gene Ontology Terms

    E-print Network

    Spang, Rainer

    ­ Decorrelating the GO (elim, weight), Alexa et al. (2006) ­ Parent-child approach, Grossmann et al. (2007 the genes of its child ­ a node can contain genes that are not found in the children · a subset of genes that we call significant genes (differentially expressed genes) Goal: · find the nodes from the graph

  2. Stability of transcription complexes on class II genes

    SciTech Connect

    VanDyke, M.W.; Sawadogo, M.; Roeder, R.G.

    1989-01-01

    Commitment of a TATA box-driven class II gene to transcription requires binding of only one transcription factor, TFIID. Additional factors (TFIIB,TFIIE, and RNA polymerase II) do not remain associated with the TFIID-promoter complex during the course of transcription. This indicates that there are two intermediates along the transcription reaction pathway which may be potential targets for the regulation of gene expression.

  3. MED 263: Microarray Data Analysis II Gene Classifier Construction

    E-print Network

    Gleeson, Joseph G.

    predicts clinical outcome of breast cancer. Nature, 2002. 415(6871): p. 530-6. 5.McHale, CM, et al cancer: Oncotype DX (Genomic Health Inc., Redwood City, CA) The Breast Cancer Gene Expression RatioMED 263: Microarray Data Analysis II Gene Classifier Construction #12;What you will learn

  4. Transcription arrest relief by S-II\\/TFIIS during gene expression in erythroblast differentiation

    Microsoft Academic Search

    Makiko Nagata; Takahiro Ito; Nagisa Arimitsu; Hiroshi Koyama; Kazuhisa Sekimizu

    2009-01-01

    Transcription stimulator S-II relieves RNA polymerase II (RNAPII) from transcription elongation arrest. Mice lacking the S-II gene (S-II KO mice) die at mid-gestation with impaired erythroblast differentiation, and have decreased expression of the Bcl-x gene. To understand a role of S-II in Bcl-x gene expression, we examined the distribution of transcription complex on the Bcl-x gene in S-II KO mice.

  5. Overview of BioCreative II gene normalization

    PubMed Central

    Morgan, Alexander A; Lu, Zhiyong; Wang, Xinglong; Cohen, Aaron M; Fluck, Juliane; Ruch, Patrick; Divoli, Anna; Fundel, Katrin; Leaman, Robert; Hakenberg, Jörg; Sun, Chengjie; Liu, Heng-hui; Torres, Rafael; Krauthammer, Michael; Lau, William W; Liu, Hongfang; Hsu, Chun-Nan; Schuemie, Martijn; Cohen, K Bretonnel; Hirschman, Lynette

    2008-01-01

    Background: The goal of the gene normalization task is to link genes or gene products mentioned in the literature to biological databases. This is a key step in an accurate search of the biological literature. It is a challenging task, even for the human expert; genes are often described rather than referred to by gene symbol and, confusingly, one gene name may refer to different genes (often from different organisms). For BioCreative II, the task was to list the Entrez Gene identifiers for human genes or gene products mentioned in PubMed/MEDLINE abstracts. We selected abstracts associated with articles previously curated for human genes. We provided 281 expert-annotated abstracts containing 684 gene identifiers for training, and a blind test set of 262 documents containing 785 identifiers, with a gold standard created by expert annotators. Inter-annotator agreement was measured at over 90%. Results: Twenty groups submitted one to three runs each, for a total of 54 runs. Three systems achieved F-measures (balanced precision and recall) between 0.80 and 0.81. Combining the system outputs using simple voting schemes and classifiers obtained improved results; the best composite system achieved an F-measure of 0.92 with 10-fold cross-validation. A 'maximum recall' system based on the pooled responses of all participants gave a recall of 0.97 (with precision 0.23), identifying 763 out of 785 identifiers. Conclusion: Major advances for the BioCreative II gene normalization task include broader participation (20 versus 8 teams) and a pooled system performance comparable to human experts, at over 90% agreement. These results show promise as tools to link the literature with biological databases. PMID:18834494

  6. Homology-dependent DNA Transfer from Plants to a Soil Bacterium Under Laboratory Conditions: Implications in Evolution and Horizontal Gene Transfer

    Microsoft Academic Search

    David Tepfer; Rolando Garcia-Gonzales; Hounayda Mansouri; Martina Seruga; Brigitte Message; Francesca Leach; Mirna Curkovic Perica

    2003-01-01

    DNA transfer was demonstrated from six species of donor plants to the soil bacterium, Acinetobacter spp. BD413, using neomycin phosphotransferase (nptII) as a marker for homologous recombination. These laboratory results are compatible with, but do not prove, DNA transfer in nature. In tobacco carrying a plastid insertion of nptII, transfer was detected with 0.1 g of disrupted leaves and in

  7. Co-transformation of grapevine somatic embryos to produce transgenic plants free of marker genes.

    PubMed

    Dutt, Manjul; Li, Zhijian T; Dhekney, Sadanand A; Gray, Dennis J

    2012-01-01

    A cotransformation system using somatic embryos was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. "Thompson Seedless" somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bidirectional dual promoter complex. Our technique included a short positive selection phase of cotransformed somatic embryos on liquid medium containing 100 mg/L kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg/L 5-fluorocytosine. PMID:22351010

  8. Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene.

    PubMed

    Singh, Deepali; Ambroise, Annick; Haicour, Robert; Sihachakr, Darasinh; Rajam, Manchikatla Venkat

    2014-04-01

    The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5-7 days in the disease development as compared to wild-type plants. PMID:24757318

  9. Screening of three Usher syndrome type II candidate genes

    SciTech Connect

    Bloemker, B.K. [Boys Town National Research hospital, Omaha, NE (United States); Swaroop, A. [Univ. of Michigan, Ann Arbor, MI (United States); Kimberling, W.J. [Yale medical School, New Haven, CT (United States)

    1994-09-01

    Usher syndrome type II (US2) is an autosomal recessive disorder that results in blindness due to retinitis pigmentosa and congenital hearing loss. The disease affects approximately 1 in 20,000 individuals in the general population and is responsible for over 50% of all cases of deafness with blindness. The underlying US2 defect is unknown. The US2 gene has been localized to the 1q41 region of chromosome 1 by linkage studies. Three genes previously localized to 1q were analyzed to assess their candidacy as the US2 gene. These were evaluated by PCR assays using DNA from a YAC contig spanning the US2 region on chromosome 1. The first gene evaluated was the human choroideremia-like gene (hCHML), which had been mapped to chromosome 1q. The sequence on 1q is a homologue of the human choroideremia gene on chromosome X. Choroideremia is a degenerative disorder causing ocular pathology similar to that observed in US2 patients. Therefore, hCHML is a candidate for the US2 gene. Two cDNAs (A and B) from an enriched human retinal pigment epithelium library have been mapped to 1q41 by in situ hybridization. Both cDNAs are considered good candidates. The hCHML and cDNA A were ruled out as candidates for the US2 gene based on negative results from PCR assays performed on YACs spanning the US2 region. cDNA B could not be ruled out as a candidate for the US2 gene by these assays. Answers to many clinical questions regarding US2 will only be resolved after the gene is identified and characterized. Eventually, understanding the function and expression of the US2 gene will provide a basis for the development of therapy.

  10. Enhanced etoposide sensitivity following adenovirus-mediated human topoisomerase II ? gene transfer is independent of topoisomerase II ?

    PubMed Central

    Zhou, Z; Zwelling, L A; Ganapathi, R; Kleinerman, E S

    2001-01-01

    The roles that the ? and ? isoforms of topoisomerase II (topo II) play in anticancer drug action were determined using MDA-VP etoposide-resistant human breast cancer cells and a newly constructed adenoviral vector containing the topo II? gene (Ad-topo II?). MDA-VP cells were more resistant to etoposide than to amsacrine and had more resistance to etoposide than did MDA-parental cells. MDA-VP cells also expressed lower topo II? RNA and protein levels than parental cells but had comparable topo II? levels. After infection with Ad-topo II?, topo II?, RNA and protein levels increased significantly, as did the cells' sensitivity to etoposide. In contrast, topo II? levels remained constant with little alteration in the cells' sensitivity to amsacrine. Band-depletion immunoblotting assays indicated that topo II? was depleted in etoposide-treated, Ad-topo II?-transduced MDA-VP cells but not in amsacrine-treated cells. Topo II? was depleted in amsacrine-treated, Ad-topo II?-MDA-VP cells, with little change in the topo II? levels. These results suggest that topo II? gene transfer does not alter topo II? expression and that enhanced sensitivity to etoposide is therefore secondary to change in topo II? levels. These studies support the theory that etoposide preferentially targets topo II?, while amsacrine targets topo II?. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11531262

  11. Screening of three Usher syndrome type II candidate genes

    Microsoft Academic Search

    B. K. Bloemker; A. Swaroop; W. J. Kimberling

    1994-01-01

    Usher syndrome type II (US2) is an autosomal recessive disorder that results in blindness due to retinitis pigmentosa and congenital hearing loss. The disease affects approximately 1 in 20,000 individuals in the general population and is responsible for over 50% of all cases of deafness with blindness. The underlying US2 defect is unknown. The US2 gene has been localized to

  12. Head and neck cancer: gene therapy approaches. Part II: genes delivered.

    PubMed

    Nemunaitis, John; O'Brien, John

    2002-03-01

    In Part I, the review summarised the safety of adenoviral vectors and provided insight into approaches being undertaken to improve the specificity, durability and potency of adenoviral delivery vehicles. In Part II, brief discussions are held regarding results of preclinical and clinical trials with a variety of different genes, which have demonstrated antitumour activity in squamous cell carcinoma of the head and neck region (HNSCC). Studies have been performed with a variety of immune modulatory genes. Preliminary results demonstrate activity with several cytokine genes, tumour antigen genes and co-stimulatory molecule genes. Despite only preliminary results, thus far, a theoretical attractive feature for the use of gene therapy for the enhancement of immune modulation is that local injection of the gene product appears to be well tolerated. It is also successful in inducing systemic immune response, potentially providing effect to metastatic sites distal from the injected site. Animal studies have confirmed efficacy in the use of specific targeting of molecules regulating cancer growth (EGF receptor [EGFR], super oxide dismutase [SOD], cyclin D1, E1A and Bcl-2). These approaches are discussed. However, the most significant clinical advances for the use of gene therapy in advanced HNSCC involves two agents: Adp53 and ONYX-015. Preliminary Phase I and II results suggest evidence of efficacy and justify accrual Phase III trials, which are currently ongoing. PMID:11890870

  13. Molecular characterization of chicken class II transactivator gene.

    PubMed

    Nikbakht Brujeni, Gholamreza; Khosravi, Mohammad

    2015-01-01

    Class II transactivator (CIITA) is an effective transcriptional factor regulating various genes in the immune system. Since the detection of CIITA in 1993, there has been considerable progress toward understanding its role as an activator of MHC II genes in human and mouse; however, there is little knowledge of this gene in other animals such as chicken. Molecular characterization of the chicken CIITA gene transcript was performed to determine its sequence and expression in different tissues. The CIITA cDNA was first generated through reverse transcriptase-polymerase chain reaction (RT-PCR) from Cobb chicken spleen cell RNA, using oligonucleotide primers based on the predicted cDNA sequence. The effect of the immune system stimulation on the CIITA gene expression in kidney, liver, thymus, and spleen were assessed by semi-quantitative RT-PCR analysis. A partial cDNA sequence (1,688 bp) encoding part of the NACHT domain followed by seven of the transactivator and one of the NLS domains were obtained. Comparison of the deduced amino acid sequence with other CIITAs reveals high level of similarities in amino acid composition, secondary structure and phosphorylation sites. Furthermore, in comparison to the Red Jungle Fowl (RJF) sequence, we found 17 single nucleotide polymorphisms in Cobb broiler chicken, ten of which were reported for the first time. Gene expression analysis indicated that CIITA RNA amounts increased in all the examined tissues following stimulation with Brucella antigen. This investigation may indicate that CIITA molecule has an important role in the chicken immune responses as well as human and other animals. PMID:25339383

  14. [Genetic transformation of flax (Linum usitatissimum L.) with chimeric GFP-TUA6 gene for visualisation of microtubules].

    PubMed

    Shisha, E N; Korkhovo?, V I; Baer, G Ia; Guzenko, E V; Lemesh, V A; Kartel', N A; Emets, A I; Blium, Ia B

    2013-01-01

    The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind. PMID:23745358

  15. Identification of the Gene at the pmg Locus, Encoding System II, the General Amino Acid

    E-print Network

    Bowman, Barry

    Identification of the Gene at the pmg Locus, Encoding System II, the General Amino Acid Transporter of the gene at the pmg locus, encoding system II, the general amino acid transporter in Neurospora crassa a deficiency in a transport system for a broad range of amino acids. We have isolated a gene that encodes

  16. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

    DOEpatents

    Ingram, Lonnie O. (Gainesville, FL); Conway, Tyrrell (Gainesville, FL)

    1992-01-01

    The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

  17. From Gene Trees to Species Trees II: Species Tree Inference by Minimizing Deep

    E-print Network

    Zhang, Louxin

    From Gene Trees to Species Trees II: Species Tree Inference by Minimizing Deep Coalescence Events Louxin Zhang Abstract--When gene copies are sampled from various species, the resulting gene tree might disagree with the containing species tree. The primary causes of gene tree and species tree discord include

  18. Unsuccessful search for DNA transfer from transgenic plants to bacteria in the intestine of the tobacco horn worm, Manduca sexta

    Microsoft Academic Search

    Jamal Deni; Brigitte Message; Maurizio Chioccioli; David Tepfer

    2005-01-01

    DNA transfer from transgenic plants to native intestinal bacteria and introduced Acinetobacter BD413 was assessed in the gut of the tobacco horn worm (Manduca sexta). The marker was kanamycin resistance gene (nptII), and tobacco carrying the nptII gene in the chloroplasts served as the donor. We detected neither whole gene transfer to native bacteria, nor transfer of fragments of nptII

  19. Trans-activator gene of HTLV-II induces IL-2 receptor and IL-2 cellular gene expression.

    PubMed

    Greene, W C; Leonard, W J; Wano, Y; Svetlik, P B; Peffer, N J; Sodroski, J G; Rosen, C A; Goh, W C; Haseltine, W A

    1986-05-16

    The human T-lymphotropic viruses types I and II (HTLV-I and -II) have been etiologically linked with certain T-cell leukemias and lymphomas that characteristically display membrane receptors for interleukin-2. The relation of these viruses to this growth factor receptor has remained unexplained. It is demonstrated here that introduction of the trans-activator (tat) gene of HTLV-II into the Jurkat T-lymphoid cell line results in the induction of both interleukin-2 receptor and interleukin-2 gene expression. The coexpression of these cellular genes may play a role in the altering T-cell growth following retroviral infection. PMID:3010456

  20. Cloning of genes for production of Escherichia coli Shiga-like toxin type II.

    PubMed Central

    Newland, J W; Strockbine, N A; Neill, R J

    1987-01-01

    Genes controlling production of Shiga-like toxin type II (SLT-II) in Escherichia coli were cloned from the SLT-II-converting bacteriophage 933W and compared with the Shiga-like toxin type I (SLT-I) genes previously isolated and described from phage 933J. Subcloning analysis identified a region within the 4.9-kilobase EcoRI fragment of phage 933W that was associated with SLT-II production. Experiments with E. coli minicells containing these subclones demonstrated that the 4.9-kilobase EcoRI fragment encodes the structural genes for SLT-II. These experiments additionally showed the genetic organization of the SLT-II genes to be the same as that of the SLT-I genes, with the coding sequence for the large A subunit adjacent to that for the smaller B subunit. The mobilities of the SLT-II subunits in sodium dodecyl sulfate-polyacrylamide gels were slightly greater than those determined for the SLT-I subunits. Although apparent processing of the SLT-I subunits was observed with polymyxin B treatment of the labeled minicells, no processing of the SLT-II subunits was detected. Southern blot hybridization studies suggested that the DNA fragment carrying the SLT-II structural genes shares approximately 50 to 60% homology with the DNA of the SLT-I structural genes. Images PMID:2822579

  1. Serial Analysis of Gene Expression Identifies Metallothionein-II as Major Neuroprotective Gene in Mouse Focal Cerebral Ischemia

    Microsoft Academic Search

    George Trendelenburg; Konstantin Prass; Josef Priller; Krisztian Kapinya; Andreas Polley; Claudia Muselmann; Karsten Ruscher; Ute Kannbley; Armin O. Schmitt; Stefanie Castell; Frank Wiegand; Andreas Meisel; Ulrich Dirnagl

    2002-01-01

    We applied serial analysis of gene expression (SAGE) to study differentially expressed genes in mouse brain 14 hr after the induction of focal cerebral ischemia. Analysis of 60,000 tran- scripts revealed 83 upregulated and 94 downregulated tran- scripts (more than or equal to eightfold). Reproducibility was demonstrated by performing SAGE in duplicate on the same starting material. Metallothionein-II (MT-II) was

  2. Transcriptional analysis of the gene for glutamine synthetase II and two upstream genes in Streptomyces coelicolor A3(2).

    PubMed

    Weisschuh, N; Fink, D; Vierling, S; Bibb, M J; Wohlleben, W; Engels, A

    2000-11-01

    The glutamine synthetase II (GSII, encoded by glnII) activity detectable in crude extracts from Streptomyces coelicolor is low compared to the activity of glutamine synthetase I (GSI, encoded by glnA) and to that of GSII from S. viridochromogenes. We have identified and sequenced a 3.9-kb BglII-BamHI fragment carrying the glutamine synthetase II gene (glnII) from S. coelicolor. Besides glnII, this region contains four ORFs (orf1-orf4). While homologues of orf1 and orf2 were also found in the glnII region of the S. viridochromogenes chromosome, this was not the case for orf3 and orf4, which encode a putative hydrolase and a transcriptional regulator (Ptr) of the MarR family, respectively. High-resolution S1 nuclease mapping showed that the S. coelicolor glnII gene is expressed from two overlapping promoters. The first comprises a vegetative promoter sequence and the second contains sequence elements that are recognized by Esigma31. Similar promoter structures were found upstream of the S. viridochromogenes glnII gene. The involvement of ptr in glnII regulation was studied by gel retardation assays. Recombinant Ptr interacted with the upstream region of ptr, but not with the promoter region of glnII. A ptr gene replacement mutant (S. coelicolor IP) was also constructed. RT-PCR analysis of RNA from wild-type S. coelicolor and the IP mutant demonstrated that expression of orf3 depends on Ptr. Thus, the difference in gene organization between S. coelicolor and S. viridochromogenes is not responsible for the difference in GSII activity. PMID:11129050

  3. Analysis of the syntenic relationship of bovine thyroglobulin, carbonic anhydrase II, and c-mos genes

    E-print Network

    Faber, Laura Kristen

    1988-01-01

    and subcloned. DNA was extracted from each of the subcloned hybrid cells and transferred to nylon filters that were exposed to radioactive cDNA sequences of the human carbonic anhydrase II gene and the bovine thyroglobulin gene. Each clone was scored... discovered. A small but random sample of the cattle population was analyzed for restriction fragment length polymorphisms (RFLPs) for the carbonic anhydrase II gene. Two blots, each containing DNA samples from twenty cattle, were digested with one of four...

  4. Molecular cloning, expression, and evolution analysis of type II CHI gene from peanut (Arachis hypogaea L.).

    PubMed

    Liu, Yu; Zhao, Shuzhen; Wang, Jiangshan; Zhao, Chuanzhi; Guan, Hongshan; Hou, Lei; Li, Changsheng; Xia, Han; Wang, Xingjun

    2015-01-01

    Chalcone isomerase (CHI) plays critical roles in plant secondary metabolism, which is important for the interaction between plants and the environment. CHI genes are widely studied in various higher plants. However, little information about CHI genes is available in peanut. Based on conservation of CHI gene family, we cloned the peanut type II CHI gene (AhCHI II) cDNA and genome sequence. The amino acid sequence of peanut CHI II was highly homologous to type II CHI from other plant species. qRT-PCR results showed that peanut CHI II is mainly expressed in roots; however, peanut CHI I is mainly expressed in tissues with high content of anthocyanin. Gene duplication and gene cluster analysis indicated that CHI II was derived from CHI I 65 million years ago approximately. Our gene structure analysis results are not in agreement with the previous hypothesis that CHI II was derived from CHI I by the insertion of an intron into the first exon. Moreover, no positive selection pressure was found in CHIs, while, 32.1 % of sites were under neutral selection, which may lead to mutation accumulation and fixation during great changes of environment. PMID:25608978

  5. Major histocompatibility complex class II A gene polymorphism in the striped bass

    SciTech Connect

    Hardee, J.J.; Godwin, U.; Benedetto, R.; McConnell, T.J. [East Carolina Univ., Greenville, NC (United States)

    1995-02-01

    Adaptions of the polymerase chain reaction were used to isolate cDNA sequences encoding the Major histocompatibility complex (Mhc) class II A gene(s) of the striped bass (Morone saxatilis). Four complete Mhc class II A genes were cloned and sequenced from a specimen originating on the Roanoke River, North Carolina, and another three A genes from a specimen originating from the Santee-Cooper Reservoir, South Carolina, identifying a total of seven unique sequences. The sequence suggests the presence of at least two Mhc class II A loci. The extensive sequence variability observed between the seven different Mhc class II clones was concentrated in the {alpha}1 encoding domain. The encoded {alpha}2, transmembrane, and cytoplasmic regions of all seven striped bass genes correlated well with those of known vertebrate Mhc class II proteins. Overall, the striped bass sequences showed greatest similarity to the Mhc class II A genes of the zebrafish. Southern blot analysis demonstrated extensive polymorphism in the Mhc class II A genes in members of a Roanoke river-caught population of striped bass versus a lesser degree of polymorphism in an aquacultured Santee-Cooper population of striped bass. 55 refs., 5 figs., 1 tab.

  6. Isolation and partial characterization of the entire human pro alpha 1(II) collagen gene.

    PubMed Central

    Sangiorgi, F O; Benson-Chanda, V; de Wet, W J; Sobel, M E; Tsipouras, P; Ramirez, F

    1985-01-01

    Using a cDNA probe specific for the bovine Type II procollagen, a series of overlapping genomic clones containing 45 kb of contiguous human DNA have been isolated. Sequencing of a 54 bp exon, number 29, provided direct evidence that the recombinant clones bear human Type II collagen sequences. Localization of the 5' and 3' ends of the gene indicated that the human Type II collagen gene is 30 kb in size. This value is significantly higher than that of the homologous avian gene. The segregation of a polymorphic restriction site in informative families conclusively demonstrated that the Type II gene is found in a single copy in the human haploid genome. Finally, sequencing of a triple helical domain exon has confirmed that a rearrangement leading to the fusion of two exons occurred in the pro alpha 1(I) gene, following the divergence of the fibrillar collagens. Images PMID:2987845

  7. Type I MADS-box genes have experienced faster birth-and-death evolution than type II MADS-box genes in angiosperms

    PubMed Central

    Nam, Jongmin; Kim, Joonyul; Lee, Shinyoung; An, Gynheung; Ma, Hong; Nei, Masatoshi

    2004-01-01

    Plant MADS-box genes form a large gene family for transcription factors and are involved in various aspects of developmental processes, including flower development. They are known to be subject to birth-and-death evolution, but the detailed features of this mode of evolution remain unclear. To have a deeper insight into the evolutionary pattern of this gene family, we enumerated all available functional and nonfunctional (pseudogene) MADS-box genes from the Arabidopsis and rice genomes. Plant MADS-box genes can be classified into types I and II genes on the basis of phylogenetic analysis. Conducting extensive homology search and phylogenetic analysis, we found 64 presumed functional and 37 nonfunctional type I genes and 43 presumed functional and 4 nonfunctional type II genes in Arabidopsis. We also found 24 presumed functional and 6 nonfunctional type I genes and 47 presumed functional and 1 nonfunctional type II genes in rice. Our phylogenetic analysis indicated there were at least about four to eight type I genes and ?15–20 type II genes in the most recent common ancestor of Arabidopsis and rice. It has also been suggested that type I genes have experienced a higher rate of birth-and-death evolution than type II genes in angiosperms. Furthermore, the higher rate of birth-and-death evolution in type I genes appeared partly due to a higher frequency of segmental gene duplication and weaker purifying selection in type I than in type II genes. PMID:14764899

  8. Contrasting histories of avian and mammalian Mhc genes revealed by class II B sequences from songbirds.

    PubMed

    Edwards, S V; Wakeland, E K; Potts, W K

    1995-12-19

    To explore the evolutionary dynamics of genes in the major histocompatibility complex (Mhc) in nonmammalian vertebrates, we have amplified complete sequences of the polymorphic second (beta1) and third (beta2) exons of class II beta chain genes of songbirds. The pattern of nucleotide substitution in the antigen-binding site of sequences cloned from three behaviorally and phylogenetically divergent songbirds [scrub jays Aphelocoma coerulescens), red-winged blackbirds (Agelaius phoeniceus), and house finches (Carpodacus mexicanus) reveals that class II B genes of songbirds are subject to the same types of diversifying forces as those observed at mammalian class II loci. By contrast, the tree of avian class II B genes reveals that orthologous relationships have not been retained as in placental mammals and that, unlike class II genes in mammals, genes in songbirds and chickens have had very recent common ancestors within their respective groups. Thus, whereas the selective forces diversifying class II B genes of birds are likely similar to those in mammals, their long-term evolutionary dynamics appear to be characterized by much higher rates of concerted evolution. PMID:8618869

  9. Chromosomal localization and structure of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.; Huberman, E. [Chicago Univ., IL (United States). Dept. of Molecular Genetics and Cell Biology]|[Argonne National Lab., IL (United States); Collart, F. [Argonne National Lab., IL (United States); Varkony, T.; Drabkin, H. [Colorado Univ., Denver, CO (United States). Health Sciences Center

    1994-05-01

    We determined the chromosomal localization and structure of the gene encoding human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205), an enzyme associated with cellular proliferation, malignant transformation, and differentiation. Using polymerase chain reaction (PCR) primers specific for type II IMPDH, we screened a panel of human-Chinese hamster cell somatic hybrids and a separate deletion panel of chromosome 3 hybrids and localized the gene to 3p21.1{yields}p24.2. Two overlapping yeast artificial chromosome clones containing the full gene for type II IMPDH were isolated and a physical map of 117 kb of human genomic DNA in this region of chromosome 3 was constructed. The gene for type II IMPDH was localized and oriented on this map and found to span no more than 12.5 kb.

  10. Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II

    Microsoft Academic Search

    Hendrik J. D. Bussink; Frank P. Buxton; Jaap Visser

    1991-01-01

    The structure and expression of the polygalacturonase-encoding pgaII genes of two recently recognized species, Aspergillus niger and Aspergillus tubigensis, was investigated. While the structure of the pgaII genes is very similar, showing 83% DNA sequence identity and 94% identity at the amino acid level, they have diverged significantly. The NH2-terminal sequence suggests that these PGs are made as pre pro-proteins

  11. Characterization and Evolution of MHC Class II B Genes in Ardeid Birds

    Microsoft Academic Search

    Li Li; Xiaopin Zhou; Xiaolin Chen

    Major histocompatibility complex (MHC) is a multi-gene family that is very suitable to investigate a wide range of open questions\\u000a in evolutionary ecology. In this study, we characterized two expressed MHC class II B genes (DAB1 and DAB2) in the Grey Heron\\u000a (Aves: Ardea cinerea). We further developed the primer pairs to amplify and sequence two MHC class II B

  12. THE RNA POLYMERASE II GENE ENCODING THE SECOND LARGEST PEPTIDE SUBUNIT (RPB2) IN PHAEOSPHAERIA NODORUM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 5586-bp sequence (accession No. DQ278491) which contains the DNA-dependent RNA polymerase II gene (RPB2) encoding the second largest protein subunit (Rpb2) was obtained from the wheat-biotype Phaeosphaeria nodorum by PCR amplification. The RPB2 gene structure and its associated signals were analyz...

  13. Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase

    SciTech Connect

    Lacks, S.A.; Springhorn, S.S.

    1984-03-01

    The gene coding for the pneumococcal DNA adenine methylase that recognizes the sequence 5'-GATC-3' was cloned in a strain of Streptococcus pneumoniae that lacked both restriction endonucleases DpnI and DpnII. The gene was cloned as a 3.7-kilobase fragment of chromosomal DNA from a DpnII-containing strain inserted in both possible orientations in the multicopy plasmid vector pMP5 to give recombinant plasmids pMP8 and pMP10. Recombinant plasmids were selected by their resistance to DpnII cleavage. Cells carrying the recombinant plasmids modified phage in vivo so that it was restricted by DpnI- but not DpnII-containing hosts. They also showed levels of DNA methylase activity five times higher than that in cells of the original DpnII strain. No DpnII activity was observed in the clones; therefore, it was concluded that the insert did not contain an intact DpnII endonuclease gene and that methylation of host DNA did not turn on a latent form of the gene. 16 references, 1 figure, 2 tables.

  14. Resistance Genes of Aminocoumarin Producers: Two Type II Topoisomerase Genes Confer Resistance against Coumermycin A1 and Clorobiocin

    PubMed Central

    Schmutz, Elisabeth; Mühlenweg, Agnes; Li, Shu-Ming; Heide, Lutz

    2003-01-01

    The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A1, and clorobiocin were investigated. All three clusters contained a gyrBR resistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A1 clusters were found to contain an additional, similar gene, named parYR. Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression of gyrBR and likewise of parYR in Streptomyces lividans TK24 resulted in resistance against novobiocin and coumermycin A1, suggesting that both gene products are able to function as aminocoumarin-resistant B subunits of gyrase. Southern hybridization experiments showed that the genome of all three antibiotic producers and of Streptomyces coelicolor contained two additional genes which hybridized with either gyrBR or parYR and which may code for aminocoumarin-sensitive GyrB and ParY proteins. Two putative transporter genes, novA and couR5, were found in the novobiocin and the coumermycin A1 cluster, respectively. Expression of these genes in S. lividans TK24 resulted in moderate levels of resistance against novobiocin and coumermycin A1, suggesting that these genes may be involved in antibiotic transport. PMID:12604514

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

    PubMed

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

    2015-05-01

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

  16. Organization of genes required for the oxidation of methanol to formaldehyde in three type II methylotrophs

    SciTech Connect

    Bastien, C.; Machlin, S.; Zhang, Y.; Donaldson, K.; Hanson, R.S. (Univ. of Minnesota, Navarre (USA))

    1989-12-01

    Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome c{sub L}, an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closely linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome c{sub L} structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed.

  17. Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker.

    PubMed

    Yemets, Alla; Radchuk, Vladimir; Bayer, Oleg; Bayer, Galina; Pakhomov, Alexey; Vance Baird, W; Blume, Yaroslav B

    2008-05-01

    A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation. PMID:18180180

  18. Gene and MicroRNA Transcriptional Signatures of Angiotensin II in Endothelial Cells

    PubMed Central

    Mehta, Jawahar L.; Mercanti, Federico; Stone, Annjannette; Wang, Xianwei; Ding, Zufeng; Romeo, Francesco

    2015-01-01

    Abstract: Growth of atherosclerotic plaque requires neovascularization (angiogenesis). To elucidate the involvement of angiotensin II (Ang II) in angiogenesis, we performed gene microarray and microRNA (miRNA) polymerase chain reaction array analyses on human coronary artery endothelial cells exposed to moderate concentration of Ang II for 2 and 12 hours. At 12, but not 2, hours, cultures treated with Ang II exhibited shifts in transcriptional activity involving 267 genes (>1.5-fold difference; P < 0.05). Resulting transcriptome was most significantly enriched for genes associated with blood vessel development, angiogenesis, and regulation of proliferation. Majority of upregulated genes implicated in angiogenesis shared a commonality of being either regulators (HES1, IL-18, and CXCR4) or targets (ADM, ANPEP, HES1, KIT, NOTCH4, PGF, and SOX18) of STAT3. In line with these findings, STAT3 inhibition attenuated Ang II–dependent stimulation of tube formation in Matrigel assay. Expression analysis of miRNAs transcripts revealed that the pattern of differential expression for miRNAs was largely consistent with proangiogenic response with a prominent theme of upregulation of miRs targeting PTEN (miR-19b-3p, miR-21-5p, 23b-3p, and 24-3p), many of which are directly or indirectly STAT3 dependent. We conclude that STAT3 signaling may be an intrinsic part of Ang II–mediated proangiogenic response in human endothelial cells. PMID:24853489

  19. Gene and microRNA transcriptional signatures of angiotensin II in endothelial cells.

    PubMed

    Mehta, Jawahar L; Mercanti, Federico; Stone, Annjannette; Wang, Xianwei; Ding, Zufeng; Romeo, Francesco; Khaidakov, Magomed

    2015-02-01

    Growth of atherosclerotic plaque requires neovascularization (angiogenesis). To elucidate the involvement of angiotensin II (Ang II) in angiogenesis, we performed gene microarray and microRNA (miRNA) polymerase chain reaction array analyses on human coronary artery endothelial cells exposed to moderate concentration of Ang II for 2 and 12 hours. At 12, but not 2, hours, cultures treated with Ang II exhibited shifts in transcriptional activity involving 267 genes (>1.5-fold difference; P < 0.05). Resulting transcriptome was most significantly enriched for genes associated with blood vessel development, angiogenesis, and regulation of proliferation. Majority of upregulated genes implicated in angiogenesis shared a commonality of being either regulators (HES1, IL-18, and CXCR4) or targets (ADM, ANPEP, HES1, KIT, NOTCH4, PGF, and SOX18) of STAT3. In line with these findings, STAT3 inhibition attenuated Ang II-dependent stimulation of tube formation in Matrigel assay. Expression analysis of miRNAs transcripts revealed that the pattern of differential expression for miRNAs was largely consistent with proangiogenic response with a prominent theme of upregulation of miRs targeting PTEN (miR-19b-3p, miR-21-5p, 23b-3p, and 24-3p), many of which are directly or indirectly STAT3 dependent. We conclude that STAT3 signaling may be an intrinsic part of Ang II-mediated proangiogenic response in human endothelial cells. PMID:24853489

  20. Isolation, characterization and evolution of ovine major histocompatibility complex class II DRA and DQA genes.

    PubMed

    Fabb, S A; Maddox, J F; Gogolin-Ewens, K J; Baker, L; Wu, M J; Brandon, M R

    1993-08-01

    Four full-length ovine major histocompatibility complex (MHC) class II A cDNA clones coding for new alleles of DRA, DQA1 and DQA2 genes were isolated from two ovine lambda gt10 cDNA libraries. The derived amino acid sequences of these clones resemble class II A molecules from other species in both size and structure. Restriction fragment length polymorphism analysis, using an Ovar-DRA probe on DNA from Merino and Romney sheep revealed only limited polymorphism in contrast to the high levels of polymorphism revealed by Ovar-DQA probes. Comparison of the predicted amino acid sequences for the three ovine A genes with class II A genes from five other species revealed that the most variable region of the molecule is the signal peptide. Although virtually every amino acid site shows variation, within or between species, there are some blocks of highly conserved residues. Within gene comparisons of nucleotide differences reveal that the greatest number of changes is found between the alleles of Ovar-DQA1 and -DQA2 genes and the least between Ovar-DRA1 alleles. Phylogenetic analysis of class II A sequences from several species place DRA and DQA genes on two distinct branches, with Ovar-DRA1 and BOLA-DRA, and Ovar-DQA1 and BOLA-DQA being most similar on their respective branches. PMID:7902039

  1. GeneXpert Testing: Applications for Clinical Microbiology, Part II

    Microsoft Academic Search

    Elizabeth M. Marlowe; Donna M. Wolk

    2008-01-01

    The impact of rapid polymerase chain reaction (PCR) technology on infectious-disease testing is continuing to evolve outside the realm of a centralized laboratory. The GeneXpert Dx system is the first unit dose, near-point-of-care, molecular device commercially available. Part I of this two-part article addressed the use of the GeneXpert system for the detection of group B Streptococcus, enterovirus, and methicillin-resistant

  2. Sequence organisation and transcriptional regulation of the mouse elastase II and trypsin genes.

    PubMed Central

    Stevenson, B J; Hagenbüchle, O; Wellauer, P K

    1986-01-01

    Elastase II and trypsin mRNAs were cloned in form of their cDNAs from pancreas of strain A/J mice, and their complete nucleotide sequences were determined. The elastase II mRNA is 912 nucleotides long and encodes a protein of 271 amino acids. The cloned trypsin mRNA species is 814 nucleotides long and encodes a protein of 246 amino acids. The elastase II gene, which exists as a single copy in the haploid mouse genome, measures 11.2 kb from cap to poly(A) site and is interrupted by at least seven introns. Between 5 and 10 trypsin genes exist in the mouse genome. Five different trypsin genes, two of which are closely linked in a tail-to-tail manner, were studied in detail. They vary in size between 3.4 and 4.0kb, and all are interrupted by four introns. DNA sequence comparison of the elastase II, trypsin and Amy-2a alpha-amylase genes reveals a conserved 13 nucleotide motif in their 5'-flanking regions. The differential accumulation of the elastase II and trypsin mRNAs in the cytoplasm of the acinar pancreatic cell is regulated predominantly at the transcriptional level. Images PMID:3641189

  3. Acetylation of RNA Polymerase II Regulates Growth-Factor-Induced Gene Transcription in Mammalian Cells

    PubMed Central

    Schröder, Sebastian; Herker, Eva; Itzen, Friederike; He, Daniel; Thomas, Sean; Gilchrist, Daniel A.; Kaehlcke, Katrin; Cho, Sungyoo; Pollard, Katherine S.; Capra, John A.; Schnölzer, Martina; Cole, Philip A.; Geyer, Matthias; Bruneau, Benoit G.; Adelman, Karen; Ott, Melanie

    2014-01-01

    SUMMARY Lysine acetylation regulates transcription by targeting histones and nonhistone proteins. Here we report that the central regulator of transcription, RNA polymerase II, is subject to acetylation in mammalian cells. Acetylation occurs at eight lysines within the C-terminal domain (CTD) of the largest polymerase subunit and is mediated by p300/KAT3B. CTD acetylation is specifically enriched downstream of the transcription start sites of polymerase-occupied genes genome-wide, indicating a role in early stages of transcription initiation or elongation. Mutation of lysines or p300 inhibitor treatment causes the loss of epidermal growth-factor-induced expression of c-Fos and Egr2, immediate-early genes with promoter-proximally paused polymerases, but does not affect expression or polymerase occupancy at housekeeping genes. Our studies identify acetylation as a new modification of the mammalian RNA polymerase II required for the induction of growth factor response genes. PMID:24207025

  4. Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene

    PubMed Central

    Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

    2012-01-01

    Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line. PMID:23136521

  5. Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms' tumour

    Microsoft Academic Search

    Osamu Ogawa; Michael R. Eccles; Jenny Szeto; Leslie A. McNoe; Kankatsu Yun; Marion A. Maw; Peter J. Smith; Anthony E. Reeve

    1993-01-01

    GENOMIC imprinting has been implicated in the onset of several embryonal tumours but the mechanism is not well understood1-3. Maternal chromosome 11p15 loss of heterozygosity4 and paternal chromosome 11 isodisomy5,6 suggest that imprinted genes are involved in the onset of Wilms' tumour and the Beckwith-Wiedemann syndrome. The insulin-like growth factor II (IGF2) gene located at 11pl5.5 has been put forward

  6. Angiotensin II Type 1 Receptor Gene Polymorphisms in Human Essential Hypertension

    Microsoft Academic Search

    Alain Bonnardeaux; Eleanor Davies; Xavier Jeunemaitre; Isabelle Fery; Anne Charru; Eric Clauser; Laurence Tiret; Francois Cambien; Pierre Corvol; Florent Soubrier

    Abstract We conducted ,the present study to determine whether the angiotensin II type I receptor (AT,) gene might beimplicated,in human ,essential hypertension ,by using case-control and ,linkage studies. The entire coding ,and 3' untranslated regions of the AT, receptor gene (2.2 kb) were amplified by polymerase ,chain reaction and ,submitted ,to single-strand conformation ,polymorphism ,in 60 hypertensive

  7. Gene targeting in embryonic stem cells, II: conditional technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  8. Characterization of BoLA class II genes

    E-print Network

    Sherwood, Sidney James

    1989-01-01

    or growth of the tumors was attributed to a genetically inherited system of about fifteen dominant genes. The last major step leading to Borer defining of the mouse MHC was the work done on human blood groups by Landsteiner (1901) and others.... Landsteiner, using blood from himself and his assistants used a simple hemagglutination assay and described the human ABQ blood groups. This showed the presence of serum ant ibodies that could detect antigenic substances on red blood cell surfaces. Von...

  9. Identification and isolation of the cytochrome oxidase subunit II gene in mitochondria of the yeast Hansenula saturnus

    Microsoft Academic Search

    Janet E. Lawson; Donald W. Deters

    1985-01-01

    Mitochondrial DNA from the petite negative yeast Hansenula saturnus has been isolated and sized by digestion with restriction enzymes. The size of the mitochondrial genome is approximately 47 kb. The gene for subunit II of cytochrome oxidase was localized in the genome by Southern blotting using a [32P]-labeled probe containing the subunit II gene of the yeast Saccharomyces cerevisiae. The

  10. Recent duplication and inter-locus gene conversion in major histocompatibility class II genes in a teleost, the three-spined stickleback.

    PubMed

    Reusch, Thorsten B H; Schaschl, Helmut; Wegner, K Mathias

    2004-09-01

    Using a bacterial artificial chromosome (BAC) library, we analysed a 99.5 kb genomic segment containing the major histocompatibility class II genes of a teleost, the three-spined stickleback Gasterosteus aculeatus. Experiments with G. aculeatus have provided direct evidence for balancing selection by pathogens and mate choice driving MH class II beta polymorphism. Two sets of paralogous class II alpha genes and beta genes in a tandem arrangement were identified, designated Gaac-DAA/DAB and Gaac-DBA/DBB. Expression analysis of the beta genes using single-strand conformation polymorphism revealed that both gene copies are expressed. Based on an analysis of pairwise nucleotide polymorphisms, we estimate that the duplication into two paralogous class II loci occurred only 1.2-2.4 million years ago, 1-2 orders of magnitude more recently than in other fish, bird or mammalian species. At the 3'-direction of the classical MH loci, we identified another seven genes or gene fragments, two of which (small inducible cytokine, complement regulatory factor) are related to immune function in other vertebrates. None of these genes were associated with MH class II genes in zebrafish, suggesting a markedly different organisation of the MH class II region in sticklebacks, and presumably, across bony fishes. When the nucleotide substitution pattern of the novel class II beta genes was analysed together with a representative sequence sample isolated from fish in northern Germany (n=27), we found that the peptide binding region of the Gaac-DAB and Gaac-DBB loci had undergone an inter-locus gene conversion (P=0.007). In accordance, we found a 10- to 20-fold higher frequency of CpG-islands on the MH class II segment compared to other species, a feature that may be conducive for inter-locus recombination. PMID:15322775

  11. Molecular Phylogeny of the Chipmunks Inferred from Mitochondrial Cytochrome b and Cytochrome Oxidase II Gene Sequences

    Microsoft Academic Search

    Antoinette J. Piaggio; Greg S. Spicer

    2001-01-01

    There are currently 25 recognized species of the chipmunk genus Tamias. In this study we sequenced the complete mitochondrial cytochrome b (cyt b) gene of 23 Tamias species. We analyzed the cyt b sequence and then analyzed a combined data set of cyt b along with a previous data set of cytochrome oxidase subunit II (COII) sequence. Maximum-likelihood was used

  12. Proteinase inhibitor II gene in transgenic poplar: Chemical and biological assays

    Microsoft Academic Search

    Ned B. Klopfenstein; Kurt K. Allen; Francisco J. Avila; Scott A. Heuchelin; Jimmy Martinez; Richard C. Carman; Richard B. Hall; Elwood R. Hart; Harold S. McNabb

    1997-01-01

    Transgenic poplar lines were developed to investigate the role of a proteinase inhibitor in pest resistance of woody plants. Using an Agrobacterium binary vector system, the clone ‘Hansen’ (Populus alba L. × P. grandidentata Michx.) was transformed with chimeric genes containing the coding region of potato proteinase inhibitor II (PIN2) linked to either a bacterial nopaline synthase (nos) or a

  13. A family of regulatory genes associated with type II restriction-modification systems.

    PubMed Central

    Tao, T; Bourne, J C; Blumenthal, R M

    1991-01-01

    Restriction-modification systems must be regulated to avoid autorestriction and death of the host cell. An open reading frame (ORF) in the PvuII restriction-modification system appears to code for a regulatory protein from a previously unrecognized family. First, interruptions of this ORF result in a nonrestricting phenotype. Second, this ORF can restore restriction competence to such interrupted mutants in trans. Third, the predicted amino acid sequence of this ORF resembles those of known DNA-binding proteins and includes a probable helix-turn-helix motif. A survey of unattributed ORFs in 15 other type II restriction-modification systems revealed three that closely resemble the PvuII ORF. All four members of this putative regulatory gene family have a common position relative to the endonuclease genes, suggesting a common regulatory mechanism. PMID:1995588

  14. Role of the Bradyrhizobium japonicum ntrC gene product in differential regulation of the glutamine synthetase II gene (glnII)

    SciTech Connect

    Martin, G.B.; Chapman, K.A.; Chelm, B.K. (Michigan State Univ., East Lansing (USA))

    1988-12-01

    We isolated the ntrC gene from Bradyrhizobium japonicum, the endosymbiont of soybean (Glycine max), and examined its role in regulating nitrogen assimilation. Two independent ntrC mutants were constructed by gene replacement techniques. One mutant was unable to produce NtrC protein, while the other constitutively produced a stable, truncated NtrC protein. Both ntrC mutants were unable to utilize potassium nitrate as a sole nitrogen source. In contrast to wild-type B. japonicum, the NtrC null mutant lacked glnII transcripts in aerobic, nitrogen-starved cultures. However, the truncated-NtrC mutant expressed glnII in both nitrogen-starved and nitrogen-excess cultures. Both mutants expressed glnII under oxygen-limited culture conditions and in symbiotic cells. These results suggest that nitrogen assimilation in B. japonicum is regulated in response to both nitrogen limitation and oxygen limitation and that separate regulatory networks exist in free-living and symbiotic cells.

  15. Horticultural characteristics of transgenic tobacco expressing the rolC gene from Agrobacterium rhizogenes

    SciTech Connect

    Scorza, R.; Zimmerman, T.W.; Cordts, J.M.; Footen, K.J. (Dept. of Agriculture, Kearneysville, WV (United States)); Ravelonandro, M. (Inst. National Recherche Agronomique, Villenave d'Ornon (France). Station de Pathologie Vegetale)

    1994-09-01

    Wisconsin 38 tobacco (Nicotiana tabacum L.) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying the rolC gene from A. rhizogenes and NPT II and GUS genes. Shoots that regenerated on kanamycin-containing medium were confirmed as transgenic through GUS assays, polymerase chain reaction (PCR), Southern blot analyses, and transmission of the foreign genes through the sexual cycle. Transgenic plants were as short as half the height of control plants; were earlier flowering by up to 35 days; and had smaller leaves, shorter internodes, smaller seed capsules, fewer seeds, smaller flowers, and reduced pollen viability. The number of seed capsules, leaf number, and specific root length were similar between transgenic and control plants. Transgenic clones varied in the expression of the rolC-induced growth alterations as did the first generation of seedlings from these clones. Such differences suggested the potential for selecting for different levels of expression. Transformation with the rolC gene presents a potentially useful method of genetically modifying horticultural crops, particularly for flowering date, height, and leaf and flower size. Chemical names used: neomycin phosphotransferase (NPTII), [beta]-glucuronidase (GUS).

  16. Ginseng Berry Extract Prevents Atherogenesis via Anti-Inflammatory Action by Upregulating Phase II Gene Expression

    PubMed Central

    Kim, Chun-Ki; Cho, Dong Hui; Lee, Kyu-Sun; Lee, Dong-Keon; Park, Chan-Woong; Kim, Wan Gi; Lee, Sang Jun; Ha, Kwon-Soo; Goo Taeg, Oh; Kwon, Young-Guen; Kim, Young-Myeong

    2012-01-01

    Ginseng berry possesses higher ginsenoside content than its root, which has been traditionally used in herbal medicine for many human diseases, including atherosclerosis. We here examined the antiatherogenic effects of the Korean ginseng berry extract (KGBE) and investigated its underlying mechanism of action in vitro and in vivo. Administration of KGBE decreased atherosclerotic lesions, which was inversely correlated with the expression levels of phase II genes to include heme oxygenase-1 (HO-1) and glutamine-cysteine ligase (GCL). Furthermore, KGBE administration suppressed NF-?B-mediated expression of atherogenic inflammatory genes (TNF-?, IL-1?, iNOS, COX-2, ICAM-1, and VCAM-1), without altering serum cholesterol levels, in ApoE?/? mice fed a high fat-diet. Treatment with KGBE increased phase II gene expression and suppressed lipopolysaccharide-induced reactive oxygen species production, NF-?B activation, and inflammatory gene expression in primary macrophages. Importantly, these cellular events were blocked by selective inhibitors of HO-1 and GCL. In addition, these inhibitors reversed the suppressive effect of KGBE on TNF-?-mediated induction of ICAM-1 and VCAM-1, resulting in decreased interaction between endothelial cells and monocytes. These results suggest that KGBE ameliorates atherosclerosis by inhibiting NF-?B-mediated expression of atherogenic genes via upregulation of phase II enzymes and thus has therapeutic or preventive potential for atherosclerosis. PMID:23243449

  17. Extensive MHC class II B gene duplication in a passerine, the common Yellowthroat (Geothlypis trichas).

    PubMed

    Bollmer, Jennifer L; Dunn, Peter O; Whittingham, Linda A; Wimpee, Charles

    2010-01-01

    The major histocompatibility complex (MHC) is characterized by a birth and death model of evolution involving gene duplication, diversification, loss of function, and deletion. As a result, gene number varies across taxa. Birds have between one and 7 confirmed MHC class II B genes, and the greatest diversity appears to occur in passerines. We used multiple primer sets on both genomic DNA (gDNA) and complementary DNA (cDNA) to characterize the range of class II B genes present in a passerine, the common yellowthroat (Geothlypis trichas). We confirmed 39 exon 2 sequences from gDNA in a single individual, indicating the presence of at least 20 class II B loci. From a second individual, we recovered 16 cDNA sequences belonging to at least 8 transcribed loci. Phylogenetic analysis showed that common yellowthroat sequences fell into subgroups consisting of classical loci, as well as at least 4 different clusters of sequences with reduced sequence variability that may represent pseudogenes or nonclassical loci. Data from 2 additional common yellowthroats demonstrated high interindividual variability. Our results reveal that some passerines possess an extraordinary diversity of MHC gene duplications, including both classical and nonclassical loci. PMID:20200139

  18. StructureActivity Study of the Lantibiotic Mutacin II from Streptococcus mutans T8 by a Gene Replacement Strategy

    Microsoft Academic Search

    PING CHEN; JAN NOVAK; MARION KIRK; STEPHEN BARNES; FENGXIA QI; PAGE W. CAUFIELD

    1998-01-01

    Mutacin II, elaborated by group II Streptococcus mutans, is a ribosomally synthesized and posttranslationally modified polypeptide antibiotic containing unusual thioether and didehydro amino acids. To ascertain the role of specific amino acid residues in mutacin II antimicrobial activity, we developed a streptococcal expression system that facilitates the replacement of the mutA gene with a single copy of a mutated variant

  19. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells

    Microsoft Academic Search

    D. S. Gilmour; J. T. Lis

    1986-01-01

    By using a protein-DNA cross-linking method, we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end

  20. Movement of coat protein genes from a commercial virus-resistant transgenic squash into a wild relative.

    PubMed

    Fuchs, Marc; Chirco, Ellen M; Gonsalves, Dennis

    2004-01-01

    We monitored pollen-mediated transgene dissemination from commercial transgenic squash CZW-3 into its wild relative Cucurbita pepo ssp. ovifera var. texana (C. texana). Transgenic squash CZW-3 expresses the neomycin phosphotransferase II (nptII) gene and the coat protein (CP) genes of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), and Watermelon mosaic virus (WMV); thereby, it is resistant to these three aphid-borne viruses. The rate of NPT II and CP transgene introgression increased with overlapping flowering patterns and a high ratio of transgenic F1 hybrids (C. texana x CZW-3) to C. texana. Transgene transfer also readily occurred from transgenic F1 hybrids into C. texana over three generations in field settings where test plants grew sympatrically and viruses were not severely limiting the growth, and fruit and seed production of C. texana. In contrast, introgression of the transgenes into C. texana was not sustained under conditions of high viral disease pressure. As expected, C. texana progeny that acquired the CP transgenes exhibited resistance to CMV, ZYMV, and WMV. This is the first report on transgene dissemination from a transgenic crop that exhibits disease resistance and hybridizes with a wild plant species without loss of fertility. PMID:15612351

  1. Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio).

    PubMed

    Li, Shuangfei; Hu, Wei; Wang, Yaping; Zhu, Zuoyan

    2004-08-01

    Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator. PMID:15493476

  2. Escherichia coli BL21(DE3) chromosome contains a group II capsular gene cluster.

    PubMed

    Andreishcheva, Ekaterina N; Vann, Willie F

    2006-12-15

    During our study of de novo synthesis of Escherichia coli K1 capsular polysaccharides, we found that E. coli BL21(DE3) has a capsular gene cluster, similar to those of group II capsular E. coli strains. Analysis of the nucleotide sequence of the E. coli BL21(DE3) gene cluster showed homologues to all group II regions 1 and 3 genes and the presence of an IS1 element in one of the region 2 ORFs, which likely prevents capsule expression. Complementation analysis showed that region 1 and 3 genes encode functional proteins that are sufficient for the export of newly synthesized polysaccharide. The gene products of Bl21(DE3) kpsC and kpsS supported in vitro de novo synthesis of K1 polysaccharide when co-expressed with K1 NeuE and NeuS. Sequence homology between BL21(DE3) region 2 open reading frames and capsule-related genes in other bacteria such as Haemophilus influenzae serotype b, suggests that the encapsulated ancestor of BL21(DE3) may have produced a ribose/ribitol-phosphate containing polysaccharide. PMID:16959439

  3. Role of the major histocompatibility complex class II Ea gene in lupus susceptibility in?mice

    PubMed Central

    Ibnou-Zekri, Nabila; Iwamoto, Masahiro; Fossati, Liliane; McConahey, Patricia J.; Izui, Shozo

    1997-01-01

    The gene(s) encoded within major histocompatibility complex (MHC) act as one of the major genetic elements contributing to the susceptibility of murine systemic lupus erythematosus (SLE). We have recently demonstrated that lupus susceptibility is more closely linked to the I-E? H-2b haplotype than to the I-E+ H-2d haplotype in lupus-prone BXSB and (NZB × BXSB)F1 hybrid mice. To investigate whether the reduced susceptibility to SLE in H-2d mice is related to the expression of the MHC class II Ea gene (absent in H-2b mice), we determined the possible role of the Ea gene as a lupus protective gene in mice. Our results showed that (i) the development of SLE was almost completely prevented in BXSB (H-2b) mice expressing two copies of the Ead transgene at the homozygous level as well as in BXSB H-2k (I-E+) congenic mice as for H-2d BXSB mice, and (ii) the expression of two functional Ea (transgenic and endogenous) genes in either H-2d/b (NZB × BXSB)F1 or H-2k/b (MRL × BXSB)F1 mice provided protection from SLE at levels comparable to those conferred by the H-2d/d or H-2k/k haplotype. In addition, the level of the Ea gene-mediated protection appeared to be dependent on the genetic susceptibility to SLE in individual lupus-prone mice. Our results indicate that the reduced susceptibility associated with the I-E+ H-2d and H-2k haplotypes (versus the I-E? H-2b haplotype) is largely, if not all, contributed by the apparent autoimmune suppressive effect of the Ea gene, independently of the expression of the I-A or other MHC-linked genes. PMID:9405668

  4. The organization of the keratin I and II gene clusters in placental mammals and marsupials show a striking similarity

    Microsoft Academic Search

    Alexander Zimek; Klaus Weber

    2006-01-01

    The genomic database for a marsupial, the opossum Monodelphis domestica, is highly advanced. This allowed a complete analysis of the keratin I and keratin II gene cluster with some 30 genes in each cluster as well as a comparison with the human keratin clusters. Human and marsupial keratin gene clusters have an astonishingly similar organization. As placental mammals and marsupials

  5. Developmental extinction of major histocompatibility complex class II gene expression in plasmocytes is mediated by silencing of the transactivator gene CIITA

    PubMed Central

    1994-01-01

    Constitutive major histocompatibility complex (MHC) class II gene expression is tightly restricted to antigen presenting cells and is under developmental control. Cells of the B cell lineage acquire the capacity to express MHC class II genes early during ontogeny and lose this property during terminal differentiation into plasma cells. Cell fusion experiments have suggested that the extinction of MHC class II expression in plasma cells is due to a dominant repression, but the underlying mechanisms are not understood. CIITA was recently identified as an MHC class II transactivator that is essential for MHC class II expression in B lymphocytes. We show here that inactivation of MHC class II genes in plasmocytes is associated with silencing of the CIITA gene. Moreover, experimentally induced expression of CIITA in plasmocytes leads to reexpression of MHC class II molecules to the same level as that observed on B lymphocytes. We therefore conclude that the loss of MHC class II expression observed upon terminal differentiation of B lymphocytes into plasmocytes results from silencing of the transactivator gene CIITA. PMID:7931066

  6. Identification of differentially expressed genes induced by angiotensin II in rat cardiac fibroblasts.

    PubMed

    Wang, X F; Gao, G D; Liu, J; Guo, R; Lin, Y X; Chu, Y L; Han, F C; Zhang, W H; Bai, Y J

    2006-01-01

    1. Cardiac fibroblasts play an important regulatory role in cardiac remodelling by undergoing proliferation, differentiation and upregulating various gene products, including some cytokines and extracellular matrix (ECM) proteins. A highly potent mediator of cardiac remodelling is angiotensin (Ang) II. 2. In the present study, the suppression subtractive hybridization method was used to identify differentially expressed cDNAs in adult rat cardiac fibroblasts induced by AngII. 3. Following mRNA isolation of non-stimulated and AngII-stimulated cells, cDNAs of both populations were prepared and subtracted by suppression polymerase chain reaction. Sequencing of the partially enriched cDNAs identified 36 genes differentially expressed, including ECM proteins (pro-alpha(1) collagen type III, fibronectin), structural protein (spectrin), enzyme (GTP-specific succinyl-CoA synthetase), transcriptional regulators (glucocorticoid-induced leucine zipper, inhibitor of DNA binding 3) and proteins involved in cell division control (cdc2) or cell signalling (insulin-like growth factor binding protein-3, mutant p53-binding protein, grp75, CGI-121, protein phosphatase type 2A, tspan-2 and Sam68). 4. The diversity of genes identified in the present study further emphasises the central role of AngII in the regulation of cardiac remodelling. PMID:16445697

  7. Dentin phosphoprotein gene locus is not associated with dentinogenesis imperfecta types II and III

    SciTech Connect

    MacDougall, M.; Zeichner-David, M.; Davis, A.; Slavkin, H. (Univ. of Southern California, Los Angeles (United States)); Murray, J. (Univ. of Iowa, Iowa City (United States)); Crall, M. (Ohio State Univ., Columbus (United States))

    1992-01-01

    Dentinogenesis imperfecta (DGI) is an autosomal dominant inherited dental disease which affects dentin production and mineralization. Genetic linkage studies have been performed on several multigeneration informative kindreds. These studies determined linkage between DGI types II and III and group-specific component (vitamin D-binding protein). This gene locus has been localized to the long arm of human chromosome 4 in the region 4q11-q21. Although this disease has been mapped to chromosome 4, the defective gene product is yet to be determined. Biochemical studies have suggested abnormal levels of dentin phosphoprotein (DPP) associated with DGI type II. This highly acidic protein is the major noncollagenous component of dentin, being solely expressed by the ectomesenchymal derived odontoblast cells of the tooth. The purpose of the present study was to establish whether DPP is associated with DGI types II and III, by using molecular biology techniques. The results indicated that DPP is not localized to any region of human chromosome 4, thus suggesting that the DPP gene is not directly associated with DGI type II or DGI type III. The data do not exclude the possibility that other proteins associated with DPP posttranslational modifications might be responsible for this genetic disease.

  8. Immunoglobulin allotype gene polymorphisms in systemic sclerosis: interactive effect of MHC class II and KM genes on anticentromere antibody production

    PubMed Central

    Kameda, H.; Pandey, J.; Kaburaki, J.; Inoko, H.; Kuwana, M.

    1998-01-01

    OBJECTIVE—To examine potential interactions between immunoglobulin (Ig)allotype gene polymorphisms and susceptibility to systemic sclerosis (SSc) as well as serological expression in SSc patients.?METHODS—IgG heavy chain allotypes G1M(f, z), G2M(n+, n-), G3M(b, g) and Ig light chain allotype KM(1, (1, 2), 3) were genotyped in 105 Japanese SSc patients and 47 race matched normal controls using polymerase chain reaction (PCR) based methods. Associations of each Ig allotype with SSc related antinuclear antibodies were examined in combination with or without MHC class II alleles.?RESULTS—GM/KM genotypic and allelic frequencies were similar in SSc patients and in normal controls. Frequencies of G1M(f) and G2M(n+) were significantly decreased in anticentromere antibody (ACA) positive SSc patients compared with ACA negative SSc patients (p = 0.04 and 0.02, respectively). Conversely, the presence of DQB1*0501 and KM(1, 2) significantly increased the risk of ACA positivity.?CONCLUSION—Ig allotype gene polymorphisms were not associated with susceptibility to SSc. Instead, the results suggested that MHC class II and KM genes are associated with autoimmune responses by interactively promoting the production of ACA.?? Keywords: autoantibody; immunoglobulin allotype; major histocompatibility complex; scleroderma PMID:9771212

  9. Casein genes of Bos taurus. II. Isolation and characterization of the /beta/-casein gene

    SciTech Connect

    Gorodetskii, S.I.; Tkach, T.M.; Kapelinskaya, T.V.

    1988-11-01

    The expression of the casein genes in the cells of the mammary gland is regulated by peptide and steroid hormones. In order to study the controlling mechanisms we have isolated and characterized the /beta/-casein gene. The gene is 8.6 kb long and exceeds by a factor of 7.8 the length of the corresponding mRNA which is encoded by nine exons. The genomic clones incorporate in addition 8.5 kb and 4.5 kb of the 5/prime/- and 3/prime/-flanking regions. We have determined the sequence of the 5- and 3-terminals of the gene and have performed a comparative analysis of the corresponding regions of the rat /beta/-casein gene. Furthermore we have identified the conversed sequences identical or homologous to the potential sections of binding to the nuclear factor CTF/NF-1 by glucocorticoid and progesterone receptors. The regulatory region of the bovine casein gene contains two variants of the TATA signal, flanking the duplication section in the promoter region.

  10. Evolution of major histocompatibility complex class I and class II genes in the brown bear

    PubMed Central

    2012-01-01

    Background Major histocompatibility complex (MHC) proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. Results We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Conclusions Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South–north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia. PMID:23031405

  11. Structure of the human gene encoding the invariant gamma-chain of class II histocompatibility antigens.

    PubMed Central

    Kudo, J; Chao, L Y; Narni, F; Saunders, G F

    1985-01-01

    The primary structures of a cDNA and the genomic DNA of a gene selectively expressed in chronic lymphocytic leukemia were determined. A computer search of the nucleotide sequence data bank identified this gene as the invariant gamma-chain associated with class II histocompatibility antigens. The invariant gamma-chain genomic sequence spans about 11 kilobases, with eight exons and seven introns. Three of the introns contain members of the Alu repeat family. A putative cap site and promoter sequence were identified at the 5' end of the gene. One or two copies of the gene is present in each haploid genome, and no evidence for amplification or polymorphism was obtained. Images PMID:3001652

  12. Active suppression of major histocompatibility complex class II gene expression during differentiation from B cells to plasma cells

    SciTech Connect

    Latron, F.; Maffei, A.; Scarpellino, L.; Bernard, M.; Accolla, R.S. (Ludwig Institute for Cancer Research, Epalinges (Switzerland)); Jotterand-Bellomo, M. (Centre Hospitalier Universitaire Vaudois, Lausanne (Switzerland)); Strominger, J.L. (Harvard Univ., Cambridge, MA (USA))

    1988-04-01

    Constitutive expression of major histocompatibility complex class II genes is acquired very early in B-cell ontogeny and is maintained up to the B-cell blast stage. Terminal differentiation in plasma cells is, however, accompanied by a loss of class II gene expression. In B cells this gene system is under the control of several loci encoding transacting factors with activator function, one of which, the aIr-1 gene product, operates across species barriers. In this report human class II gene expression is shown to be extinguished in somatic cell hybrids between the human class II-positive B-cell line Raji and the mouse class-II negative plasmacytoma cell line P3-U1. Since all murine chromosomes are retained in these hybrids and no preferential segregation of a specific human chromosome is observed, the results are compatible with the presence of suppressor factors of mouse origin, operating across species barriers and inhibiting class II gene expression. Suppression seems to act at the level of transcription or accumulation of class II-specific mRNA, since no human, and very few murine, class II transcripts are detectable in the hybrids.

  13. Characterisation of a Plancitoxin-1-Like DNase II Gene in Trichinella spiralis

    PubMed Central

    Liu, Mingyuan; Liu, Pan; Wang, Xuelin; Li, Tingting; Tang, Bin; Gao, He; Sun, Qingsong; Liu, Xidong; Zhao, Ying; Wang, Feng; Wu, Xiuping; Boireau, Pascal; Liu, Xiaolei

    2014-01-01

    Background Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain. Methodology/Principal Findings In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C. Conclusions This study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity. PMID:25165857

  14. Inflammatory bowel disease associations with HLA Class II genes

    SciTech Connect

    Castro, R. [Cedars-Sinai Medical Center, Los Angeles, CA (United States); Yang, H.; Targan, S. [Roche Molecular Systems, Inc., Alameda, CA (United States)] [and others

    1994-09-01

    A PCR-SSOP assay has been used to analyze HLA-Class II DRB1 and DQB1 alleles in 378 Caucasians from a population in Southern California. The data has been analyzed separately for the Ashkenasi Jews and non-Jewish patients (n=286) and controls (n=92). Two common clinical forms of inflammatory bowel disease (IBD) have been studied: ulcerative colitis (UC) and Crohn`s disease (CD). In CD, we observed a susceptible effect with the rare DR1 allele - DRB*0103 [O.R.=4.56; 95% CI (0.96, 42.97); p=0.03]; a trend for an increase in DRB1*0103 was also observed in UC patients. A susceptible effect with DRB1*1502 [O.R.=5.20; 95% CI (1.10, 48.99); p=0.02] was observed in non-Jewish UC patients. This susceptible effect was restricted to UC ANCA-positive (antineutrophil cytoplasmic antibodies) patients. In addition, a significant association with DRB1*1101-DQB1*0301 [O.R.=9.46; 95% CI (1.30, 413.87); p=0.01] was seen with UC among non-Jewish patients: this haplotype was increased with CD among non-Jewish patients. Two protective haplotypes were detected among CD non-Jewish patients: DRB1*1301-DQB1*0603 [O.R.=0.34; 95% CI (0.09, 1.09); p=0.04], and DRB*0404-DQB1*0302 [O.R.=<0.08; 95% CI (0.0, 0.84); p=0.01]. When the same data were analyzed at the serology level, we observed a positive association in UC with DR2 [O.R.6.77; 95% CI (2.47, 22.95); p=2 x 10{sup -4}], and a positive association in CD with DR1 [O.R.=2.63; 95% CI (1.14, 6.62); p=0.01] consistent with previous reports. Thus, some IBD disease associations appear to be common to both UC and CD, while some are unique to one disease.

  15. BglII gene polymorphism of the a2ß1 integrin gene is a risk factor for diabetic retinopathy in Caucasians with type 2 diabetes

    Microsoft Academic Search

    MojcaGlobo?nik Petrovi?; Marko Hawlina; Borut Peterlin; Daniel Petrovi?

    2003-01-01

    Platelets are thought to be involved in the pathogenesis of diabetic retinopathy. The BglII gene polymorphism of the a2ß1 integrin, which is a platelet collagen receptor, has been suggested as a genetic risk factor for diabetic retinopathy in Japanese subjects. The aim of this study was to look for a relationship between the BglII gene polymorphism of the a2ß1 integrin

  16. BglII gene polymorphism of the ?2?1 integrin gene is a risk factor for diabetic retinopathy in Caucasians with type 2 diabetes

    Microsoft Academic Search

    Mojca Globo?nik Petrovi?; Marko Hawlina; Borut Peterlin; Daniel Petrovi?

    2003-01-01

    Platelets are thought to be involved in the pathogenesis of diabetic retinopathy. The BglII gene polymorphism of the ?2?1 integrin, which is a platelet collagen receptor, has been suggested as a genetic risk factor for diabetic retinopathy in Japanese subjects. The aim of this study was to look for a relationship between the BglII gene polymorphism of the ?2?1 integrin

  17. Coordinate amplification of metallothionein I and II gene sequences in cadmium-resistant CHO variants

    SciTech Connect

    Hildebrand, C.E.; Crawford, B.D.; Enger, M.D.

    1983-01-01

    Cadmium-resistanc (Cd/sup r/) variants of the Chinese hamster cell line, CHO, have been derived by stepwise selection for growth in medium containing CdCl/sub 2/. These variants show coordinately increased production of both metallothionein (MT) I and II and were stably resistant to Cd/sup 2 +/ in the absence of continued selection. Genomic DNAs from these Cd/sup r/ sublines were analyzed for both MT gene copy number and MT gene organization, using cDNA sequence probes specific for each of the two Chinese hamster isometallothioneins. These analyses revealed coordinate amplification of MT I and II genes in all Cd/sup r/ variants which had increased copies of MT-encoding sequences. In situ hybridization of an MT-encoding probe to mitotic chromosomes of a Cd/sup r/ variant, which has amplified MT genes at least 14-fold, revealed a single chromosomal site of hybridization. These results suggest that the isoMTs constitute a functionally related gene cluster which amplifies coordinately in response to toxic metal stress.

  18. Characterization and evolution of MHC class II B genes in Ardeid birds.

    PubMed

    Li, Li; Zhou, Xiaopin; Chen, Xiaolin

    2011-06-01

    Major histocompatibility complex (MHC) is a multi-gene family that is very suitable to investigate a wide range of open questions in evolutionary ecology. In this study, we characterized two expressed MHC class II B genes (DAB1 and DAB2) in the Grey Heron (Aves: Ardea cinerea). We further developed the primer pairs to amplify and sequence two MHC class II B loci in ten ardeid birds. Phylogenetic analysis revealed that different parts of the genes showed different evolutionary patterns. The exon 2 sequences tended to cluster two gene-specific lineages. In each lineage, exon 2 sequences from several species showed closer relationships than sequences within species, and two shared identical alleles were found between species (Egretta sacra and Nycticorax nycticorax; Egretta garzetta and Bubulcus ibis), supporting the hypothesis of trans-species polymorphism. In contrast, the species-specific intron 2 plus partial exon 3 tree suggested that DAB1 and DAB2 were subject to concerted evolution. GENECONV analyses showed the gene exchange played an important role in the ardeid MHC evolution. PMID:21590337

  19. Cloning, characterization, and regulation of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.A.; Huberman, E. [Argonne National Lab., IL (United States). Center for Mechanistic Biology and Biotechnology]|[Univ. of Chicago, IL (United States)

    1997-01-01

    Human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. Regulated IMPDH activity is associated with cellular proliferation, transformation, and differentiation. The authors cloned and sequenced the entire gene for type II IMPDH and here provide details regarding the organization of the gene and the characterization of its promoter. The gene spans approximately 5 kb and is disrupted by 12 introns. The transcriptional start sites were determined by S1 nuclease mapping to be somewhat heterogeneous but predominated at 102 and 85 nucleotides from the translational initiation codon. Through the use of heterologous gene constructs and transient transfection assays, a minimal promoter from {minus}206 to {minus}85 was defined. This promoter is TATA-less and contains several transcription factor motifs including four potential Sp 1 binding sites. The minimal promoter is GC-rich (69%) and resembles a CpG island. Through the use of gel mobility shift assays, nuclear proteins were shown to specifically interact with this minimal promoter. Stable transfectants were used to demonstrate that the down-regulation of IMPDH gene expression in response to reduced cellular proliferation occurs by a transcriptional mechanism.

  20. DNA typing of HLA class II genes in native inhabitants of Chukotka

    SciTech Connect

    Krylov, M.Yu.; Erdesz, S.; Alexeeva, L.I. [Institute of Rheumatology, Moscow (Russian Federation)

    1995-06-01

    Polymorphism of HLA class II genes was studied in native Chukotka inhabitants with the use of DNA oligotyping. The characteristics of the distribution of allelic variants of the loci HLA-DRB1, -DQA1, -DQB1, and -DPB1 were revealed; they were similar to those of other Subarctic Mongoloid populations and different from those for comparable populations of other climatic and geographic zones. Our data suggest that the specific features found for the distributions of some alleles of the loci examined are related to the geographic variation in the HLA gene system studied. 20 refs., 4 tabs.

  1. Neferine inhibits angiotensin II-induced rat aortic smooth muscle cell proliferation predominantly by downregulating fractalkine gene expression

    PubMed Central

    ZHENG, LULU; CAO, YONGWEN; LIU, SHAO; PENG, ZHENYU; ZHANG, SAIDAN

    2014-01-01

    Neferine inhibits the angiotensin II (AngII)-induced proliferation of vascular smooth muscle cells (SMCs), but the underlying mechanism is unclear. The aim of this study was to explore the mechanism underlying the effect of neferine on the proliferation of vascular SMCs. Rat aortic SMCs (RASMCs) were used and fractalkine (Fkn) gene expression was measured by quantitative polymerase chain reaction and western blot analysis. The proliferation of RASMCs was analyzed by MTT assay and flow cytometry. It was revealed that AngII induced Fkn expression in a dose- and time-dependent manner. Fkn-knockdown with small interfering RNA attenuated the AngII-induced RASMC proliferation. Furthermore, neferine inhibited Fkn expression and attenuated the AngII-induced RASMC proliferation. These findings suggest that the Fkn gene may play an important role in AngII-induced RASMC proliferation and that neferine acts to attenuate AngII-induced RASMC proliferation by inhibiting Fkn expression. PMID:25289057

  2. Differential accumulation of Phytophthora cambivora cox II gene transcripts in infected chestnut tissue.

    PubMed

    Vannini, Andrea; Tomassini, Alessia; Bruni, Natalia; Vettraino, Anna M

    2014-04-01

    This study provides a novel qRT-PCR protocol for specific detection and proof of viability of Phytophthora in environmental samples based on differential accumulation of cox II transcripts. Chemical and physical treatments were tested for their ability to induce in vitro the accumulation of cytochrome oxidase genes encoding subunits II (cox II) transcripts in Phytophthora cambivora. Glucose 170 mM, KNO3 0.25 mM and K3 PO3 0.5 and 0.8 mM induced the transcription of cox II in P. cambivora living mycelium while no transcription was observed in mycelium previously killed with 0.5% (p/v) RidomilGold(®) R WG. Living chestnut tissue was artificially infected with P. cambivora and treated with inducers. In vivo experiments confirmed the ability of glucose to induce the accumulation of P. cambivora cox II transcripts. Based on these results, pretreatment of environmental samples with glucose prior to nucleic acid extraction increased the accumulation of specific cox II transcripts, and therefore the sensitivity of qRT-PCR assay for detection of P. cambivora in living tissues. Furthermore, differential accumulation of transcripts between treated and untreated samples represents an unequivocal proof of inoculum viability. PMID:24527950

  3. Gene Duplication and the Evolution of Group II Chaperonins: Implications for Structure and Function

    Microsoft Academic Search

    John M. Archibald; Christian Blouin; W. Ford Doolittle

    2001-01-01

    Chaperonins are multisubunit protein-folding assemblies. They are composed of two distinct structural classes, which also have a characteristic phylogenetic distribution. Group I chaperonins (called GroEL\\/cpn60\\/hsp60) are present in Bacteria and eukaryotic organelles while group II chaperonins are found in Archaea (called the thermosome or TF55) and the cytoplasm of eukaryotes (called CCT or TriC). Gene duplication has been an important

  4. PapilioPhylogeny Based on Mitochondrial Cytochrome Oxidase I and II Genes

    Microsoft Academic Search

    Michael S. Caterino; Felix A. H. Sperling

    1999-01-01

    Butterflies of the genusPapiliohave served as the basis for numerous studies in insect physiology, genetics, and ecology. However, phylogenetic work on relationships among major lineages in the genus has been limited and inconclusive. We have sequenced 2.3 kb of DNA from the mitochondrial cytochrome oxidase I and II genes (COI and COII) for 23Papiliotaxa and two outgroups,Pachliopta neptunusandEurytides marcellus,in order

  5. MHC class II ? genes in the endangered Hainan Eld's deer (Cervus eldi hainanus).

    PubMed

    Liu, Hong-Yi; Xue, Fei; Wan, Qiu-Hong; Ge, Yun-Fa

    2013-01-01

    Contrary to neutral markers, the major histocompatibility complex (MHC) can reflect the fitness and adaptive potential of a given species due to its association with the immune system. For this reason, the use of MHC in endangered wildlife management has increased greatly in recent years. Here, we isolated complementary DNA (cDNA) and genomic DNA (gDNA) sequences to characterize the MHC class II ? genes in Hainan Eld's deer (Cervus eldi hainanus), a highly endangered cervid, which recovered from a severe population bottleneck consisting of 26 animals. Analysis of 7 individuals revealed the presence of 3 DRB and 3 DQB putatively functional gDNA sequences. The Ceel-DRB and DQB sequences displayed high variability in exon 2, and most nonsynonymous substitutions were detected in this region. Phylogenetic analysis indicated that trans-species evolution of MHC class II ? might occur in the Cervinae subfamily. Comparison of the number of sequences between gDNA and cDNA revealed that all sequences isolated from the genome were detectable in the cDNA libraries derived from different tissues (including the liver, kidney, and spleen), suggesting none of these sequences were derived from silent genes or pseudogenes. Characterization of the MHC class II ? genes may lay the foundation for future studies on genetic structure, mate choice, and viability analysis in Hainan Eld's deer. PMID:24078679

  6. Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

    PubMed Central

    Galli, Giorgio Giacomo; Honnens de Lichtenberg, Kristian; Carrara, Matteo; Hans, Wolfgang; Wuelling, Manuela; Mentz, Bettina; Multhaupt, Hinke Arnolda; Fog, Cathrine Kolster; Jensen, Klaus Thorleif; Rappsilber, Juri; Vortkamp, Andrea; Coulton, Les; Fuchs, Helmut; Gailus-Durner, Valérie; Hrab? de Angelis, Martin; Calogero, Raffaele Adolfo; Couchman, John Robert; Lund, Anders Henrik

    2012-01-01

    PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix. PMID:22589746

  7. Evolution of Type II Antifreeze Protein Genes in Teleost Fish: A Complex Scenario Involving Lateral Gene Transfers and Episodic Directional Selection

    PubMed Central

    Sorhannus, Ulf

    2012-01-01

    I examined hypotheses about lateral transfer of type II antifreeze protein (AFP) genes among “distantly” related teleost fish. The effects of episodic directional selection on amino acid evolution were also investigated. The strict consensus results showed that the type II AFP and type II antifreeze-like protein genes were transferred from Osmerus mordax to Clupea harengus, from the ancestral lineage of the Brachyopsis rostratus—Hemitripterus americanus clade to the ancestor of the Hypomesus nipponensis—Osmerus mordax group and from the ancestral lineage of Brachyopsis rostratus—Hemitripterus americanus—Siniperca chuatsi—Perca flavescens to Perca flavescens. At the present time, the available evidence is more consistent with the LGT hypothesis than with other alternative explanations. The overall results indicate that evolutionary history of the type II AFP gene is complex, and that episodic directional selection was instrumental in the evolution of this freeze-preventing protein from a C-type lectin precursor. PMID:23032610

  8. Evolution of Type II Antifreeze Protein Genes in Teleost Fish: A Complex Scenario Involving Lateral Gene Transfers and Episodic Directional Selection.

    PubMed

    Sorhannus, Ulf

    2012-01-01

    I examined hypotheses about lateral transfer of type II antifreeze protein (AFP) genes among "distantly" related teleost fish. The effects of episodic directional selection on amino acid evolution were also investigated. The strict consensus results showed that the type II AFP and type II antifreeze-like protein genes were transferred from Osmerus mordax to Clupea harengus, from the ancestral lineage of the Brachyopsis rostratus-Hemitripterus americanus clade to the ancestor of the Hypomesus nipponensis-Osmerus mordax group and from the ancestral lineage of Brachyopsis rostratus-Hemitripterus americanus-Siniperca chuatsi-Perca flavescens to Perca flavescens. At the present time, the available evidence is more consistent with the LGT hypothesis than with other alternative explanations. The overall results indicate that evolutionary history of the type II AFP gene is complex, and that episodic directional selection was instrumental in the evolution of this freeze-preventing protein from a C-type lectin precursor. PMID:23032610

  9. MHC class II transactivator CIITA is a recurrent gene fusion partner in lymphoid cancers.

    PubMed

    Steidl, Christian; Shah, Sohrab P; Woolcock, Bruce W; Rui, Lixin; Kawahara, Masahiro; Farinha, Pedro; Johnson, Nathalie A; Zhao, Yongjun; Telenius, Adele; Neriah, Susana Ben; McPherson, Andrew; Meissner, Barbara; Okoye, Ujunwa C; Diepstra, Arjan; van den Berg, Anke; Sun, Mark; Leung, Gillian; Jones, Steven J; Connors, Joseph M; Huntsman, David G; Savage, Kerry J; Rimsza, Lisa M; Horsman, Douglas E; Staudt, Louis M; Steidl, Ulrich; Marra, Marco A; Gascoyne, Randy D

    2011-03-17

    Chromosomal translocations are critically involved in the molecular pathogenesis of B-cell lymphomas, and highly recurrent and specific rearrangements have defined distinct molecular subtypes linked to unique clinicopathological features. In contrast, several well-characterized lymphoma entities still lack disease-defining translocation events. To identify novel fusion transcripts resulting from translocations, we investigated two Hodgkin lymphoma cell lines by whole-transcriptome paired-end sequencing (RNA-seq). Here we show a highly expressed gene fusion involving the major histocompatibility complex (MHC) class II transactivator CIITA (MHC2TA) in KM-H2 cells. In a subsequent evaluation of 263 B-cell lymphomas, we also demonstrate that genomic CIITA breaks are highly recurrent in primary mediastinal B-cell lymphoma (38%) and classical Hodgkin lymphoma (cHL) (15%). Furthermore, we find that CIITA is a promiscuous partner of various in-frame gene fusions, and we report that CIITA gene alterations impact survival in primary mediastinal B-cell lymphoma (PMBCL). As functional consequences of CIITA gene fusions, we identify downregulation of surface HLA class II expression and overexpression of ligands of the receptor molecule programmed cell death 1 (CD274/PDL1 and CD273/PDL2). These receptor-ligand interactions have been shown to impact anti-tumour immune responses in several cancers, whereas decreased MHC class II expression has been linked to reduced tumour cell immunogenicity. Thus, our findings suggest that recurrent rearrangements of CIITA may represent a novel genetic mechanism underlying tumour-microenvironment interactions across a spectrum of lymphoid cancers. PMID:21368758

  10. Overexpression of Ran gene from Lepidium latifolium L. (LlaRan) renders transgenic tobacco plants hypersensitive to cold stress.

    PubMed

    Sinha, Vimlendu Bhushan; Grover, Atul; Singh, Sadhana; Pande, Veena; Ahmed, Zakwan

    2014-09-01

    Ran is a multifunctional small GTPase involved in important cellular activities like nucleocytoplasmic transport, mitotic spindle assembly, nuclear envelope formation, etc., but is also known to be differentially expressed in response to abiotic stress, particularly low temperature. We have over-expressed Lepidium latifolium (Fam. Brassicaceae) Ran gene in tobacco to study the response of the plants to cold stress (24 h; 4 °C). Transformation of the tobacco plants was verified using PCR targeting Ran gene and co-transformed selectable marker gene nptII. Segregation in Mendelian ratios was validated in five transgenic lines by germination of T1 and T2 seeds on moist filter papers containing 150 mg/l kanamycin. Higher levels of electrolyte leakage and lipid peroxidation pointed towards hypersensitivity of plants. Similarly, lesser proline accumulation compared to wild types also indicated susceptibility of plants to death under chilling conditions. Specific activity of antioxidant enzymes superoxide dismutase and glutathione reductase was also measured under stressed and control conditions. A variation was observed across the different lines, and four out of five lines showed lesser specific activity compared to wild type plants, thus indicating reduced capability of scavenging free radicals. In totality, a strong evidence on induced hypersensitivity to cold stress has been collected which may further be helpful in designing appropriate strategies for engineering crop plants for survival under cold stress conditions. PMID:24973880

  11. Investigation of the polymorphic Ava II site by a PCR-based assay at the human CD18 gene locus

    Microsoft Academic Search

    Shinya Mastuura; Fumio Kishi

    1994-01-01

    The AvaII polymorphic site within the human CD18 gene was investigated in the Japanese population. A distinct distribution pattern is observed in this population. This polymorphism provides a new genetic marker for the long arm of chromosome 21 and should be a useful marker of leukocyte adhesion deficiency caused by mutations of the CD18 gene.

  12. How GeneChip® was developed (Part II), Stephen FodorSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-03-26

    Interviewee: Stephen Fodor DNAi Location:Applications>Genes and medicine>genetic profiling>Stephen Fodor How the chip was developed (Part II) Stephen Fodor continues his discussion of the experiments that laid the groundwork for GeneChip® technology.

  13. A new point mutation in the ND1 mitochondrial gene identified in a type II diabetic patient

    SciTech Connect

    Kalinin, V.N. [Research Center of Medical Genetics, Moscow (Russian Federation); Schmidt, W.; Olek, K. [Institut fuer Molekularbiologische Diagnostik, Bonn (Germany)] [and others

    1995-08-01

    A novel mutation in a mitochondrial gene was identified in a patient with type II diabetes mellitus. G-to-A transition was localized at the nt3316 position of gene ND1 and resulted in alanine threonine replacement at position 4 of mitochondrial NAD-H-dehydrogenase. 6 refs., 2 figs.

  14. Gene required for normal MHC class II expression and function is localized to approximately 45 kb of DNA in the class II region of the MHC.

    PubMed

    Ceman, S; Petersen, J W; Pinet, V; DeMars, R

    1994-03-15

    In certain mutant human B cell lines, MHC-encoded class II molecules displayed at the cell surface have an abnormal conformation and are unstable in the presence of SDS. The mutants cannot present exogenous protein Ags to T cells but elicit responses with exogenous antigenic peptides; thus, formation of intracellular complexes between antigenic peptides and class II molecules is impaired. Previous analysis of LCL deletion mutants, .82, .174, and 5.2.4, showed that genes needed for this function must be present in approximately 230 kb of DNA in the class II region of the MHC. We now describe a new deletion mutant, .61, which has normal class II-mediated Ag processing/presentation. The TAP1, TAP2, LMP2, and LMP7 genes are deleted from .61, demonstrating that those genes are not needed for normal formation of intracellular class II/peptide complexes. The genes in question must be located in DNA that is present in .61 and .82 (both normal) and absent from .174 and 5.2.4. (both defective). Mapping of the deletion breakpoints indicates that genes needed for normal class II-associated Ag processing/presentation are either: 1) in an approximately 40 kb L DNA segment located between the DMB and LMP2 loci or 2) in an R region between the DQA2 and DQB1 loci and are completely included on a 5.1-kb fragment formed by joining of DNA that flanks the deletion in .61. The evidence favors location of the genes in the L DNA segment. PMID:8144887

  15. Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Ma, Ruiqin; He, Feng; Wen, Haishen; Li, Jifang; Shi, Bao; Shi, Dan; Liu, Miao; Mu, Weijie; Zhang, Yuanqing; Hu, Jian; Han, Weiguo; Zhang, Jianan; Wang, Qingqing; Yuan, Yuren; Liu, Qun

    2012-03-01

    As a specific gene of fish, cytochrome P450c17-II ( CYP17-II) gene plays a key role in the growth, development an reproduction level of fish. In this study, the single-stranded conformational polymorphism (SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-II gene in a population of 75 male Japanese flounder ( Paralichthys olivaceus). Three single nucleotide polymorphisms (SNPs) were identified in CYP17-II gene of Japanese flounder. They were c.G594A (p.G188R), c.G939A and c.G1502A (p.G490D). SNP1 (c.G594A), located in exon 4 of CYP17-II gene, was significantly associated with gonadosomatic index (GSI). Individuals with genotype GG of SNP1 had significantly lower GSI ( P < 0.05) than those with genotype AA or AG. SNP2 (c.G939A) located at the CpG island of CYP17-II gene. The mutation changed the methylation of exon 6. Individuals with genotype AA of SNP2 had significantly lower serum testosterone (T) level and hepatosomatic index (HSI) compared to those with genotype GG. The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder. However, the SNP3 (c.G1502A) located in exon 9 did not affect the four measured reproductive traits. This study showed that CYP17-II gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.

  16. Persistent Ehrlichia chaffeensis infection occurs in the absence of functional major histocompatibility complex class II genes

    NASA Technical Reports Server (NTRS)

    Ganta, Roman Reddy; Wilkerson, Melinda J.; Cheng, Chuanmin; Rokey, Aaron M.; Chapes, Stephen K.

    2002-01-01

    Human monocytic ehrlichiosis is an emerging tick-borne disease caused by the rickettsia Ehrlichia chaffeensis. We investigated the impact of two genes that control macrophage and T-cell function on murine resistance to E. chaffeensis. Congenic pairs of wild-type and toll-like receptor 4 (tlr4)- or major histocompatibility complex class II (MHC-II)-deficient mice were used for these studies. Wild-type mice cleared the infection within 2 weeks, and the response included macrophage activation and the synthesis of E. chaffeensis-specific Th1-type immunoglobulin G response. The absence of a functional tlr4 gene depressed nitric oxide and interleukin 6 secretion by macrophages and resulted in short-term persistent infections for > or =30 days. In the absence of MHC-II alleles, E. chaffeensis infections persisted throughout the entire 3-month evaluation period. Together, these data suggest that macrophage activation and cell-mediated immunity, orchestrated by CD4(+) T cells, are critical for conferring resistance to E. chaffeensis.

  17. Histone acetylation facilitates RNA polymerase II transcription of the Drosophila hsp26 gene in chromatin.

    PubMed Central

    Nightingale, K P; Wellinger, R E; Sogo, J M; Becker, P B

    1998-01-01

    A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for those acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell-free system to reconstitute chromatin with increased histone acetylation levels, we directly tested for a causal role of histone acetylation in transcription by RNA pol II. Chromatin, containing either control or acetylated histones, was reconstituted to comparable nucleosome densities and characterized by electron microscopy after psoralen cross-linking as well as by in vitro transcription. While H1-containing control chromatin severely repressed transcription of our model hsp26 gene, highly acetylated chromatin was significantly less repressive. Acetylation of histones, and particularly of histone H4, affected transcription at the level of initiation. Monitoring the ability of the transcription machinery to associate with the promoter in chromatin, we found that heat shock factor, a crucial regulator of heat shock gene transcription, profited most from histone acetylation. These experiments demonstrate that histone acetylation can modulate activator access to their target sites in chromatin, and provide a causal link between histone acetylation and enhanced transcription initiation of RNA pol II in chromatin. PMID:9582280

  18. Androgen-dependent expression, gene structure, and molecular evolution of guinea pig caltrin II, a WAP-motif protein.

    PubMed

    Furutani, Yutaka; Kato, Akira; Kawai, Ryoji; Fibriani, Azzania; Kojima, Soichi; Hirose, Shigehisa

    2004-11-01

    We determined the cDNA and gene structures of guinea pig caltrin II, a unique member of the calcium transporter inhibitors containing a whey acidic protein (WAP) motif, and we established that it is a secretory protein with a potential 21-amino acid signal peptide in its N-terminus. Northern blot analysis and in situ hybridization histochemistry indicated that the expression of caltrin II is restricted to luminal epithelial cells in the seminal vesicles. Its message levels markedly decreased either after castration (and were restored by simultaneous administration of testosterone) or after treatment of the animals with estradiol, suggesting that the expression of caltrin II is androgen-dependent. Recombinant caltrin II had an elastase-inhibitor activity. Comparison of sequence between the caltrin II and related genes and their molecular evolutionary analyses revealed that caltrin II and seminal vesicle secretory proteins (SVPs) appear to be evolved from a common ancestor gene that is made by the fusion of semenogelin and trappin genes. Caltrin II and SVPs lost the transglutaminase substrate domain and the WAP motif, respectively, within a single exon, resulting in the exertion of different functions. PMID:15240421

  19. Parental allele specific methylation of the human insulin-like growth factor II gene and Beckwith-Wiedemann syndrome.

    PubMed Central

    Schneid, H; Seurin, D; Vazquez, M P; Gourmelen, M; Cabrol, S; Le Bouc, Y

    1993-01-01

    In an attempt to elucidate the role of methylation in parental imprinting at the IGF-II gene locus, for which imprinting has already been described in the mouse, we undertook an allele specific methylation study of the human IGF-II gene (mapped to 11p15.5) in a control population and in patients with Beckwith-Wiedemann syndrome. In control leucocyte DNA (16 unrelated adults and eight families), the maternal allele of the IGF-II gene was specifically hypomethylated, whereas no such allele specific methylation was found for either the insulin or the calcitonin genes which are located in 11p15.5 and 11p15.1, respectively. Furthermore, the IGF-II gene specific hypomethylation was localised on the 5' portion of exon 9. In the patients with Beckwith-Wiedemann syndrome in which the IGF-II gene is thought to be involved and where paternal isodisomy has been described, hypomethylation of the maternal allele was conserved in leucocyte DNA, but abnormal methylation was detected in malformed tissues where the paternal allele was also demethylated. Some specific mechanism linked to methylation therefore seems to be involved in the pathogenesis of Beckwith-Wiedemann syndrome. Images PMID:8320696

  20. Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

    PubMed Central

    Lauder, S; Bankmann, M; Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1996-01-01

    Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected. PMID:8943333

  1. Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

    PubMed

    Lauder, S; Bankmann, M; Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1996-12-01

    Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected. PMID:8943333

  2. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani

    PubMed Central

    McNeil, Bonnie A.; Simon, Dawn M.; Zimmerly, Steven

    2014-01-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5? splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5? exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns. PMID:24214997

  3. Inhibition of major histocompatibility complex class II gene transcription by nitric oxide and antioxidants.

    PubMed

    Grimm, Michael; Spiecker, Martin; De Caterina, Raffaele; Shin, Wee Soo; Liao, James K

    2002-07-19

    Interferon (IFN)-gamma facilitates cellular immune response, in part, by inducing the expression of major histocompatibility complex class II (MHC-II) molecules. We demonstrate that IFN-gamma induces the expression of HLA-DRA in vascular endothelial cells via mechanisms involving reactive oxygen species. IFN-gamma-induced HLA-DRA expression was inhibited by nitric oxide (NO) and antioxidants such as superoxide dismutase, catalase, pyrrolidine dithiocarbamate, and N-acetylcysteine. Nuclear run-on assays demonstrated that NO and antioxidants inhibited IFN-gamma-induced HLA-DRA gene transcription. Transient transfection studies using a fully functional HLA-DRA promoter construct ([-300]DR alpha.CAT) showed that inhibition of endogenous NO synthase activity by N(omega)-monomethyl-l-arginine or addition of exogenous hydrogen peroxide (H(2)O(2)) augmented basal and IFN-gamma-stimulated [-300]DR alpha.CAT activity. However, H(2)O(2) and N(omega)-monomethyl-l-arginine could induce HLA-DRA expression suggesting that H(2)O(2) is a necessary but not a sufficient mediator of IFN-gamma-induced HLA-DRA expression. Electrophoretic mobility shift assay and Western blotting demonstrated that NO and antioxidants had little or no effect on IFN-gamma-induced IRF-1 activation or MHC-II transactivator (CIITA) expression but did inhibit IFN-gamma-induced activation of STAT1 alpha (p91) and Y box transcription factors, NF-Y(A) and NF-Y(B). These results indicate that NO and antioxidants may attenuate vascular inflammation by antagonizing the effects of intracellular reactive oxygen species generation by IFN-gamma, which is necessary for MHC-II gene transcription. PMID:12006557

  4. Nucleotide sequence of the Dpn II DNA methylase gene of Streptococcus pneumoniae and its relationship to the dam gene of Escherichia coli

    SciTech Connect

    Mannarelli, B.M.; Balganesh, T.S.; Greenberg, B.; Springhorn, S.S.; Lacks, S.A.

    1985-07-01

    The structural gene (dpnM) for the Dpn II DNA methylase of Streptococcus pneumoniae, which is part of the Dpn II restriction system and methylates adenine in the sequence 5'-G-A-T-C-3', was identified by subcloning fragments of a chromosomal segment from a Dpn II-producing strain in an S. pneumoniae host/vector cloning system and demonstrating function of the gene also in Bacillus subtilis. Determination of the nucleotide sequence of the gene and adjacent DNA indicates that it encodes a polypeptide of 32,903 daltons. A putative promoter for transcription of the gene lies within a hundred nucleotides of the polypeptide start codon. Comparison of the coding sequence to that of the dam gene of Escherichia coli, which encodes a similar methylase, revealed 30% of the amino acid residues in the two enzymes to be identical. This homology presumably reflects a common origin of the two genes prior to the divergence of Gram-positive and Gram-negative bacteria. It is suggested that the restriction function of the gene is primitive, and that the homologous restriction system in E. coli has evolved to play an accessory role in heteroduplex DNA base mismatch repair.

  5. DMA and DMB are the only genes in the class II region of the human MHC needed for class II-associated antigen processing.

    PubMed

    Ceman, S; Rudersdorf, R A; Petersen, J M; DeMars, R

    1995-03-15

    Previous studies have shown that homozygous mutations between the LMP2 and DNA loci in the human MHC cause class II molecules to be abnormally conformed and unstable in the presence of SDS at low temperature, and impede class II-associated Ag processing and presentation. These abnormalities result from impaired ability to form intracellular class II/peptide complexes that predominate in normal cells. We show in this work that this defect results from deficient expression of either the DMA or the DMB gene. Human B-LCL.174 (DR3) cells, which have a deletion of all known expressible genes in the class II region, express transgene-encoded HLA-DR3, but have the abnormalities. Transfer of cosmid HA14, which contains the DMA and DMB genes, into .174 (DR3) cells restored normal DR3 conformation, stability in 0.4% SDS at 0 degree, and ability to process and present tetanus toxoid, but only when both DMA and DMB mRNAs were present. The requirement for both genetic expressions in engendering normal phenotypes was confirmed by transferring the cloned genes into .174 (DR3) cells separately or together. Because normal phenotypes were fully restored in transferent cells expressing DMA plus DMB, other genes in the approximately 1-mb homozygous class II region deletion in .174 (DR3) cells either do not participate in or are dispensable for apparently normal production of intracellular class II/peptide complexes. The properties of DM-deficient EBV-transformed B lymphoblastoid cell lines (LCLs) suggest ways of identifying humans in whom DM deficiency contributes to congenital immunodeficiency and malignancy. PMID:7876531

  6. Expressed MHC class II genes in sea otters (Enhydra lutris) from geographically disparate populations

    USGS Publications Warehouse

    Bowen, L.; Aldridge, B.M.; Miles, A.K.; Stott, J.L.

    2006-01-01

    The major histocompatibility complex (MHC) is central to maintaining the immunologic vigor of individuals and populations. Classical MHC class II genes were targeted for partial sequencing in sea otters (Enhydra lutris) from populations in California, Washington, and Alaska. Sequences derived from sea otter peripheral blood leukocyte mRNAs were similar to those classified as DQA, DQB, DRA, and DRB in other species. Comparisons of the derived amino acid compositions supported the classification of these as functional molecules from at least one DQA, DQB, and DRA locus and at least two DRB loci. While limited in scope, phylogenetic analysis of the DRB peptide-binding region suggested the possible existence of distinct clades demarcated by geographic region. These preliminary findings support the need for additional MHC gene sequencing and expansion to a comprehensive study targeting additional otters. ?? 2006 Blackwell Munksgaard.

  7. Positive selection on MHC class II DRB and DQB genes in the bank vole (Myodes glareolus).

    PubMed

    Scherman, Kristin; Råberg, Lars; Westerdahl, Helena

    2014-05-01

    The major histocompatibility complex (MHC) class IIB genes show considerable sequence similarity between loci. The MHC class II DQB and DRB genes are known to exhibit a high level of polymorphism, most likely maintained by parasite-mediated selection. Studies of the MHC in wild rodents have focused on DRB, whilst DQB has been given much less attention. Here, we characterised DQB genes in Swedish bank voles Myodes glareolus, using full-length transcripts. We then designed primers that specifically amplify exon 2 from DRB (202 bp) and DQB (205 bp) and investigated molecular signatures of natural selection on DRB and DQB alleles. The presence of two separate gene clusters was confirmed using BLASTN and phylogenetic analysis, where our seven transcripts clustered according to either DQB or DRB homologues. These gene clusters were again confirmed on exon 2 data from 454-amplicon sequencing. Our DRB primers amplify a similar number of alleles per individual as previously published DRB primers, though our reads are longer. Traditional d N/d S analyses of DRB sequences in the bank vole have not found a conclusive signal of positive selection. Using a more advanced substitution model (the Kumar method) we found positive selection in the peptide binding region (PBR) of both DRB and DQB genes. Maximum likelihood models of codon substitutions detected positively selected sites located in the PBR of both DQB and DRB. Interestingly, these analyses detected at least twice as many positively selected sites in DQB than DRB, suggesting that DQB has been under stronger positive selection than DRB over evolutionary time. PMID:24748547

  8. Sheep exhibit novel variations in the organization of the mammalian type II gonadotropin-releasing hormone receptor gene.

    PubMed

    Gault, Paula M; Morgan, Kevin; Pawson, Adam J; Millar, Robert P; Lincoln, Gerald A

    2004-05-01

    Species-specific differences in genes encoding type II GnRH receptor indicate that a functional hepta-helical receptor is produced in monkeys but not in rodents, cows, chimpanzees, or humans. To further investigate the extent of evolutionary differences, we sequenced the type II GnRH receptor gene from wild-type Soay sheep. The gene was isolated by long-distance PCR using primers to PEX11beta and RBM8A genes known to flank type II GnRH receptor gene homologues. The gene spans 5.7-kb DNA and was sequenced after shot-gun subcloning. Its novel features include absence of a Pit-1 transcription factor binding site, a premature stop codon (TAG) in exon 1, an in-frame deletion of 51 bp (17 codons) in exon 2, and several nonconservative codon changes. Sheep breed variation in the gene was assessed using genomic DNA in PCR-restriction digest assays for the premature stop codon and in a PCR assay for the deletion. Both characteristics were present in all 15 breeds tested. Receptor gene expression was investigated using poly-A(+) RNA Northern analysis, RT-PCR, and in situ hybridization. An oligonucleotide probe to exon 1 revealed an alternative transcript in testis but not in pituitary gland. No transcripts in testis or pituitary were detectable using an exon 2-3 probe. All tissues examined including multiple brain areas and gonadotrope-enriched cell cultures were negative for type II GnRH receptor in RT-PCR. Testis and pituitary sections were negative with exon 1 riboprobes and exon 1 or 2-3 oligonucleotide probes in in situ hybridization. A hepta-helical type II GnRH receptor is therefore not expressed from this sheep gene. PMID:14749360

  9. Population genetic structure of malaria vector Anopheles stephensi using mitochondrial cytochrome oxidase II gene in Indian populations.

    PubMed

    Sharma, Arvind; Deshmukh, Arunaditya; Sharma, Richa; Kumar, Ashwani; Mukherjee, Sayantan; Chandra, G C; Gakhar, S K

    2014-10-01

    The genetic differentiation in A. stephensi based on haplotype diversity using Restriction Fragment Length Polymorphism and bysequencing of CO II gene across different localities in India has been analyzed. The presence of only one DraI restriction site in CO II gene conferred to haplotype B indicating that the gene is very much conserved and the gene flow is not affected even by a major geographical distance barrier. The sequencing and analysisof various population parameters revealed seven haplotypes in all populations. The West Bengal population was found to be more genetically diverse than others. The geographic distance between populations was found to be contributing to the genetic differentiation. The sign of demographic expansion were found in three of the five populations. The local geographic barriers were found to be ineffective in prevention of gene flow. PMID:25345249

  10. Differential expression of secretogranin II and chromogranin A genes in the female rat pituitary through sexual maturation and estrous cycle

    SciTech Connect

    Anouar, Y.; Duval, J. (C.U.R.A. 256, Rennes (France))

    1991-03-01

    Secretogranin II (SgII) is a protein of pituitary secretory granules released by LHRH-stimulated gonadotrope cells. Estrogens and androgens are modulators of SgII release. Experiments were performed to determine the regulation of expression of the SgII gene in the female rat pituitary, during sexual maturation and according to the estrous cycle. Age- and cycle-related changes in SgII mRNA content were estimated through cytoplasmic slot blot; SgII content was determined by western blotting; maturation of the protein was controlled through (35S)sulfate labeling. Variations in chromogranin A (CgA), another protein of secretory granules, were analyzed in the same experimental conditions to assess the specificity of SgII regulation. The pituitary SgII concentration increased between days 7 and 21 (2.2-fold) and then declined to the initial 7-day-old value. Simultaneously, the CgA concentration went through a maximum between days 14 and 21 and then strongly dropped to barely detectable levels in the adult pituitary. The SgII mRNA concentration followed roughly the same pattern as the protein. Moreover, the sulfation level remained constant between days 14 and 60. These results demonstrated a regulatory mechanism operating, during sexual maturation, on the SgII gene and not on the protein processing or on storage/release steps. In the 4-day cycling females, the pituitary SgII mRNA and protein contents were the lowest during estrus. They then increased to their highest values in diestrus II. Moreover, the sulfation level of SgII was significantly higher during estrus than during any other stage. Due to its low content level, variations in pituitary CgA could not be demonstrated during the cycle.

  11. Association of angiotensin-converting enzyme and angiotensin II type I receptor gene polymorphisms with extreme obesity in Polish individuals.

    PubMed

    Pacholczyk, Marta; Ferenc, Tomasz; Kowalski, Jan; Adamczyk, Przemys?aw; Chojnowski, Jacek; Ponikowska, Irena

    2013-08-01

    There is strong evidence for the presence of a functional renin-angiotensin system in human adipose tissue. The aim of our study was to investigate the association of polymorphic variants of angiotensin-converting enzyme gene (ACE I/D) and angiotensin II type I receptor gene (AGTR1 A1166C) with extreme obesity and obesity-associated type 2 diabetes mellitus (T2DM) and to examine their combined effect on extremely obese patients. Overall, no significant associations were detected between ACE and AGTR1 gene polymorphisms and extreme obesity. However, extremely obese patients with T2DM showed an increased frequency of ACE II genotype compared with controls (p<0.05) and with non-diabetic extremely obese patients (p<0.01). The results suggest that II genotype of ACE was a significant contributor to extreme obesity in AA homozygotes of AGTR1 gene, regardless of the presence of T2DM. Moreover, the analysis of genetic polymorphisms demonstrated that ACE II and AGTR1 AC genotypes were most frequently observed in patients with extreme obesity and T2DM. On the basis of our results, we suggest that ACE II homozygosity may be a significant predictor of extreme obesity and T2DM and that the interaction between ACE and AGTR1 genes may be considered a predisposing factor for extreme obesity and extreme obesity-associated T2DM development. PMID:23745680

  12. Identification of forensically important Sarcophagidae (Diptera) based on partial mitochondrial cytochrome oxidase I and II genes.

    PubMed

    Aly, Sanaa Mohamed; Wen, Jifang; Wang, Xiang

    2013-06-01

    Entomological evidence is of great importance in forensic cases for postmortem interval calculation. The use of Sarcophagidae (Diptera) for postmortem interval estimation is limited because morphological determination is often hampered because of similar characteristics in the larval, pupal, and even adult stage. To make the species identification more accurate and reliable, DNA-based identification is considered. In this study, we assessed the use of partial mitochondrial cytochrome oxidase I and II genes for discrimination of forensically important Sarcophagidae from Egypt and China [Sarcophaga argyrostoma (Robineau-Desvoidy), Sarcophaga dux (Thomson), Sarcophaga albiceps (Meigen), and Wohlfahrtia nuba (Wiedemann)]. This region was amplified using polymerase chain reaction followed by direct sequencing of the amplification products and using restriction enzymes HinfI and MfeI. Nucleotide sequence divergences were calculated using the Kimura 2-parameter distance model, and a neighbor-joining phylogenetic tree was generated. All examined specimens were assigned to the correct species. Combinations of the restriction enzymes HinfI and MfeI provide different restriction fragment length polymorphism profiles even among 3 sympatric species that belong to the Sarcophaga genus. Therefore, this study demonstrates that the studied partial mitochondrial cytochrome oxidase I and II genes were found to be instrumental for the molecular identification of these forensically important flesh fly species. PMID:23629402

  13. Microarray analysis of altered gene expression in murine fibroblasts transformed by nickel(II) to nickel(II)-resistant malignant phenotype

    SciTech Connect

    Kowara, Renata [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States)]. E-mail: Renata.Kowara@nrc-cnrc.gc.ca; Karaczyn, Aldona [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Cheng, Robert Y.S. [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Salnikow, Konstantin [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States); Kasprzak, Kazimierz S. [Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702 (United States)

    2005-05-15

    B200 cells are Ni(II)-transformed mouse BALB/c-3T3 fibroblasts displaying a malignant phenotype and increased resistance to Ni(II) toxicity. In an attempt to find genes whose expression has been altered by the transformation, the Atlas Mouse Stress/Toxicology cDNA Expression Array (Clontech Laboratories, Inc., Palo Alto, CA) was used to analyze the levels of gene expression in both parental and Ni(II)-transformed cells. Comparison of the results revealed a significant up- or downregulation of the expression of 62 of the 588 genes present in the array (approximately 10.5%) in B200 cells. These genes were assigned to different functional groups, including transcription factors and oncogenes (9/14; fractions in parentheses denote the number of up-regulated versus the total number of genes assigned to this group), stress and DNA damage response genes (11/12), growth factors and hormone receptors (6/9), metabolism (7/7), cell adhesion (2/7), cell cycle (3/6), apoptosis (3/4), and cell proliferation (2/3). Among those genes, overexpression of beta-catenin and its downstream targets c-myc and cyclin D1, together with upregulated cyclin G, points at the malignant character of B200 cells. While the increased expression of glutathione (GSH) synthetase, glutathione-S-transferase A4 (GSTA4), and glutathione-S-transferase theta (GSTT), together with high level of several genes responding to oxidative stress, suggests the enforcement of antioxidant defenses in Ni-transformed cells.

  14. Identification and mapping of two divergent, unlinked major histocompatibility complex class II B genes in Xiphophorus fishes.

    PubMed Central

    McConnell, T J; Godwin, U B; Norton, S F; Nairn, R S; Kazianis, S; Morizot, D C

    1998-01-01

    We have isolated two major histocompatibility complex (MHC) class II B genes from the inbred fish strain Xiphophorus maculatus Jp 163 A. We mapped one of these genes, designated here as DXB, to linkage group III, linked to a malic enzyme locus, also syntenic with human and mouse MHC. Comparison of genomic and cDNA clones shows the gene consists of six exons and five introns. The encoded beta1 domain has three amino acids deleted and a cytoplasmic tail nine amino acids longer than in other teleost class II beta chains, more similar to HLA-DRB, clawed frog Xela-F3, and nurse shark Gici-B. Key residues for disulfide bonds, glycosylation, and interaction with alpha chains are conserved. These same features are also present in a swordtail (Xiphophorus helleri) genomic DXB PCR clone. A second type of class II B clone was amplified by PCR from X. maculatus and found to be orthologous to class II genes identified in other fishes. This DAB-like gene is 63% identical to the X. maculatus DXB sequence in the conserved beta2-encoding exon and was mapped to new unassigned linkage group LG U24. The DXB gene, then, represents an unlinked duplicated locus not previously identified in teleosts. PMID:9691047

  15. Isolation and characterization of major histocompatibility complex (MHC) class II B genes in the Barn owl (Aves: Tyto alba).

    PubMed

    Burri, Reto; Niculita-Hirzel, Hélène; Roulin, Alexandre; Fumagalli, Luca

    2008-09-01

    We isolated major histocompatibility complex class II B (MHCIIB) genes in the Barn owl (Tyto alba). A PCR-based approach combined with primer walking on genomic and complementary DNA as well as Southern blot analyses revealed the presence of two MHCIIB genes, both being expressed in spleen, liver, and blood. Characteristic structural features of MHCIIB genes as well as their expression and high non-synonymous substitution rates in the region involved in antigen binding suggest that both genes are functional. MHC organization in the Barn owl is simple compared to passerine species that show multiple duplications, and resembles the minimal essential MHC of chicken. PMID:18548243

  16. A Serine Protease-Encoding Gene (aprII) of Alteromonas sp. Strain O-7 Is Regulated by the Iron Uptake Regulator (Fur) Protein

    Microsoft Academic Search

    HIROSHI TSUJIBO; KATSUSHIRO MIYAMOTO; TAKASHI OKAMOTO; HIDEYUKI ORIKOSHI; YOSHIHIKO INAMORI

    2000-01-01

    The ferric uptake regulator (Fur) box-like sequence was located upstream of the serine protease-encoding gene (aprII) from a marine bacterium, Alteromonas sp. strain O-7. To clarify whether the production of AprII (the gene product of aprII) is regulated by the environmental iron concentrations, this strain was cultured under iron-depleted or iron-rich conditions and the level of AprII in the culture

  17. Chicken MHC class I and II gene effects on antibody response kinetics in adult chickens.

    PubMed

    Zhou, Huaijun; Lamont, Susan J

    2003-06-01

    The major histocompatibility complex (MHC) plays an important role in regulation of the immune response. The MHC class I and II genes were selected as candidates to investigate associations with vaccine response to Salmonella enteritidis and kinetics of antibody response to sheep red blood cell (SRBC) and Brucella abortus. Primary antibody response after S. enteritidis vaccination at day 10, and antibody response to SRBC and killed B. abortus after immunization at 19 and 22 weeks were measured in an F2 population. The resource population was derived from males of two highly inbred MHC-congenic Fayoumi chicken lines (M5.1 and M15.2) mated with highly inbred G-B1 Leghorn line hens. Secondary phase parameters of minimum titers ( Y(min)), maximum titers ( Y(max)), and time needed to achieve Y(min) ( t(min)) and Y(max) ( t(max)) were estimated from post-secondary titers by using a non-linear regression model. Associations of single nucleotide polymorphisms (SNPs) in MHC class I and II genes with antibody response parameters were determined by a general linear model. Significant associations were found primarily in the M15.2 grandsire haplotype. There were significant associations between MHC class I alpha(1) and alpha(2) SNPs and antibody response to S. enteritidis, primary antibody response to B. abortus, Y(min) to SRBC, and Y(max) to both SRBC and B. abortus. There were significant effects of the MHC class II beta(1) domain SNP on S. enteritidis antibody and Y(max) to SRBC. The results suggest that the characterized SNPs might be used in future applications by marker-assisted selection to improve vaccine response and immunocompetence in chickens. PMID:12743657

  18. Early diagnosis of and surgical strategy for adrenal medullary disease in MEN II gene carriers

    SciTech Connect

    Jansson, S.; Tisell, L.E.; Fjaelling, M.L.; Lindberg, S.; Jacobsson, L.; Zachrisson, B.F.

    1988-01-01

    Sixteen multiple endocrine neoplasia type II (MEN II) gene carriers--12 who had undergone thyroidectomy because of medullary carcinoma of the thyroid and 4 whose thyroid glands had been removed because of C cell hyperplasia--were examined for the presence of pheochromocytomas. No patient had sought medical advice for pheochromocytoma symptoms. Fourteen patients had MEN IIa syndromes, one patient had a MEN IIb and another patient had a mixed syndrome of von Recklinghausen's neurofibromatosis and MEN II. Eight patients had undergone unilateral adrenalectomy for pheochromocytoma 11 +/- 4 years before. The patients underwent clinical examination, determination of the urinary excretion of catecholamines and metabolites, and /sup 131/I-metaiodobenzylguanidine (/sup 131/I-MIBG) and CAT scans. /sup 131/I-MIBG scanning was performed with images 1, 4, and 7 days after the radionuclide injection. In seven of eight patients who had undergone unilateral adrenalectomies, the /sup 131/I-MIBG scans showed accumulation of the radionuclide in the remaining adrenal gland. Bilateral adrenal accumulation of the radionuclide was demonstrated in seven of eight MEN IIa gene carriers who had not undergone adrenalectomy. Five patients, two of whom had undergone adrenalectomy, were found to have unilateral pheochromocytomas less than 2 cm in diameter. Only one of these five patients had an elevated excretion of urinary catecholamines. Between day 4 and day 7 after /sup 131/I-MIBG injection, adrenal glands with pheochromocytomas increased their relative accumulation of the radionuclide significantly more (p less than 0.02) than did adrenal glands without any demonstrable pheochromocytomas. All the pheochromocytomas were viewed by means of CAT scans.

  19. Genetic Diversity of the Flagellin Genes of Clostridium botulinum Groups I and II

    PubMed Central

    Woudstra, Cedric; Lambert, Dominic; Anniballi, Fabrizio; De Medici, Dario; Austin, John

    2013-01-01

    Botulinum neurotoxins (BoNTs) are produced by phenotypically and genetically different Clostridium species, including Clostridium botulinum and some strains of Clostridium baratii (serotype F) and Clostridium butyricum (serotype E). BoNT-producing clostridia responsible for human botulism encompass strains of group I (secreting proteases, producing toxin serotype A, B, or F, and growing optimally at 37°C) and group II (nonproteolytic, producing toxin serotype E, B, or F, and growing optimally at 30°C). Here we report the development of real-time PCR assays for genotyping C. botulinum strains of groups I and II based on flaVR (variable region sequence of flaA) sequences and the flaB gene. Real-time PCR typing of regions flaVR1 to flaVR10 and flaB was optimized and validated with 62 historical and Canadian C. botulinum strains that had been previously typed. Analysis of 210 isolates of European origin allowed the identification of four new C. botulinum flaVR types (flaVR11 to flaVR14) and one new flaVR type specific to C. butyricum type E (flaVR15). The genetic diversity of the flaVR among C. botulinum strains investigated in the present study reveals the clustering of flaVR types into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolytic Clostridium botulinum, subgroup 2 is made up of nonproteolytic C. botulinum only, and subgroup 5 is specific to C. butyricum type E. The genetic variability of the flagellin genes carried by C. botulinum and the possible association of flaVR types with certain geographical areas make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology of C. botulinum. PMID:23603687

  20. Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme

    PubMed Central

    2013-01-01

    Background Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II). Results Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria. Conclusions Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes an exceptional model of acquisition and maintenance of redundant gene functions between non-homologous genes as a result of convergent evolution. Subsequently, although old episodic events of HGT could not be excluded, the results supported a prevalent role of gene loss in explaining the distribution of DXR-II in specific pathogenic eubacteria. Our results highlight the importance of the functional characterization of evolutionary shortcuts in isoprenoid biosynthesis for screening specific antibacterial drugs and for regulating the production of isoprenoids of human interest. PMID:24004839

  1. pSiM24 Is a Novel Versatile Gene Expression Vector for Transient Assays As Well As Stable Expression of Foreign Genes in Plants

    PubMed Central

    Sahoo, Dipak Kumar; Dey, Nrisingha; Maiti, Indu Bhushan

    2014-01-01

    We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3?rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and ?-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses. PMID:24897541

  2. Depletion of REF/Aly alters gene expression and reduces RNA polymerase II occupancy

    PubMed Central

    Stubbs, Sarah H.; Conrad, Nicholas K.

    2015-01-01

    Pre-mRNA processing is mechanistically linked to transcription with RNA pol II serving as a platform to recruit RNA processing factors to nascent transcripts. The TREX complex member, REF/Aly, has been suggested to play roles in transcription and nuclear RNA stability in addition to its more broadly characterized role in mRNA export. We employed RNA-seq to identify a subset of transcripts with decreased expression in both nuclear and cytoplasmic fractions upon REF/Aly knockdown, which implies that REF/Aly affects their expression upstream of its role in mRNA export. Transcription inhibition experiments and metabolic labeling assays argue that REF/Aly does not affect stability of selected candidate transcripts. Instead, ChIP assays and nuclear run-on analysis reveal that REF/Aly depletion diminishes the transcription of these candidate genes. Furthermore, we determined that REF/Aly binds directly to candidate transcripts, supporting a direct effect of REF/Aly on candidate gene transcription. Taken together, our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of transcription. Our data are consistent with the model that REF/Aly is involved in linking splicing with transcription efficiency. PMID:25477387

  3. Depletion of REF/Aly alters gene expression and reduces RNA polymerase II occupancy.

    PubMed

    Stubbs, Sarah H; Conrad, Nicholas K

    2015-01-01

    Pre-mRNA processing is mechanistically linked to transcription with RNA pol II serving as a platform to recruit RNA processing factors to nascent transcripts. The TREX complex member, REF/Aly, has been suggested to play roles in transcription and nuclear RNA stability in addition to its more broadly characterized role in mRNA export. We employed RNA-seq to identify a subset of transcripts with decreased expression in both nuclear and cytoplasmic fractions upon REF/Aly knockdown, which implies that REF/Aly affects their expression upstream of its role in mRNA export. Transcription inhibition experiments and metabolic labeling assays argue that REF/Aly does not affect stability of selected candidate transcripts. Instead, ChIP assays and nuclear run-on analysis reveal that REF/Aly depletion diminishes the transcription of these candidate genes. Furthermore, we determined that REF/Aly binds directly to candidate transcripts, supporting a direct effect of REF/Aly on candidate gene transcription. Taken together, our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of transcription. Our data are consistent with the model that REF/Aly is involved in linking splicing with transcription efficiency. PMID:25477387

  4. Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation

    PubMed Central

    Tummala, Padmaja; Mali, Raghuveer S.; Guzman, Eduardo; Zhang, Xiao

    2010-01-01

    Purpose During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites. Methods High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10–12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples. FLT-3 Interacting Zinc-finger-1 (FIZ1), a zinc-finger protein that associates with active promoter complexes of photoreceptor-specific genes, provided an additional ChIP marker to highlight genes activated in the mature neural retina. To demonstrate the use of ChIP-on-chip predictions to find novel gene activation events, four additional genes were selected for quantitative PCR analysis (qRT–PCR analysis); these four genes have human homologs located in unidentified retinal disease regions: Solute carrier family 25 member 33 (Slc25a33), Lysophosphatidylcholine acyltransferase 1 (Lpcat1), Coiled-coil domain-containing 126 (Ccdc126), and ADP-ribosylation factor-like 4D (Arl4d). Results ChIP-on-chip Pol-II peak signal ratios >1.8 predicted increased amounts of transcribing Pol-II and increased expression with an estimated 97% accuracy, based on analysis of the validation gene set. Using this threshold ratio, 1,101 genes were predicted to experience increased binding of Pol-II in their promoter regions during terminal maturation of the neural retina. Over 800 of these gene activations were additional to those previously reported by microarray analysis. Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation. Expression of all four genes was diminished in adult retinas lacking rod photoreceptors (Rd1 mice) compared to normal retinas (90% loss for Ccdc126 and Arl4d). For rhodopsin (Rho), a marker of photoreceptor maturation, two regions of maximum Pol-II signal corresponded to the upstream rhodopsin enhancer region and the rhodopsin proximal promoter region. Conclusions High-resolution maps of Pol-II binding around transcription start sites were generated for the postnatal mouse retina; which can predict activation increases for a specific gene of interest. Novel gene activation predictions are enriched for biologic functions relevant to vision, neural function, and chromatin regulation. Use of the data set to detect novel activation increases was demonstrated by expression analysis for several genes that have human homologs located within unidentified retinal disease regions: Slc25a33, Lpcat1, Ccdc126, and Arl4d. Analysis of photoreceptor-deficient retinas indicated that all four genes are expressed in photoreceptors. Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI). Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene’s promoter complex. PMID:20161818

  5. A new set of differentially expressed signaling genes is early expressed in coffee leaf rust race II incompatible interaction.

    PubMed

    Diola, Valdir; Brito, Giovani G; Caixeta, Eveline T; Pereira, Luiz F P; Loureiro, Marcelo E

    2013-08-01

    New races of coffee rust are overcoming resistance genes available in germplasm and cultivated cultivars and bringing recently some coffee-producing countries in severe economic challenge. The objective of this study was to identify the genes that are linked to host resistance to the major coffee rust race II. In our study, we have identified and studied a segregating population that has a single monogenic resistant gene to coffee rust. Coffee leaves of parents, resistant, and susceptible genotypes of the F2 generation plants were inoculated with pathogen spores. A differential analysis was performed by combined cDNA-AFLP and bulk segregant analysis (BSA) in pooled samples collected 48 and 72 h postinoculation, increasing the selectiveness for differential gene expression. Of 108 differential expressed genes, between 33,000 gene fragments analyzed, 108 differential expressed genes were identified in resistant plants. About 20 and 22 % of these resistant-correlated genes are related to signaling and defense genes, respectively. Between signaling genes, the major subclass corresponds to receptor and resistant homolog genes, like nucleotide-binding site leucine-rich repeat (NBS-LRR), Pto-like, RLKs, Bger, and RGH1A, all not previously described in coffee rust responses. The second major subclass included kinases, where two mitogen-activated kinases (MAPK) are identified. Further gene expression analysis was performed for 21 selected genes by real-time PCR gene expression analysis at 0, 12, 24, 48, and 72 h postinoculation. The expression of genes involved in signaling and defense was higher at 24 and 72 h after inoculation, respectively. The NBS-LRR was the more differentially expressed gene between the signaling genes (four times more expressed in the resistant genotype), and thraumatin (PR5) was the more expressed between all genes (six times more expressed). Multivariate analysis reinforces the significance of the temporal separation of identified signaling and defense genes: early expression of signaling genes support the hypothesis that higher expression of the signaling components up regulates the defense genes identified. Additionally the increased gene expression of these two gene sets is associated with a single monogenic resistance trait to to leaf coffee rust in the interaction characterized here. PMID:23835851

  6. Population genetics and natural selection in the gene encoding the Duffy binding protein II in Iranian Plasmodium vivax wild isolates.

    PubMed

    Valizadeh, Vahideh; Zakeri, Sedigheh; Mehrizi, Akram Abouie; Djadid, Navid Dinparast

    2014-01-01

    Region II of Duffy binding protein (PvDBP-II) is one of the most promising blood-stage vaccine candidate antigens against Plasmodium vivax and having knowledge of the nature and genetic polymorphism of PvDBP-II among global P. vivax isolates is important for developing a DBP-based vaccine. By using PCR and sequencing, the present molecular population genetic approach was carried out to investigate sequence diversity and natural selection of dbp-II gene in 63 P. vivax isolates collected from unstable and low transmission malaria-endemic areas of Iran during 2008-2012. Also, phylogenetic analysis, the diversifying natural selection, and recombination across the pvdbp-II gene, including regions containing B-cell epitopes were analyzed using the DnaSP and MEGA4 programs. Twenty two single nucleotide polymorphisms (SNPs, including 20 non-synonymous and 2 synonymous) were identified in PvDBP-II, resulting in 16 different PvDBP-II haplotypes among the Iranian P. vivax isolates. High binding inhibitory B-cell epitope (H3) overlapping with intrinsically unstructured/disordered region (aa: 384-392) appeared to be highly polymorphic (D384G/E385K/ K386N/Q/R390H), and positive selective pressure acted on this region. Most of the polymorphic amino acids, which are located on the surface of the protein, are under selective pressure that implies increased recombination events and exposure to the human immune system. In summary, PvDBP-II gene displays genetic polymorphism among Iranian P. vivax isolates and it is under selective pressure. Mutations, recombination, and positive selection seem to play a role in the resulting genetic diversity, and phylogenetic analysis of DNA sequences demonstrates that Iranian isolates represent a sample of the global population. These results are useful for understanding the nature of the P. vivax population in Iran and also for development of PvDBP-II-based malaria vaccine. PMID:24384095

  7. Type II Transmembrane Serine Protease Gene Variants Associate with Breast Cancer

    PubMed Central

    Luostari, Kaisa; Hartikainen, Jaana M.; Tengström, Maria; Palvimo, Jorma J.; Kataja, Vesa

    2014-01-01

    Type II transmembrane serine proteases (TTSPs) are related to tumor growth, invasion, and metastasis in cancer. Genetic variants in these genes may alter their function, leading to cancer onset and progression, and affect patient outcome. Here, 464 breast cancer cases and 370 controls were genotyped for 82 single-nucleotide polymorphisms covering eight genes. Association of the genotypes was estimated against breast cancer risk, breast cancer–specific survival, and survival in different treatment groups, and clinicopathological variables. SNPs in TMPRSS3 (rs3814903 and rs11203200), TMPRSS7 (rs1844925), and HGF (rs5745752) associated significantly with breast cancer risk (Ptrend?=?0.008–0.042). SNPs in TMPRSS1 (rs12151195 and rs12461158), TMPRSS2 (rs2276205), TMPRSS3 (rs3814903), and TMPRSS7 (rs2399403) associated with prognosis (P?=?0.004–0.046). When estimating the combined effect of the variants, the risk of breast cancer was higher with 4–5 alleles present compared to 0–2 alleles (P?=?0.0001; OR, 2.34; 95% CI, 1.39–3.94). Women with 6–8 survival-associating alleles had a 3.3 times higher risk of dying of breast cancer compared to women with 1–3 alleles (P?=?0.001; HR, 3.30; 95% CI, 1.58–6.88). The results demonstrate the combined effect of variants in TTSPs and their related genes in breast cancer risk and patient outcome. Functional analysis of these variants will lead to further understanding of this gene family, which may improve individualized risk estimation and development of new strategies for treatment of breast cancer. PMID:25029565

  8. Ancestral Polymorphism of Mhc Class II Genes in Mice: Implications for Balancing Selection and the Mammalian Molecular Clock

    PubMed Central

    Edwards, S. V.; Chesnut, K.; Satta, Y.; Wakeland, E. K.

    1997-01-01

    To investigate the evolutionary dynamics at Mhc class II DR genes of mice (genus Mus), we sequenced the peptide binding regions (PBRs) of 41 DRB (=E?) genes and eight DRA (=E?) genes from 15 strains representing eight species. As expected trees of these PBR sequences imply extensive maintenance of ancestral DRB alleles across species. We use a coalescent simulation model to show that the number of interspecific coalescent events (c) observed on these trees was higher than the number expected for neutral genealogies and similar sample sizes and is more consistent with balancing selection than with neutrality. Patterns of ancestral polymorphism in mouse DRB alleles were also used to examine the tempo of synonymous substitution in the PBR of mouse class II genes. Both absolute and relative rate tests on DRA and DRB genes imply increased substitution rates at two- and fourfold degnerate sites of mice and rats relative to primates, and decreased rates for the DRB genes of primates relative to ungulate and carnivore relatives. Thus rates of synonymous substitution at Mhc DR genes in mammals appear to be subject to generation time effects in ways similar to those found at other mammalian genes. PMID:9178014

  9. Role of Reactive Oxygen Species-Sensitive Extracellular Signal-Regulated Kinase Pathway in Angiotensin II-Induced Endothelin1 Gene Expression in Vascular Endothelial Cells

    Microsoft Academic Search

    Yung-Ho Hsu; Jin-Jer Chen; Nen-Chung Chang; Cheng-Hsien Chen; Ju-Chi Liu; Tso-Hsiao Chen; Cherng-Jye Jeng; Hung-Hsing Chao; Tzu-Hurng Cheng

    2004-01-01

    Background: Circulating angiotensin II (Ang II) increases vascular endothelin-1 (ET-1) tissue levels, which in turn mediate a major part of Ang II-stimulated vascular growth and hypertension in vivo. Ang II also stimulates the generation of reactive oxygen species (ROS) within vascular endothelial cells. However, whether ROS are involved in Ang II-induced ET-1 gene expression, and the related intracellular mechanisms occurring

  10. Estrogen receptor ? gene PvuII polymorphism and coronary artery disease: a meta-analysis of 21 studies*

    PubMed Central

    Ding, Jie; Xu, Hui; Yin, Xiang; Zhang, Fu-rong; Pan, Xiao-ping; Gu, Yi-an; Chen, Jun-zhu; Guo, Xiao-gang

    2014-01-01

    The association between the estrogen receptor ? gene (ESR1) PvuII polymorphism (c.454-397T>C) and coronary artery disease (CAD) is controversial. Thus, we conducted a meta-analysis to evaluate the relationship. Data were collected from 21 studies encompassing 9926 CAD patients and 16 710 controls. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the relationship between PvuII polymorphism and CAD. The polymorphism in control populations in all studies followed Hardy-Weinberg equilibrium. We found a significant association between ESR1 PvuII polymorphism and CAD risk in all subjects. When the data were stratified by region, a significant association between ESR1 PvuII polymorphism and CAD risk was observed in Asian populations but not in Western populations. The current study suggests that ESR1 PvuII polymorphism has an important role in CAD susceptibility. PMID:24599688

  11. ANGIOTENSIN II INDUCED CARDIOVASCULAR LOAD REGULATES CARDIAC REMODELING AND RELATED GENE EXPRESSION IN LATE GESTATION FETAL SHEEP

    PubMed Central

    Scholz, Thomas D.; Peterson, Emily S.; Volk, Ken A.; Segar, Jeffrey L.

    2014-01-01

    Background Angiotensin II (ANG II) stimulates fetal heart growth, though little is known regarding changes in cardiomyocyte endowment or the molecular pathways mediating the response. We measured cardiomyocyte proliferation and morphology in ANG II treated fetal sheep and assessed transcriptional pathway responses in ANG II and losartan (an ANG II type 1 receptor antagonist) treated fetuses. Methods In twin gestation pregnant sheep, one fetus received ANG II (50 ?g/kg/min iv) or losartan (20 mg/kg/d iv) for 7 days; non-instrumented twins served as controls. Results ANG II produced increases in heart mass, cardiomyocyte area (left ventricle (LV) and right ventricle mononucleated and LV binucleated cells) and the percentage of Ki-67-positive mononucleated cells in the LV (all p< 0.05). ANG II and losartan produced generally opposing changes in gene expression, affecting an estimated 55% of the represented transcriptome. The most prominent significantly effected biological pathways included those involved in cytoskeletal remodeling and cell cycle activity. Conclusion ANG II produces an increase in fetal cardiac mass via cardiomyocyte hypertrophy and likely hyperplasia, involving transcriptional responses in cytoskeletal remodeling and cell cycle pathways. PMID:24614802

  12. A transcriptionally active human type II gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the antisense orientation on chromosome 1q.12.

    PubMed

    Morgan, Kevin; Conklin, Darrell; Pawson, Adam J; Sellar, Robin; Ott, Thomas R; Millar, Robert P

    2003-02-01

    GnRH-II peptide hormone exhibits complete sequence conservation across vertebrate species, including man. Type-II GnRH receptor genes have been characterized recently in nonhuman primates, but the human receptor gene homolog contains a frameshift, a premature stop codon (UGA), and a 3' overlap of the RBM8A gene on chromosome 1q.12. A retrotransposed pseudogene, RBM8B, retains partial receptor sequence. In this study, bioinformatics show that the human receptor gene promoter overlaps the peroxisomal protein 11-beta gene promoter and the premature UGA is positionally conserved in chimpanzee. A CGA [arginine (Arg)] occurs in porcine DNA, but UGA is shifted one codon to the 5' direction in bovine DNA, suggesting independent evolution of premature stop codons. In contrast to marmoset tissue RNA, exon- and strand-specific probes are required to distinguish differently spliced human receptor gene transcripts in cell lines (HP75, IMR-32). RBM8B is not transcribed. Sequencing of cDNAs for spliced receptor mRNAs showed no evidence for alteration of the premature UGA by RNA editing, but alternative splicing circumvents the frameshift to encode a two-membrane-domain protein before this UGA. A stem-loop motif resembling a selenocysteine insertion sequence and a potential alternative translation initiation site might enable expression of further proteins involved in interactions within the GnRH system. PMID:12538601

  13. Interfacial stress affects rat alveolar type II cell signaling and gene expression

    PubMed Central

    Hobi, Nina; Ravasio, Andrea

    2012-01-01

    Previous work from our group (Ravasio A, Hobi N, Bertocchi C, Jesacher A, Dietl P, Haller T. Am J Physiol Cell Physiol 300: C1456–C1465, 2011.) showed that contact of alveolar epithelial type II cells with an air-liquid interface (IAL) leads to a paradoxical situation. It is a potential threat that can cause cell injury, but also a Ca2+-dependent stimulus for surfactant secretion. Both events can be explained by the impact of interfacial tensile forces on cellular structures. Here, the strength of this mechanical stimulus became also apparent in microarray studies by a rapid and significant change on the transcriptional level. Cells challenged with an IAL in two different ways showed activation/inactivation of cellular pathways involved in stress response and defense, and a detailed Pubmatrix search identified genes associated with several lung diseases and injuries. Altogether, they suggest a close relationship of interfacial stress sensation with current models in alveolar micromechanics. Further similarities between IAL and cell stretch were found with respect to the underlying signaling events. The source of Ca2+ was extracellular, and the transmembrane Ca2+ entry pathway suggests the involvement of a mechanosensitive channel. We conclude that alveolar type II cells, due to their location and morphology, are specific sensors of the IAL, but largely protected from interfacial stress by surfactant release. PMID:22610352

  14. Discrete gene loci regulate neurodegeneration, lymphocyte infiltration, and major histocompatibility complex class II expression in the CNS.

    PubMed

    Lidman, Olle; Swanberg, Maria; Horvath, Linn; Broman, Karl W; Olsson, Tomas; Piehl, Fredrik

    2003-10-29

    Neurodegeneration and inflammation are fundamental aspects of many neurological diseases. A genome-wide scan of the response to ventral root avulsion (VRA) in a rat F2 cross discloses specific gene regions that regulate these processes. Two gene loci displayed linkage to neurodegeneration and T cell infiltration, respectively, and a single locus displayed extreme linkage to VRA-induced major histocompatibility complex class II expression on microglia. The demonstration that polymorphic genes in different loci control neurodegeneration and CNS inflammation has implications for various experimental rodent nervous system paradigms and potentially for genetically regulated susceptibility to a variety of human CNS diseases. PMID:14586010

  15. Genetic and expression studies of SMN2 gene in Russian patients with spinal muscular atrophy type II and III

    PubMed Central

    2011-01-01

    Background Spinal muscular atrophy (SMA type I, II and III) is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron gene (SMN1). SMN2 is a centromeric copy gene that has been characterized as a major modifier of SMA severity. SMA type I patients have one or two SMN2 copies while most SMA type II patients carry three SMN2 copies and SMA III patients have three or four SMN2 copies. The SMN1 gene produces a full-length transcript (FL-SMN) while SMN2 is only able to produce a small portion of the FL-SMN because of a splice mutation which results in the production of abnormal SMN?7 mRNA. Methods In this study we performed quantification of the SMN2 gene copy number in Russian patients affected by SMA type II and III (42 and 19 patients, respectively) by means of real-time PCR. Moreover, we present two families consisting of asymptomatic carriers of a homozygous absence of the SMN1 gene. We also developed a novel RT-qPCR-based assay to determine the FL-SMN/SMN?7 mRNA ratio as SMA biomarker. Results Comparison of the SMN2 copy number and clinical features revealed a significant correlation between mild clinical phenotype (SMA type III) and presence of four copies of the SMN2 gene. In both asymptomatic cases we found an increased number of SMN2 copies in the healthy carriers and a biallelic SMN1 absence. Furthermore, the novel assay revealed a difference between SMA patients and healthy controls. Conclusions We suggest that the SMN2 gene copy quantification in SMA patients could be used as a prognostic tool for discrimination between the SMA type II and SMA type III diagnoses, whereas the FL-SMN/SMN?7 mRNA ratio could be a useful biomarker for detecting changes during SMA pharmacotherapy. PMID:21762474

  16. Agrobacterium-mediated transformation of `Alpine' Fragaria vesca, and transmission of transgenes to R1 progeny

    Microsoft Academic Search

    K. M. Haymes; T. M. Davis

    1998-01-01

    Agrobacterium-mediated transformation was used to stably introduce ?-glucuronidase (gus) and neomycin phosphotransferase (nptII) marker genes into `Alpine' Fragaria vesca FRA 197, a diploid (2n = 2x = 14) strawberry. R0 generation transformants derived from a single clump of kanamycin-resistant\\u000a callus were vegetatively propagated. The presence of the gus and nptII genes in five clonal R0 runner plants was confirmed by

  17. Cloning and characterization of SnRK2 subfamily II genes from Nicotiana tabacum.

    PubMed

    Zhang, Hongying; Jia, Hongfang; Liu, Guoshun; Yang, Shengnan; Zhang, Songtao; Yang, Yongxia; Yang, Peipei; Cui, Hong

    2014-09-01

    SnRK2 is a plant-specific protein kinase family involved in abiotic stress signalling. In this study, NtSnRK2.1, NtSnRK2.2, and NtSnRK2.3, were cloned from tobacco by in silico cloning and reverse transcription PCR. The three protein kinases were classed into subfamily II of the SnRK2 family using a phylogenetic tree and C-terminus analysis. Subcellular localization revealed NtSnRK2s in the nuclear and cytoplasmic compartments. Dynamic expression of NtSnRK2s in tobacco plants that were exposed to drought, salt, or cold stressors were characterised using quantitative real-time PCR. It was revealed that the three genes showed similar patterns of transcription under abiotic stress responses; there was evidence NtSnRK2s participated in abscisic acid-dependent signalling pathways. NtSnRK2.1-3 responded much faster to drought and salt than to cold stress. To investigate the role of NtSnRK2s under abiotic stresses, NtSnRK2.1 gene was over-expressed in tobacco. A stress tolerance assay showed that tobacco plants that over-expressed NtSnRK2.1 plants had greater salt tolerance. The results indicate that NtSnRK2s are involved in abiotic stress response pathways. PMID:24919756

  18. Type II cytokeratin gene expression is indicative of early cell differentiation in the chick embryo

    SciTech Connect

    Charlebois, T.S.

    1988-01-01

    Embryonic development in vertebrates appears to involve a series of inductive tissue interactions that lead to regional specializations, which eventually become elaborated in the basic body plan of the embryo. The inductive interactions leading to early regionalization of the embryo are often particularly difficult to evaluate because of the absence of available morphological or biochemical evidence that such events have occurred. In the 36 hour chick embryo, the regional subdivision of the early ectoderm is evidence by a marked lens-forming bias in the head ectoderm, which is absent in the presumptive dorsal epidermis of the trunk region. As a strategy for isolating genes whose differential expression might reflect this regional subdivision, a cDNA library from 36 hour embryos was prepared and screened for differential hybridization to ({sup 32}P)cDNA probes synthesized using template RNA isolated from 36 hour head ectoderm and trunk ectoderm. A cDNA clone (T4) was isolated which hybridizes to transcripts present at much higher levels in trunk ectoderm than in head ectoderm. Partial nucleotide and deduced amino acid sequences of this clone indicate that it represents a gene encoding a type II cytokeratin. The distribution of transcripts complementary to the T4 probe was evaluated in early embryos using RNA gel blot analysis and in situ hybridization to tissue sections.

  19. IGF TYPE II RECEPTOR GENE EXPRESSION IN GRANULOSA AND THECA CELLS OF CATTLE SELECTED FOR TWIN OVULATIONS AND BIRTHS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Regulation of multiple ovulations in monotocous species such as cattle is not well understood. Therefore, gene expression of the IGF type II receptor (IGF2R) and LH receptor (LHR) in granulosa (GC) and theca (TC) cells as well as estradiol (E2) and progesterone (P4) levels in follicular fluid (FF) ...

  20. Transformation of blackgram (Vigna mungo (L.) Hepper) by barley chitinase and ribosome-inactivating protein genes towards improving resistance to Corynespora leaf spot fungal disease.

    PubMed

    Chopra, Rajan; Saini, Raman

    2014-12-01

    Blackgram (Vigna mungo (L.) Hepper), an important grain legume crop, is sensitive to many fungal pathogens including Corynespora cassiicola, the causal agent of corynespora leaf spot disease. In the present study, plasmid pGJ42 harboring neomycin phosphotransferase (nptII) a selectable marker gene, the barley antifungal genes chitinase (AAA56786) and ribosome-inactivating protein (RIP; AAA32951) were used for the transformation, to develop fungal resistance for the first time in blackgram. The presence and integration of transgene into the blackgram genome was confirmed by PCR and Southern analysis with an overall transformation frequency of 10.2 %. Kanamycin selection and PCR analysis of T0 progeny revealed the inheritance of transgene in Mendelian fashion (3:1). Transgenic plants (T1), evaluated for fungal resistance by in vitro antifungal assay, arrested the growth of C. cassiicola up to 25-40 % over the wild-type plants. In fungal bio-assay screening, the transgenic plants (T1) sprayed with C. cassiicola spores showed a delay in onset of disease along with their lesser extent in terms of average number of diseased leaves and reduced number and size of lesions. The percent disease protection among different transformed lines varies in the range of 27-47 % compare to control (untransformed) plants. These results demonstrate potentiality of chitinase and RIP from a heterologous source in developing fungal disease protection in blackgram and can be helpful in increasing the production of blackgram. PMID:25227687

  1. Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type II caused by a defect in the gene encoding. alpha. -mannosidase II

    SciTech Connect

    Fukuda, M.N.; Masri, K.A. (La Jolla Cancer Research Foundation, CA (USA)); Dell, A. (Imperial College of Science Technology and Medicine, London (England)); Luzzatto, L. (Hammersmith Hospital, London (England)); Moremen, K.W. (Massachusetts Institute of Technology, Cambridge, MA (USA))

    1990-10-01

    Congenital dyserythropoietic anemia type II, or hereditary erythroblastic multinuclearity with a positive acidified-serum-lysis test (HEMPAS), is a genetic anemia in humans inherited by an autosomally recessive mode. The enzyme defect in most HEMPAS patients has previously been proposed as a lowered activity of N-acetylglucosaminyltransferase II, resulting in a lack of polylactosamine on proteins and leading to the accumulation of polylactosaminyl lipids. A recent HEMPAS case, G.C., has now been analyzed by cell-surface labeling, fast-atom-bombardment mass spectrometry of glycopeptides, and activity assay of glycosylation enzymes. Significantly decreased glycosylation of polylactosaminoglycan proteins and incompletely processed asparagine-linked oligosaccharides were detected in the erythrocyte membranes of G.C. These results suggest that G.C. cells contain a mutation in {alpha}-ManII-encoding gene that results in inefficient expression of {alpha}-ManII mRNA, either through reduced transcription or message instability. This report demonstrates that HEMPAS is caused by a defective gene encoding an enzyme necessary for the synthesis of asparagine-linked oligosaccharides.

  2. Gene Expression Profiling Associated with Angiotensin II Type 2 Receptor-Induced Apoptosis in Human Prostate Cancer Cells

    PubMed Central

    Pei, Nana; Jie, Feilong; Luo, Jie; Wan, Renqiang; Zhang, Yanling; Chen, Xinglu; Liang, Zhibing; Du, Hongyan; Li, Andrew; Chen, Baihong; Zhang, Yi; Sumners, Colin; Li, Jinlong; Gu, Weiwang; Li, Hongwei

    2014-01-01

    Increased expression of angiotensin II type 2 receptor (AT2R) induces apoptosis in numerous tumor cell lines, with either Angiotensin II-dependent or Angiotensin II-independent regulation, but its molecular mechanism remains poorly understood. Here, we used PCR Array analysis to determine the gene and microRNA expression profiles in human prostate cancer cell lines transduced with AT2R recombinant adenovirus. Our results demonstrated that AT2R over expression leads to up-regulation of 6 apoptosis-related genes (TRAIL-R2, BAG3, BNIPI, HRK, Gadd45a, TP53BP2), 2 cytokine genes (IL6 and IL8) and 1 microRNA, and down-regulation of 1 apoptosis-related gene TNFSF10 and 2 cytokine genes (BMP6, BMP7) in transduced DU145 cells. HRK was identified as an up-regulated gene in AT2R-transduced PC-3 cells by real-time RT-PCR. Next, we utilized siRNAs to silence the up-regulated genes to further determine their roles on AT2R overexpression mediated apoptosis. The results showed downregulation of Gadd45a reduced the apoptotic effect by ?30% in DU145 cells, downregulation of HRK reduced AT2R-mediated apoptosis by more than 50% in PC-3 cells, while downregulation of TRAIL-R2 enhanced AT2R-mediated apoptosis more than 4 times in DU145 cells. We also found that the effects on AT2R-mediated apoptosis caused by downregulation of Gadd45a, TRAIL-R2 and HRK were independent in activation of p38 MAPK, p44/42 MAPK and p53. Taken together, our results demonstrated that TRAIL-R2, Gadd45a and HRK may be novel target genes for further study of the mechanism of AT2R-mediated apoptosis in prostate cancer cells. PMID:24658029

  3. Splice variants of the condensin II gene Ncaph2 include alternative reading frame translations of exon 1.

    PubMed

    Theodoratos, Angelo; Wilson, Laurence O W; Gosling, Katharine M; Fahrer, Aude M

    2012-04-01

    Condensins I and II are five-protein complexes that are important for the condensation of chromatin. They are essential for mitosis and important for regulating gene expression during interphase. Here, we investigated the transcription and translation of the mouse Ncaph2 gene, which encodes a subunit of condensin II. We identified three splice variants within the first exon, a NAGNAG splice variant at the beginning of exon 16 and alternative 3'-UTRs. In total, Ncaph2 is potentially capable of generating 12 unique mRNA transcripts and six unique proteins. We confirm that Ncaph2 can generate three different N-termini, all encoded by exon 1, one of which is translated from an alternative reading frame. This alternative reading frame splice variant appears to be a novel outcome of splicing. If this is applicable to other genes, it would account for a previously unappreciated level of eukaryotic protein diversity. PMID:22333158

  4. Analysis of the region in between two closely linked patatin genes: class II promoter activity in tuber, root and leaf.

    PubMed

    Nap, J P; Dirkse, W G; Louwerse, J; Onstenk, J; Visser, R; Loonen, A; Heidekamp, F; Stiekema, W J

    1992-11-01

    From a potato genomic library a phage lambda clone was isolated that carried nucleotide sequences of two patatin genes, thus demonstrating a close physical linkage between these two members of the patatin gene family. Sequence and restriction analysis showed the genes to be oriented in tandem. The more upstream gene was a pseudogene truncated at the 3' end, whereas the downstream gene was a class II patatin gene. In addition to a 208 bp fragment also present in patatin class I promoters, the region in between both genes contained various direct repeats also found in other patatin genes. To study the promoter activity of this intergenic region, a 2.78 kb fragment was transcriptionally fused to the beta-glucuronidase gene and reintroduced into potato cultivar Bintje. Histochemical analysis revealed expression in the outermost layer of cells of the cortex, in the tuber phellogen, in or around the root vascular system, and also in the abaxial phloem layer of the vascular bundle in leaves. PMID:1450383

  5. Feature Selection and Classification of MAQC-II Breast Cancer and Multiple Myeloma Microarray Gene Expression Data

    PubMed Central

    Liu, Qingzhong; Sung, Andrew H.; Chen, Zhongxue; Liu, Jianzhong; Huang, Xudong; Deng, Youping

    2009-01-01

    Microarray data has a high dimension of variables but available datasets usually have only a small number of samples, thereby making the study of such datasets interesting and challenging. In the task of analyzing microarray data for the purpose of, e.g., predicting gene-disease association, feature selection is very important because it provides a way to handle the high dimensionality by exploiting information redundancy induced by associations among genetic markers. Judicious feature selection in microarray data analysis can result in significant reduction of cost while maintaining or improving the classification or prediction accuracy of learning machines that are employed to sort out the datasets. In this paper, we propose a gene selection method called Recursive Feature Addition (RFA), which combines supervised learning and statistical similarity measures. We compare our method with the following gene selection methods: Support Vector Machine Recursive Feature Elimination (SVMRFE)Leave-One-Out Calculation Sequential Forward Selection (LOOCSFS)Gradient based Leave-one-out Gene Selection (GLGS) To evaluate the performance of these gene selection methods, we employ several popular learning classifiers on the MicroArray Quality Control phase II on predictive modeling (MAQC-II) breast cancer dataset and the MAQC-II multiple myeloma dataset. Experimental results show that gene selection is strictly paired with learning classifier. Overall, our approach outperforms other compared methods. The biological functional analysis based on the MAQC-II breast cancer dataset convinced us to apply our method for phenotype prediction. Additionally, learning classifiers also play important roles in the classification of microarray data and our experimental results indicate that the Nearest Mean Scale Classifier (NMSC) is a good choice due to its prediction reliability and its stability across the three performance measurements: Testing accuracy, MCC values, and AUC errors. PMID:20011240

  6. Selection and Trans-Species Polymorphism of Major Histocompatibility Complex Class II Genes in the Order Crocodylia

    PubMed Central

    Jaratlerdsiri, Weerachai; Isberg, Sally R.; Higgins, Damien P.; Miles, Lee G.; Gongora, Jaime

    2014-01-01

    Major Histocompatibility Complex (MHC) class II genes encode for molecules that aid in the presentation of antigens to helper T cells. MHC characterisation within and between major vertebrate taxa has shed light on the evolutionary mechanisms shaping the diversity within this genomic region, though little characterisation has been performed within the Order Crocodylia. Here we investigate the extent and effect of selective pressures and trans-species polymorphism on MHC class II ? and ? evolution among 20 extant species of Crocodylia. Selection detection analyses showed that diversifying selection influenced MHC class II ? diversity, whilst diversity within MHC class II ? is the result of strong purifying selection. Comparison of translated sequences between species revealed the presence of twelve trans-species polymorphisms, some of which appear to be specific to the genera Crocodylus and Caiman. Phylogenetic reconstruction clustered MHC class II ? sequences into two major clades representing the families Crocodilidae and Alligatoridae. However, no further subdivision within these clades was evident and, based on the observation that most MHC class II ? sequences shared the same trans-species polymorphisms, it is possible that they correspond to the same gene lineage across species. In contrast, phylogenetic analyses of MHC class II ? sequences showed a mixture of subclades containing sequences from Crocodilidae and/or Alligatoridae, illustrating orthologous relationships among those genes. Interestingly, two of the subclades containing sequences from both Crocodilidae and Alligatoridae shared specific trans-species polymorphisms, suggesting that they may belong to ancient lineages pre-dating the divergence of these two families from the common ancestor 85–90 million years ago. The results presented herein provide an immunogenetic resource that may be used to further assess MHC diversity and functionality in Crocodylia. PMID:24503938

  7. Selection and trans-species polymorphism of major histocompatibility complex class II genes in the order Crocodylia.

    PubMed

    Jaratlerdsiri, Weerachai; Isberg, Sally R; Higgins, Damien P; Miles, Lee G; Gongora, Jaime

    2014-01-01

    Major Histocompatibility Complex (MHC) class II genes encode for molecules that aid in the presentation of antigens to helper T cells. MHC characterisation within and between major vertebrate taxa has shed light on the evolutionary mechanisms shaping the diversity within this genomic region, though little characterisation has been performed within the Order Crocodylia. Here we investigate the extent and effect of selective pressures and trans-species polymorphism on MHC class II ? and ? evolution among 20 extant species of Crocodylia. Selection detection analyses showed that diversifying selection influenced MHC class II ? diversity, whilst diversity within MHC class II ? is the result of strong purifying selection. Comparison of translated sequences between species revealed the presence of twelve trans-species polymorphisms, some of which appear to be specific to the genera Crocodylus and Caiman. Phylogenetic reconstruction clustered MHC class II ? sequences into two major clades representing the families Crocodilidae and Alligatoridae. However, no further subdivision within these clades was evident and, based on the observation that most MHC class II ? sequences shared the same trans-species polymorphisms, it is possible that they correspond to the same gene lineage across species. In contrast, phylogenetic analyses of MHC class II ? sequences showed a mixture of subclades containing sequences from Crocodilidae and/or Alligatoridae, illustrating orthologous relationships among those genes. Interestingly, two of the subclades containing sequences from both Crocodilidae and Alligatoridae shared specific trans-species polymorphisms, suggesting that they may belong to ancient lineages pre-dating the divergence of these two families from the common ancestor 85-90 million years ago. The results presented herein provide an immunogenetic resource that may be used to further assess MHC diversity and functionality in Crocodylia. PMID:24503938

  8. Molecular polymorphism and expression analysis of MHC class II B gene from red sea bream (Chrysophrys major).

    PubMed

    Chen, Song-Lin; Zhang, Yu-Xi; Xu, Mei-Yu; Ji, Xiang-Shan; Yu, Guo-Cai; Dong, Cheng-Fang

    2006-01-01

    MHC class II (major histocompatibility complex class II) plays an important role in the immune response of vertebrates. Its function is to present antigenic peptides to the T-cell receptor. In order to study the function and molecular polymorphism of class II B gene in fish, we have isolated cDNAs encoding class II B from spleen cDNA library of red sea bream (Chrysophrys major) by using EST sequencing, and examined genomic organization, molecular polymorphism and expression of red sea bream class II B gene. As in other vertebrates, five exons and four introns were identified in red sea bream class II B gene. Seven class II B alleles were identified from seven individuals of red sea bream. The deduced amino acid sequence of red sea bream MHC class II B 1(Chma-DAB*0101) had 87.1, 85.1, 87.1, 90.4, 87.1, 90.8% identity with those of red sea bream class II B 2, 3, 4, 5, 6, 7(Chma-DAB*0201-Chma-DAB*0701), respectively, and had 75.2, 74.5, 55.9, 55.1, 34.3 and 30.4% identity with those of striped sea bass, cichlid, rainbow trout, Atlantic salmon, mouse and human, respectively. Four different class II B alleles were observed in a single individual and two different 3' untranslated region (3' UTR) sequences from this individual may infer the existence of two loci at least. Semi-quantitative RT-PCR demonstrated that high expression was detected in liver, head kidney, kidney, intestine, gill, stomach, hear and spleen, low expression in muscle and blood. Challenge of red sea bream with the pathogenic bacteria, Vibrio anguillarum, resulted in a significant decrease in the expression of MHC class II B mRNA from 5 to 72 h after infection in liver, spleen, head kidney and intestine, followed by a recovery to normal level after 96 h. PMID:16045985

  9. Engineering gibberellin metabolism in Solanum nigrum L. by ectopic expression of gibberellin oxidase genes.

    PubMed

    Bhattacharya, A; Ward, D A; Hedden, P; Phillips, A L; Power, J B; Davey, M R

    2012-05-01

    Gibberellins (GAs) control many aspects of plant development, including seed germination, shoot growth, flower induction and growth and fruit expansion. Leaf explants of Solanum nigrum (Black Nightshade; Solanaceae) were used for Agrobacterium-mediated delivery of GA-biosynthetic genes to determine the influence of their encoded enzymes on the production of bioactive GAs and plant stature in this species. Constructs were prepared containing the neomycin phosphotransferase (nptII) gene for kanamycin resistance as a selectable marker, and the GA-biosynthetic genes, their expression under the control of the CaMV 35S promoter. The GA-biosynthetic genes comprised AtGA20ox1, isolated from Arabidopsis thaliana, the product from which catalyses the formation of C(19)-GAs, and MmGA3ox1 and MmGA3ox2, isolated from Marah macrocarpus, which encode functionally different GA 3-oxidases that convert C(19)-GAs to biologically active forms. Increase in stature was observed in plants transformed with AtGA20ox1, MmGA3ox2 and MmGA3ox1 + MmGA3ox2, their presence and expression being confirmed by PCR and RT-PCR, respectively, accompanied by an increase in GA(1) content. Interestingly, MmGA3ox1 alone did not induce a sustained increase in plant height, probably because of only a marginal increase in bioactive GA(1) content in the transformed plants. The results are discussed in the context of regulating plant stature, since this strategy would decrease the use of chemicals to promote plant growth. PMID:22238061

  10. Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection.

    PubMed

    Pantazis, Christopher J; Fisk, Sarah; Mills, Kerri; Flinn, Barry S; Shulaev, Vladimir; Veilleux, Richard E; Dan, Yinghui

    2013-03-01

    KEY MESSAGE : We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics. Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12-16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1-2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying. PMID:23160638

  11. Three Classes of Plasmid (47–63 kb) Carry the Type B Neurotoxin Gene Cluster of Group II Clostridium botulinum

    PubMed Central

    Carter, Andrew T.; Austin, John W.; Weedmark, Kelly A.; Corbett, Cindi; Peck, Michael W.

    2014-01-01

    Pulsed-field gel electrophoresis and DNA sequence analysis of 26 strains of Group II (nonproteolytic) Clostridium botulinum type B4 showed that 23 strains carried their neurotoxin gene cluster on a 47–63 kb plasmid (three strains lacked any hybridization signal for the neurotoxin gene, presumably having lost their plasmid). Unexpectedly, no neurotoxin genes were found on the chromosome. This apparent constraint on neurotoxin gene transfer to the chromosome stands in marked contrast to Group I C. botulinum, in which neurotoxin gene clusters are routinely found in both locations. The three main classes of type B4 plasmid identified in this study shared different regions of homology, but were unrelated to any Group I or Group III plasmid. An important evolutionary aspect firmly links plasmid class to geographical origin, with one class apparently dominant in marine environments, whereas a second class is dominant in European terrestrial environments. A third class of plasmid is a hybrid between the other two other classes, providing evidence for contact between these seemingly geographically separated populations. Mobility via conjugation has been previously demonstrated for the type B4 plasmid of strain Eklund 17B, and similar genes associated with conjugation are present in all type B4 plasmids now described. A plasmid toxin–antitoxin system pemI gene located close to the neurotoxin gene cluster and conserved in each type B4 plasmid class may be important in understanding the mechanism which regulates this unique and unexpected bias toward plasmid-borne neurotoxin genes in Group II C. botulinum type B4. PMID:25079343

  12. Phase I\\/II trial of gene therapy with autologous tumor cells modified with tag7\\/PGRP-S gene in patients with disseminated solid tumors

    Microsoft Academic Search

    V. M. Moiseyenko; A. O. Danilov; I. A. Baldueva; A. B. Danilova; N. V. Tyukavina; S. S. Larin; S. L. Kiselev; R. V. Orlova; V. V. Anisimov; A. I. Semenova; L. A. Shchekina; G. I. Gafton; V. A. Kochnev; A. S. Barchuk; S. V. Kanaev; K. P. Hanson; G. P. Georgiev

    2005-01-01

    Background: The use of genetically modified autologous tumor cells appears to be a promising approach for cancer therapy. A phase I\\/II trial was undertaken to define the feasibility, safety and antitumor effects of the autologous vaccine prepared by transferring tag7\\/PGRP-S gene into malig- nant melanoma and renal cell carcinoma cells. Patients and methods: Twenty-one patients (17 with disseminated malignant melanoma

  13. Part II: In vitro measurement of gene Alfred O. Hero III

    E-print Network

    Hero, Alfred O.

    questions ! Gene sequencing: what is the sequence of base pairs in a DNA segment, gene, or genome? ! Gene Mapping: what are positions (loci) of genes on a chromosome? ! Gene expression profiling: what is pattern-labeled RNA from sample is hybridized to chip ! Abundance of RNA bound to each probe is laser-scanned " cDNA

  14. Comparison and Evaluation of Two Diagnostic Methods for Detection of npt II and GUS Genes in Nicotiana tabacum.

    PubMed

    Almasi, Mohammad Amin; Aghapour-Ojaghkandi, Mehdi; Bagheri, Khadijeh; Ghazvini, Mohammadreza; Hosseyni-Dehabadi, Seyed Mohammad

    2015-04-01

    To diminish the time required for some diagnostic assays including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP) and also a visual detection protocol on the basis of npt II and GUS genes in transgenic tobacco plants were used. Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum leaf discs was performed with plant transformation vector of pBI 121. From kanamycin-resistant plants selected by their antibiotic resistance, four plants were selected for DNA isolation. Presence of the transgene was confirmed in the transformants by PCR and LAMP. In this regard, all LAMP and PCR primers were designed on the basis of the gene sequences of npt II and GUS. The LAMP assay was applied for direct detection of gene marker from plant samples without DNA extraction steps (direct LAMP assay). Also, a novel colorimetric LAMP assay for rapid and easy detection of npt II and GUS genes was developed here, its potential compared with PCR assay. The LAMP method, on the whole, had the following advantages over the PCR method: easy detection, high sensitivity, high efficiency, simple manipulation, safety, low cost, and user friendly. PMID:25820356

  15. Interleukin-7 mediates glucose utilization in lymphocytes through transcriptional regulation of the hexokinase II gene.

    PubMed

    Chehtane, Mounir; Khaled, Annette R

    2010-06-01

    The cytokine interleukin-7 (IL-7) has essential growth activities that maintain the homeostatic balance of the immune system. Little is known of the mechanism by which IL-7 signaling regulates metabolic activity in support of its vital function in lymphocytes. We observed that IL-7 deprivation caused a rapid decline in the metabolism of glucose that was attributable to loss of intracellular glucose retention. To identify the transducer of the IL-7 metabolic signal, we examined the expression of three important regulators of glucose metabolism, the glucose transporter GLUT-1 and two glycolytic enzymes, hexokinase II (HXKII) and phosphofructokinase-1 (PFK-1), using an IL-7-dependent T-cell line and primary lymphocytes. We found that in lymphocytes deprived of IL-7 loss of glucose uptake correlated with decreased expression of HXKII. Readdition of IL-7 to cytokine-deprived lymphocytes restored the transcription of the HXKII gene within 2 h, but not that of GLUT-1 or PFK-1. IL-7-mediated increases in HXKII, but not GLUT-1 or PFK-1, were also observed at the protein level. Inhibition of HXKII with 3-bromopyruvate or specific small-interfering RNA decreased glucose utilization, as well as ATP levels, in the presence of IL-7, whereas overexpression of HXKII, but not GLUT-1, restored glucose retention and increased ATP levels in the absence of IL-7. We conclude that IL-7 controls glucose utilization by regulating the gene expression of HXKII, suggesting a mechanism by which IL-7 supports bioenergetics that control cell fate decisions in lymphocytes. PMID:20200205

  16. Direct demonstration of termination signals for RNA polymerase II from the sea urchin H2A histone gene.

    PubMed Central

    Briggs, D; Jackson, D; Whitelaw, E; Proudfoot, N J

    1989-01-01

    Previous studies [1,2] suggested but did not prove that the sea urchin H2A histone gene possesses strong transcriptional termination signals close to, but separate from, the 3' processing signals. In this study we have demonstrated by two independent approaches that these sequences elicit authentic transcriptional termination. First we show by nuclear run off analysis that nascent transcription terminates in the immediate 3' flanking region of the H2A gene, in an A-rich region. Second we show that these termination signals prevent transcriptional read through when placed in the intron of a globin gene. The intronic position of the termination signal rules out any effect on steady state mRNA levels. We have therefore defined DNA sequences which act as a transcription terminator when placed in heterologous RNA polymerase II genes. Images PMID:2813057

  17. Functional analysis of the class II hydrophobin gene HFB2-6 from the biocontrol agent Trichoderma asperellum ACCC30536.

    PubMed

    Huang, Ying; Mijiti, Gulijimila; Wang, Zhiying; Yu, Wenjing; Fan, Haijuan; Zhang, Rongshu; Liu, Zhihua

    2015-02-01

    A class II hydrophobin gene, HFB2-6, was cloned from Trichoderma asperellum ACCC30536 and its biocontrol function was studied. According to our previous transcriptome data, six of the eight class II hydrophobin genes were obviously differential expression in four inducing conditions, especially the gene HFB2-6. Moreover, HFB2-6 proven to be differentially transcribed under eight different treatments. HFB2-6 transcripts were up-regulated under 1% Alternaria alternata cell wall and 5% A. alternata fermentation liquid treatments, and by nutritional stress conditions, suggesting that HFB2-6 plays roles in interactions with both biotic and abiotic environmental conditions. HFB2-6 expression was down-regulated under 1% poplar leaf powder culture conditions, but its expression was up-regulated under 1% poplar root powder, indicating that HFB2-6 has a function in root colonization. Furthermore, the recombinant hydrophobin rHFB2-6 was successfully expressed in Escherichia coli BL21-HFB2-6 and purified from the recombinant strain. Genes related to both the jasmonic acid and salicylic acid signal transduction pathways were up-regulated by interaction with renatured rHFB2-6. The ORCA3 (octadecanoid-derivative responsive Catharanthus AP2-domain) gene of the poplar jasmonic acid signal transduction pathway showed a peak expression of 4.48 times at 2 h, and the peak expression of PR1 (pathogenesis-related protein gene) in the salicylic acid signal transduction pathway was 4.58 times at 72 h. Two genes, MP (monopteros) and GH3.17 (auxin original response gene), in the auxin signal transduction pathway were also up-regulated after induction with rHFB2-6, indicating that rHFB2-6 can promote poplar growth and confer broad-spectrum resistance to pathogens. PMID:25644947

  18. Transcriptional Network Analysis Reveals that AT1 and AT2 Angiotensin II Receptors Are Both Involved in the Regulation of Genes Essential for Glioma Progression

    PubMed Central

    Azevedo, Hátylas; Fujita, André; Bando, Silvia Yumi; Iamashita, Priscila; Moreira-Filho, Carlos Alberto

    2014-01-01

    Gliomas are aggressive primary brain tumors with high infiltrative potential. The expression of Angiotensin II (Ang II) receptors has been associated with poor prognosis in human astrocytomas, the most common type of glioma. In this study, we investigated the role of Angiotensin II in glioma malignancy through transcriptional profiling and network analysis of cultured C6 rat glioma cells exposed to Ang II and to inhibitors of its membrane receptor subtypes. C6 cells were treated with Ang II and specific antagonists of AT1 and AT2 receptors. Total RNA was isolated after three and six hours of Ang II treatment and analyzed by oligonucleotide microarray technology. Gene expression data was evaluated through transcriptional network modeling to identify how differentially expressed (DE) genes are connected to each other. Moreover, other genes co-expressing with the DE genes were considered in these analyses in order to support the identification of enriched functions and pathways. A hub-based network analysis showed that the most connected nodes in Ang II-related networks exert functions associated with cell proliferation, migration and invasion, key aspects for glioma progression. The subsequent functional enrichment analysis of these central genes highlighted their participation in signaling pathways that are frequently deregulated in gliomas such as ErbB, MAPK and p53. Noteworthy, either AT1 or AT2 inhibitions were able to down-regulate different sets of hub genes involved in protumoral functions, suggesting that both Ang II receptors could be therapeutic targets for intervention in glioma. Taken together, our results point out multiple actions of Ang II in glioma pathogenesis and reveal the participation of both Ang II receptors in the regulation of genes relevant for glioma progression. This study is the first one to provide systems-level molecular data for better understanding the protumoral effects of Ang II in the proliferative and infiltrative behavior of gliomas. PMID:25365520

  19. The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III.

    PubMed

    Padilla-Mejía, Norma E; Florencio-Martínez, Luis E; Moreno-Campos, Rodrigo; Vizuet-de-Rueda, Juan C; Cevallos, Ana M; Hernández-Rivas, Rosaura; Manning-Cela, Rebeca; Martínez-Calvillo, Santiago

    2015-03-01

    Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major. This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5' end of all the mRNAs. In L. major, tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5' rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5' end and a poly(A) tail at the 3' end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei. However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo, as has recently been found in other eukaryotes. PMID:25548151

  20. A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation

    PubMed Central

    George, Amee J.; Purdue, Brooke W.; Gould, Cathryn M.; Thomas, Daniel W.; Handoko, Yanny; Qian, Hongwei; Quaife-Ryan, Gregory A.; Morgan, Kylie A.; Simpson, Kaylene J.; Thomas, Walter G.; Hannan, Ross D.

    2013-01-01

    Summary The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R–EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R–EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R–EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR–EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer. PMID:24046455

  1. HLA non-class II genes may confer type I diabetes susceptibility in a Mapuche (Amerindian) affected family.

    PubMed

    Pérez-Bravo, Francisco; Martinez-Laso, Jorge; Martin-Villa, Jose M; Moscoso, Juan; Moreno, Almudena; Serrano-Vela, Juan I; Zamora, Jorge; Asenjo, Silvia; Gleisner, Andrea; Arnaiz-Villena, Antonio

    2006-01-01

    A rare case of type I diabetes is studied in an Amerindian (Mapuche) family from Chile, analyzing glutamic acid decarboxylase, islet-cell autoantibodies and human leukocyte antigen (HLA) genes. The affected sib is the only one that has one specific HLA haplotype combination that differs from the other sibs only in the HLA class I genes. It is concluded that HLA diabetes susceptibility factors may be placed outside the class II region or even that susceptibility factors do not exist in the HLA region in this Amerindian family. PMID:16473308

  2. Complete nucleotide sequence of the gene for human heparin cofactor II and mapping to chromosomal band 22q11

    SciTech Connect

    Herzog, R.; Lutz, S.; Blin, N. (Universitaet des Saarlandes, Homburg/Saar (West Germany)); Marasa, J.C.; Blinder, M.A.; Tollefsen, D.M. (Washington Univ., St. Louis, MO (USA))

    1991-02-05

    Heparin cofactor II (HCII) is a 66-kDa plasma glycoprotein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Clones comprising the entire HCII gene were isolated from a human leukocyte genomic library in EMBL-3 {lambda} phage. The sequence of the gene was determined on both strands of DNA (15,849 bp) and included 1,749 bp of 5{prime}-flanking sequence, five exons, four introns, and 476 bp of DNA 3{prime} to the polyadenylation site. Ten complete and one partial Alu repeats were identified in the introns and 5{prime}-flanking region. The HCII gene was regionally mapped on chromosome 22 using rodent-human somatic cell hybrids, carrying only parts of human chromosome 22, and the chronic myelogenous leukemia cell line K562. With the cDNA probe HCII7.2, containing the entire coding region of the gene, the HCII gene was shown to be amplified 10-20-fold in K562 cells by Southern analysis and in situ hybridization. From these data, the authors concluded that the HCII gene is localized on the chromosomal band 22q11 proximal to the breakpoint cluster region (BCR). Analysis by pulsed-field gel electrophoresis indicated that the amplified HCII gene in K562 cells maps at least 2 Mbp proximal to BCR-1. Furthermore, the HCII7.2 cDNA probe detected two frequent restriction fragment length polymorphisms with the restriction enzymes BamHI and Hind III.

  3. Transcriptional pause, arrest and termination sites for RNA polymerase II in mammalian N- and c-myc genes.

    PubMed Central

    Keene, R G; Mueller, A; Landick, R; London, L

    1999-01-01

    Using either highly purified RNA polymerase II (pol II) elongation complexes assembled on oligo(dC)-tailed templates or promoter-initiated (extract-generated) pol II elongation complexes, the precise 3" ends of transcripts produced during transcription in vitro at several human c- and N- myc pause, arrest and termination sites were determined. Despite a low overall similarity between the entire c- and N- myc first exon sequences, many positions of pol II pausing, arrest or termination occurred within short regions of related sequence shared between the c- and N- myc templates. The c- and N- myc genes showed three general classes of sequence conservation near intrinsic pause, arrest or termination sites: (i) sites where arrest or termination occurred after the synthesis of runs of uridines (Us) preceding the transcript 3" end, (ii) sites downstream of potential RNA hairpins and (iii) sites after nucleotide addition following either a U or a C or following a combination of several pyrimidines near the transcript 3" end. The finding that regions of similarity occur near the sites of pol II pausing, arrest or termination suggests that the mechanism of c- and N- myc regulation at the level of transcript elongation may be similar and not divergent as previously proposed. PMID:10454615

  4. Vra4 congenic rats with allelic differences in the class II transactivator gene display altered susceptibility to experimental autoimmune encephalomyelitis.

    PubMed

    Harnesk, Karin; Swanberg, Maria; Ockinger, Johan; Diez, Margarita; Lidman, Olle; Wallström, Erik; Lobell, Anna; Olsson, Tomas; Piehl, Fredrik

    2008-03-01

    Presentation of Ag bound to MHC class II (MHC II) molecules to CD4+ T cells is a key event in adaptive immune responses. Genetic differences in MHC II expression in the rat CNS were recently positioned to allelic variability in the CIITA gene (Mhc2ta), located within the Vra4 locus on rat chromosome 10. In this study, we have examined reciprocal Vra4-congenic strains on the DA and PVGav1 backgrounds, respectively. After experimental nerve injury the strain-specific MHC II expression on microglia was reversed in the congenic strains. Similar findings were obtained after intraparenchymal injection of IFN-gamma in the brain. Expression of MHC class II was also lower on B cells and dendritic cells from the DA.PVGav1-Vra4- congenic strain compared with DA rats after in vitro stimulation with IFN-gamma. We next explored whether Vra4 may affect the outcome of experimental autoimmune disease. In experimental autoimmune encephalomyelitis induced by immunization with myelin oligodendrocyte glycoprotein, DA.PVGav1-Vra4 rats displayed a lower disease incidence and milder disease course compared with DA, whereas both PVGav1 and PVGav1.DA-Vra4 rats were completely protected. These results demonstrate that naturally occurring allelic differences in Mhc2ta have profound effects on the quantity of MHC II expression in the CNS and on immune cells and that this genetic variability also modulates susceptibility to autoimmune neuroinflammation. PMID:18292553

  5. Construction of a standard reference plasmid containing seven target genes for the detection of transgenic cotton.

    PubMed

    Wang, Xujing; Tang, Qiaoling; Dong, Lei; Dong, Yufeng; Su, Yueyan; Jia, Shirong; Wang, Zhixing

    2014-07-01

    Insect resistance and herbicide tolerance are the dominant traits of commercialized transgenic cotton. In this study, we constructed a general standard reference plasmid for transgenic cotton detection. Target genes, including the cowpea trypsin gene cptI, the insect resistance gene cry1Ab/1Ac, the herbicide tolerance gene cp4-epsps, the Agrobacterium tumefaciens nopaline synthase (Nos) terminator that exists in transgenic cotton and part of the endogenous cotton SadI gene were amplified from plasmids pCPT1, pBT, pCP4 and pBI121 and from DNA of the nontransgenic cotton line K312, respectively. The genes cry1Ab/1Ac and cptI, as well as cp4-epsps and the Nos terminator gene, were ligated together to form the fusion genes cptI-Bt and cp4-Nos, respectively, by overlapping PCR. We checked the validity of genes Sad1, cptI-Bt and cp4-Nos by DNA sequencing. Then, positive clones of cptI-Bt, cp4-Nos and Sad1 were digested with the corresponding restriction enzymes and ligated sequentially into vector pCamBIA2300, which contains the CAMV 35S promoter and nptII gene, to form the reference plasmid pMCS. Qualitative detection showed that pMCS is a good positive control for transgenic cotton detection. Real-time PCR detection efficiencies with pMCS as a calibrator ranged from 94.35% to 98.67% for the standard curves of the target genes (R(2)?0.998). The relative standard deviation of the mean value for the known sample was 11.95%. These results indicate that the strategy of using the pMCS plasmid as a reference material is feasible and reliable for the detection of transgenic cotton. Therefore, this plasmid can serve as a useful reference tool for qualitative and quantitative detection of single or stacked trait transgenic cotton, thus paving the way for the identification of various products containing components of transgenic cotton. PMID:24929128

  6. Mutations in the VLGR1 Gene Implicate G-Protein Signaling in the Pathogenesis of Usher Syndrome Type II

    Microsoft Academic Search

    Michael D. Weston; Mirjam W. J. Luijendijk; Kurt D. Humphrey; Claes Möller; William J. Kimberling

    2004-01-01

    Usher syndrome type II (USH2) is a genetically heterogeneous autosomal recessive disorder with at least three genetic subtypes (USH2A, USH2B, and USH2C) and is classified phenotypically as congenital hearing loss and progressive retinitis pigmentosa. The VLGR1 (MASS1) gene in the 5q14.3-q21.1 USH2C locus was considered a likely candidate on the basis of its protein motif structure and expressed-sequence-tag representation from

  7. MHC class I and MHC class II DRB gene variability in wild and captive Bengal tigers ( Panthera tigris tigris )

    Microsoft Academic Search

    Ina Pokorny; Reeta Sharma; Surendra Prakash Goyal; Sudanshu Mishra; Ralph Tiedemann

    2010-01-01

    Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation\\u000a and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class\\u000a I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2\\u000a domain of MHC class

  8. Expression of the Zymomonas mobilis alcohol dehydrogenase II ( adhB ) and pyruvate decarboxylase ( pdc ) genes in Bacillus

    Microsoft Academic Search

    Maria de F. S. Barbosa; L. O. Ingram

    1994-01-01

    The genes encodingZymomonas mobilis pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were expressed inBacillus subtilis YB886(pLOI500) under the control of aBacillus SPO2 phage promoter and caused a 50% reduction of growth rate compared with the unmodified vector. Expression was further confirmed by Western blots, activity stains of native gels, and in vitro measurements of alcohol dehydrogenase activity. Additional strains

  9. Analyses of RNA Polymerase II Genes from Free-Living Protists: Phylogeny, Long Branch Attraction, and the Eukaryotic Big Bang

    Microsoft Academic Search

    Joel B. Dacks; Alexandra Marinets; W. Ford Doolittle; Thomas Cavalier-Smith; John M. Logsdon

    The phylogenetic relationships among major eukaryotic protist lineages are largely uncertain. Two significant ob- stacles in reconstructing eukaryotic phylogeny are long-branch attraction (LBA) effects and poor taxon sampling of free-living protists. We have obtained and analyzed gene sequences encoding the largest subunit of RNA Poly- merase II (RPB1) from Naegleria gruberi (a heterolobosean), Cercomonas ATCC 50319 (a cercozoan), and Och-

  10. Characterization of MHC class I and II genes in a subantarctic seabird, the blue petrel, Halobaena caerulea (Procellariiformes)

    Microsoft Academic Search

    Maria Strandh; Mimi Lannefors; Francesco Bonadonna; Helena Westerdahl

    The great polymorphism observed in the major histocompatibility complex (MHC) genes is thought to be maintained by pathogen-mediated\\u000a selection possibly combined with MHC-disassortative mating, guided by MHC-determined olfactory cues. Here, we partly characterize\\u000a the MHC class I and II B of the blue petrel, Halobaena caerulea (Procellariiformes), a bird with significant olfactory abilities that lives under presumably low pathogen burdens

  11. A 39-kb sequence around a blackbird Mhc class II gene: ghost of selection past and songbird genome architecture.

    PubMed

    Edwards, S V; Gasper, J; Garrigan, D; Martindale, D; Koop, B F

    2000-09-01

    To gain an understanding of the evolution and genomic context of avian major histocompatibility complex (Mhc) genes, we sequenced a 38.8-kb Mhc-bearing cosmid insert from a red-winged blackbird (Agelaius phoeniceus). The DNA sequence, the longest yet retrieved from a bird other than a chicken, provides a detailed view of the process of gene duplication, divergence, and degeneration ("birth and death") in the avian Mhc, as well as a glimpse into major noncoding features of a songbird genome. The peptide-binding region (PBR) of the single Mhc class II B gene in this region, Agph-DAB2, is almost devoid of polymorphism, and a still-segregating single-base-pair deletion and other features suggest that it is nonfunctional. Agph-DAB2 is estimated to have diverged about 40 MYA from a previously characterized and highly polymorphic blackbird Mhc gene, Aph-DAB1, and is therefore younger than most mammalian Mhc paralogs and arose relatively late in avian evolution. Despite its nonfunctionality, Agph-DAB2 shows very high levels of nonsynonymous divergence from Agph-DAB1 and from reconstructed ancestral sequences in antigen-binding PBR codons-a strong indication of a period of adaptive divergence preceding loss of function. We also found that the region sequenced contains very few other unambiguous genes, a partial Mhc- class II gene fragment, and a paucity of simple-sequence and other repeats. Thus, this sequence exhibits some of the genomic streamlining expected for avian as compared with mammalian genomes, but is not as densely packed with functional genes as is the chicken Mhc. PMID:10958854

  12. The evolutionarily conserved Pol II flap loop contributes to proper transcription termination on short yeast genes.

    PubMed

    Pearson, Erika; Moore, Claire

    2014-11-01

    Current models of transcription termination factor recruitment to the RNA polymerase II (Pol II) transcription complex rely exclusively on the direct interaction between the termination factor and phosphorylated isoforms of the Pol II C-terminal domain (CTD). Here, we report that the Pol II flap loop is needed for physical interaction of Pol II with the Pcf11/Clp1 subcomplex of cleavage factor IA (CF IA), which functions in both 3? end processing and Pol II termination, and for proper termination of short RNAs in vitro and in vivo. Deletion of the flap loop reduces the in vivo interaction of Pol II with CF IA but increases the association of Nrd1 during stages of the transcription cycle when the CTD is predominantly Ser5 phosphorylated. We propose a model in which the flap loop coordinates a binding equilibrium between the competing termination factors Pcf11 and Nrd1 to Pol II during termination of short RNA synthesis. PMID:25437538

  13. Successive silencing of tandem reporter genes in potato (Solanum tuberosum) over 5 years of vegetative propagation

    PubMed Central

    Nocarova, Eva; Opatrny, Zdenek; Fischer, Lukas

    2010-01-01

    Background and Aims Transgenic plants represent an excellent tool for experimental plant biology and are an important component of modern agriculture. Fully understanding the stability of transgene expression is critical in this regard. Most changes in transgene expression occur soon after transformation and thus unwanted lines can be discarded easily; however, transgenes can be silenced long after their integration. Methods To study the long-term changes in transgene expression in potato (Solanum tuberosum), the activity of two reporter genes, encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII), was monitored in a set of 17 transgenic lines over 5 years of vegetative propagation in vitro. Key Results A decrease in transgene expression was observed mainly in lines with higher initial GFP expression and a greater number of T-DNA insertions. Complete silencing of the reporter genes was observed in four lines (nearly 25 %), all of which successively silenced the two reporter genes, indicating an interconnection between their silencing. The loss of GFP fluorescence always preceded the loss of kanamycin resistance. Treatment with the demethylation drug 5-azacytidine indicated that silencing of the NPTII gene, but probably not of GFP, occurred directly at the transcriptional level. Successive silencing of the two reporter genes was also reproduced in lines with reactivated expression of previously silenced transgenes. Conclusions We suggest a hypothetical mechanism involving the successive silencing of the two reporter genes that involves the switch of GFP silencing from the post-transcriptional to transcriptional level and subsequent spreading of methylation to the NPTII gene. PMID:20829194

  14. Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences.

    PubMed

    Azevedo, M I; Botton, S A; Pereira, D I B; Robe, L J; Jesus, F P K; Mahl, C D; Costa, M M; Alves, S H; Santurio, J M

    2012-09-14

    Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum. PMID:22483240

  15. The great diversity of major histocompatibility complex class II genes in Philippine native cattle.

    PubMed

    Takeshima, S N; Miyasaka, T; Polat, M; Kikuya, M; Matsumoto, Y; Mingala, C N; Villanueva, M A; Salces, A J; Onuma, M; Aida, Y

    2014-12-01

    Bovine leukocyte antigens (BoLA) are extensively used as markers for bovine disease and immunological traits. However, none of the BoLA genes in Southeast Asian breeds have been characterized by polymerase chain reaction (PCR)-sequence-based typing (SBT). Therefore, we sequenced exon 2 of the BoLA class II DRB3 gene from 1120 individual cows belonging to the Holstein, Sahiwal, Simbrah, Jersey, Brahman, and Philippine native breeds using PCR-SBT. Several cross-breeds were also examined. BoLA-DRB3 PCR-SBT identified 78 previously reported alleles and five novel alleles. The number of BoLA-DRB3 alleles identified in each breed from the Philippines was higher (71 in Philippine native cattle, 58 in Brahman, 46 in Holstein × Sahiwal, and 57 in Philippine native × Brahman) than that identified in breeds from other countries (e.g., 23 alleles in Japanese Black and 35 in Bolivian Yacumeño cattle). A phylogenetic tree based on the DA distance calculated from the BoLA-DRB3 allele frequency showed that Philippine native cattle from different Philippine islands are closely related, and all of them are closely similar to Philippine Brahman cattle but not to native Japanese and Latin American breeds. Furthermore, the BoLA-DRB3 allele frequency in Philippine native cattle from Luzon Island, located in the Northern Philippines was different from that in cattle from Iloilo, Bohol, and Leyte Islands, which are located in the Southern Philippines. Therefore, we conclude that Philippine native cattle can be divided into two populations, North and South areas. Moreover, a neutrality test revealed that Philippine native cattle from Leyte showed significantly greater genetic diversity, which may be maintained by balancing selection. This study shows that Asian breeds have high levels of BoLA-DRB3 polymorphism. This finding, especially the identification of five novel BoLA-DRB3 alleles, will be helpful for future SBT studies of BoLA-DRB3 alleles in East Asian cattle. PMID:25606401

  16. The great diversity of major histocompatibility complex class II genes in Philippine native cattle

    PubMed Central

    Takeshima, S.N.; Miyasaka, T.; Polat, M.; Kikuya, M.; Matsumoto, Y.; Mingala, C.N.; Villanueva, M.A.; Salces, A.J.; Onuma, M.; Aida, Y.

    2014-01-01

    Bovine leukocyte antigens (BoLA) are extensively used as markers for bovine disease and immunological traits. However, none of the BoLA genes in Southeast Asian breeds have been characterized by polymerase chain reaction (PCR)-sequence-based typing (SBT). Therefore, we sequenced exon 2 of the BoLA class II DRB3 gene from 1120 individual cows belonging to the Holstein, Sahiwal, Simbrah, Jersey, Brahman, and Philippine native breeds using PCR-SBT. Several cross-breeds were also examined. BoLA-DRB3 PCR-SBT identified 78 previously reported alleles and five novel alleles. The number of BoLA-DRB3 alleles identified in each breed from the Philippines was higher (71 in Philippine native cattle, 58 in Brahman, 46 in Holstein × Sahiwal, and 57 in Philippine native × Brahman) than that identified in breeds from other countries (e.g., 23 alleles in Japanese Black and 35 in Bolivian Yacumeño cattle). A phylogenetic tree based on the DA distance calculated from the BoLA-DRB3 allele frequency showed that Philippine native cattle from different Philippine islands are closely related, and all of them are closely similar to Philippine Brahman cattle but not to native Japanese and Latin American breeds. Furthermore, the BoLA-DRB3 allele frequency in Philippine native cattle from Luzon Island, located in the Northern Philippines was different from that in cattle from Iloilo, Bohol, and Leyte Islands, which are located in the Southern Philippines. Therefore, we conclude that Philippine native cattle can be divided into two populations, North and South areas. Moreover, a neutrality test revealed that Philippine native cattle from Leyte showed significantly greater genetic diversity, which may be maintained by balancing selection. This study shows that Asian breeds have high levels of BoLA-DRB3 polymorphism. This finding, especially the identification of five novel BoLA-DRB3 alleles, will be helpful for future SBT studies of BoLA-DRB3 alleles in East Asian cattle. PMID:25606401

  17. Characterisation of class II B MHC genes from a ratite bird, the little spotted kiwi (Apteryx owenii).

    PubMed

    Miller, Hilary C; Bowker-Wright, Gemma; Kharkrang, Marie; Ramstad, Kristina

    2011-04-01

    Major histocompatibility complex (MHC) genes are important for vertebrate immune response and typically display high levels of diversity due to balancing selection from exposure to diverse pathogens. An understanding of the structure of the MHC region and diversity among functional MHC genes is critical to understanding the evolution of the MHC and species resilience to disease exposure. In this study, we characterise the structure and diversity of class II MHC genes in little spotted kiwi Apteryx owenii, a ratite bird representing the basal avian lineage (paleognaths). Results indicate that little spotted kiwi have a more complex MHC structure than that of other non-passerine birds, with at least five class II MHC genes, three of which are expressed and likely to be functional. Levels of MHC variation among little spotted kiwi are extremely low, with 13 birds assayed having nearly identical MHC genotypes (only two genotypes containing four alleles, three of which are fixed). These results suggest that recent genetic drift due to a species-wide bottleneck of at most seven birds has overwhelmed past selection for high MHC diversity in little spotted kiwi, potentially leaving the species highly susceptible to disease. PMID:21221966

  18. Attenuation of corneal myofibroblast development through nanoparticle-mediated soluble transforming growth factor-? type II receptor (sTGF?RII) gene transfer

    E-print Network

    Sharma, Ajay

    Purpose: To explore (i) the potential of polyethylenimine (PEI)-DNA nanoparticles as a vector for delivering genes into human corneal fibroblasts, and (ii) whether the nanoparticle-mediated soluble extracellular domain of ...

  19. Dynamic expression pattern of corticotropin-releasing hormone, urotensin I and II genes under acute salinity and temperature challenge during early development of zebrafish.

    PubMed

    Luo, Lei; Chen, Aqin; Hu, Chongchong; Lu, Weiqun

    2014-12-01

    Corticotropin-releasing hormone (CRH), urotensin I (UI) and urotensin II (UII) are found throughout vertebrate species from fish to human. To further understand the role of crh, uI and uII in teleosts during development, we investigated the expression pattern of crh, uI, uII? and uII? genes, and their response to acute salinity and temperature challenge during early development of zebrafish, Danio rerio. The results reveal that crh, uI, uII? and uII? mRNA are detected from 0hpf, and the expression levels increase to a maximum at 6 days post fertilization (dpf), with the exception of uII? that peak at 5dpf. Exposure of zebrafish embryos and larvae to acute osmotic (30ppt) stress for 15 min failed to modify expression levels of crh, uI, uII? and uII? mRNA from levels in control fish except at 6dpf when uII? and uII? were significantly (P < 0.05) modified. Exposure of embryos and larvae to a cold (18 °C) or hot stress (38 °C) generally down-regulated mRNA levels of crh, uI, uII? and uII? apart from at 3dpf. The results indicate that the contribution of crh, uI, uII? and uII? genes to the stress response in zebrafish may be stressor-specific during early development. Overall, the results from this study provide a basis for further research into the developmental and stressor-specific function of crh, uI, uII? and uII? in zebrafish. PMID:25154920

  20. Genomic instability in the type II TGF-beta1 receptor gene in atherosclerotic and restenotic vascular cells.

    PubMed Central

    McCaffrey, T A; Du, B; Consigli, S; Szabo, P; Bray, P J; Hartner, L; Weksler, B B; Sanborn, T A; Bergman, G; Bush, H L

    1997-01-01

    Cells proliferating from human atherosclerotic lesions are resistant to the antiproliferative effect of TGF-beta1, a key factor in wound repair. DNA from human atherosclerotic and restenotic lesions was used to test the hypothesis that microsatellite instability leads to specific loss of the Type II receptor for TGF-beta1 (TbetaR-II), causing acquired resistance to TGF-beta1. High fidelity PCR and restriction analysis was adapted to analyze deletions in an A10 microsatellite within TbetaR-II. DNA from lesions, and cells grown from lesions, showed acquired 1 and 2 bp deletions in TbetaR-II, while microsatellites in the hMSH3 and hMSH6 genes, and hypermutable regions of p53 were unaffected. Sequencing confirmed that these deletions occurred principally in the replication error-prone A10 microsatellite region, though nonmicrosatellite mutations were observed. The mutations could be identified within specific patches of the lesion, while the surrounding tissue, or unaffected arteries, exhibited the wild-type genotype. This microsatellite deletion causes frameshift loss of receptor function, and thus, resistance to the antiproliferative and apoptotic effects of TGF-beta1. We propose that microsatellite instability in TbetaR-II disables growth inhibitory pathways, allowing monoclonal selection of a disease-prone cell type within some vascular lesions. PMID:9410894

  1. Assignment of the human casein kinase II [alpha][prime] subunit gene (CSNK2A1) to chromosome 16p13. 2-p13. 3

    SciTech Connect

    Yang-Feng, T.L. (Yale Univ. School of Medicine, New Haven, CT (United States)); Naiman, T.; Kopatz, I.; Eli, D.; Dafni, N.; Canaani, D. (Tel Aviv Univ., Ramat Aviv (Israel))

    1994-01-01

    The authors have previously mapped the CK II-[beta] gene (CSNK2B) to chromosome 6p12-p21 and the CK II-[alpha] sequence to two sites, chromosomes 11p15.5-p15.4 and 20p13, the latter having been verified by other investigators. The sequencing of a genomic human DNA fragment has shown that the CK II-[alpha] gene (CSNK2A) localized to chromosome 11 is a processed (pseudo) gene and therefore the active gene is presumably on chromosome 20. The other catalytic subunit gene CK II-[alpha][prime] was localized to chromosome 16 by somatic cell hybrid analysis. The authors now report the regional mapping of the CK II-[alpha][prime] gene (CSNK2A1) to chromosome 16p13.2-p13.3. The probe used was a 414-bp fragment from the 3[prime] nontranslated region of the human CK II-[alpha][prime] cDNA. Chromosomal localization was carried out by in situ hybridization as previously described. Of 128 grains scored in 75 cells, 13 (10.2%) were located on the distal short arm of chromosome 16, bands p13.2-p13.3. No other sites were labeled above background. 7 refs., 1 fig.

  2. Iron chelation and a free radical scavenger suppress angiotensin II-induced downregulation of klotho, an anti-aging gene, in rat

    Microsoft Academic Search

    Kan Saito; Nobukazu Ishizaka; Haruo Mitani; Minoru Ohno; Ryozo Nagai

    2003-01-01

    Administration of angiotensin II to rats decreases renal expression of klotho, an aging-related gene, and also causes abnormal iron deposition in renal cells. Here we have examined the effects of iron overload and iron chelation on renal expression of klotho in untreated rats and rats treated with angiotensin II. Administration of iron–dextran caused a downregulation of klotho expression, and iron

  3. The Type F6 Neurotoxin Gene Cluster Locus of Group II Clostridium botulinum Has Evolved by Successive Disruption of Two Different Ancestral Precursors

    PubMed Central

    Carter, Andrew T.; Stringer, Sandra C.; Webb, Martin D.; Peck, Michael W.

    2013-01-01

    Genome sequences of five different Group II (nonproteolytic) Clostridium botulinum type F6 strains were compared at a 50-kb locus containing the neurotoxin gene cluster. A clonal origin for these strains is indicated by the fact that sequences were identical except for strain Eklund 202F, with 10 single-nucleotide polymorphisms and a 15-bp deletion. The essential topB gene encoding topoisomerase III was found to have been split by the apparent insertion of 34.4 kb of foreign DNA (in a similar manner to that in Group II C. botulinum type E where the rarA gene has been disrupted by a neurotoxin gene cluster). The foreign DNA, which includes the intact 13.6-kb type F6 neurotoxin gene cluster, bears not only a newly introduced topB gene but also two nonfunctional botulinum neurotoxin gene remnants, a type B and a type E. This observation combined with the discovery of bacteriophage integrase genes and IS4 elements suggest that several rounds of recombination/horizontal gene transfer have occurred at this locus. The simplest explanation for the current genotype is that the ancestral bacterium, a Group II C. botulinum type B strain, received DNA firstly from a strain containing a type E neurotoxin gene cluster, then from a strain containing a type F6 neurotoxin gene cluster. Each event disrupted the previously functional neurotoxin gene. This degree of successive recombination at one hot spot is without precedent in C. botulinum, and it is also the first description of a Group II C. botulinum genome containing more than one neurotoxin gene sequence. PMID:23645598

  4. Regulation of major histocompatibility complex class II gene expression, genetic variation and disease

    Microsoft Academic Search

    L Handunnetthi; S V Ramagopalan; G C Ebers; J C Knight

    2010-01-01

    Major histocompatibility complex (MHC) class II molecules are central to adaptive immune responses and maintenance of self-tolerance. Since the early 1970s, the MHC class II region at chromosome 6p21 has been shown to be associated with a remarkable number of autoimmune, inflammatory and infectious diseases. Given that a full explanation for most MHC class II disease associations has not been

  5. Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter.

    PubMed

    Kwon, Deug-Nam; Park, Mi-Ryung; Park, Jong-Yi; Cho, Ssang-Goo; Park, Chankyu; Oh, Jae-Wook; Song, Hyuk; Kim, Jae-Hwan; Kim, Jin-Hoi

    2011-07-01

    Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells. We also identified a new core promoter region and putative negative cis-regulatory element within a minimal promoter region. In addition, we showed that hepatocyte nuclear factor 4 (HNF4) can directly bind in the pUPII core promoter (5F-1) region, which plays a critical role in controlling promoter activity. Transient cotransfection experiments showed that HNF4 positively regulates pUPII gene promoter activity. Thus, the binding element and its binding protein, HNF4 transcription factor, may be involved in the mechanism that specifically regulates pUPII gene transcription. PMID:21658368

  6. Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes

    PubMed Central

    Stadelmayer, Bernd; Micas, Gaël; Gamot, Adrien; Martin, Pascal; Malirat, Nathalie; Koval, Slavik; Raffel, Raoul; Sobhian, Bijan; Severac, Dany; Rialle, Stéphanie; Parrinello, Hugues; Cuvier, Olivier; Benkirane, Monsef

    2014-01-01

    RNA polymerase II (RNAPII) pausing/termination shortly after initiation is a hallmark of gene regulation. Here, we show that negative elongation factor (NELF) interacts with Integrator complex subunits (INTScom), RNAPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both human immunodeficiency virus type 1 promoter and genome-wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits. Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity. Finally, INTScom target genes are enriched in human immunodeficiency virus type 1 transactivation response element/NELF binding element and in a 3' box sequence required for small nuclear RNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle. PMID:25410209

  7. Duplication, balancing selection and trans-species evolution explain the high levels of polymorphism of the DQA MHC class II gene in voles (Arvicolinae)

    Microsoft Academic Search

    J. Bryja; M. Galan; N. Charbonnel; J. F. Cosson

    2006-01-01

    Major histocompatibility complex (MHC) genes play important role in host–parasite interactions and parasites are crucial factors influencing the population dynamics of hosts. We described the structure and diversity of exon 2 of the MHC class II DQA gene in three species of voles (Arvicolinae) exhibiting regular multi-annual fluctuations of population density and analysed the processes leading to the observed MHC

  8. Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics

    Microsoft Academic Search

    C. TOLLENAERE; J. BRYJA; M. GALAN; P. CADET; J. DETER; Y. CHAVAL; K. BERTHIER; A. RIBAS SALVADOR; L. VOUTILAINEN; J. LAAKKONEN; H. HENTTONEN; J.-F. COSSON; N. CHARBONNEL

    2008-01-01

    We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homo- geneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for

  9. Human leukocyte antigen class II immune response genes, female gender, and cigarette smoking as risk and modulating factors in abdominal aortic aneurysms

    Microsoft Academic Search

    Todd E. Rasmussen; John W. Hallett Jr; Henry D. Tazelaar; Virginia M. Miller; Stephanie Schulte; W. Michael O'Fallon; Cornelia M. Weyand

    2002-01-01

    Objective: Aortic inflammation and the genes that regulate the immune response play an important role in abdominal aortic aneurysm pathogenesis. However, the modulating effects of such genetic and other environmental factors on the severity on aneurysm inflammation is not known. The objective of this study was to determine the influence of the human leukocyte antigen (HLA) class II genes, gender,

  10. Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-? family

    Microsoft Academic Search

    Jennifer R Thomson; Rajiv D Machado; Michael W Pauciulo; Neil V Morgan; Marc Humbert; Greg C Elliott; Ken Ward; Magdi Yacoub; Ghada Mikhail; Paula Rogers; John Newman; Lisa Wheeler; Timothy Higenbottam; J Simon R Gibbs; Jim Egan; Agnes Crozier; Andrew Peacock; Robert Allcock; Paul Corris; James E Loyd; Richard C Trembath; William C Nichols

    2000-01-01

    BACKGROUNDPrimary pulmonary hypertension (PPH), resulting from occlusion of small pulmonary arteries, is a devastating condition. Mutations of the bone morphogenetic protein receptor type II gene (BMPR2), a component of the transforming growth factor beta (TGF-?) family which plays a key role in cell growth, have recently been identified as causing familial PPH. We have searched for BMPR2 gene mutations in

  11. Selective modulation of protein kinase A I and II reveals distinct roles in thyroid cell gene expression and growth.

    PubMed

    Calebiro, Davide; de Filippis, Tiziana; Lucchi, Simona; Martinez, Fernando; Porazzi, Patrizia; Trivellato, Roberta; Locati, Massimo; Beck-Peccoz, Paolo; Persani, Luca

    2006-12-01

    A global gene expression profiling of TSH stimulation on differentiated (FRTL5) and partially dedifferentiated [FRT/TSHR (TSH receptor)] rat thyroid cells was performed. A total of 123 TSH-regulated genes (95 newly described) were identified in FRTL5, whereas no significant transcriptional modifications were seen in FRT/TSHR cells. Because regulatory subunit IIbeta (RIIbeta) of protein kinase A (PKA), a key element downstream of cAMP, was expressed in FRTL5 but not in cAMP-refractory FRT/TSHR cells, we hypothesized that this gene may play an important role in TSH signaling. We therefore performed a series of experiments to investigate the involvement of RIIbeta and the different PKA isoforms. A positive effect of PKA II- but not of PKA I-selective activation on gene transcription and proliferation in FRTL5 cells, as well as an impairment of TSH nuclear effects after RIIbeta silencing were observed, suggesting that PKA II plays an essential role in TSH signaling. This view was supported by the restoration of TSH nuclear effects after reexpression of RIIbeta in FRT/TSHR cells. Because PKA I stimulation could increase iodide uptake in FRTL5 cells without affecting gene transcription, PKA I may mediate TSH actions at posttranscriptional levels. Analyses on three human cancer cell lines confirmed the possible loss of RIIbeta expression and antiproliferative activity of PKA I-selective cAMP analogs ( approximately 60% at 200 microm in BRAF-mutated cells). The inhibitory effect of PKA I apparently required constitutive MAPK activation and was associated with an inhibition of ERK phosphorylation. These findings may open new therapeutic perspectives in patients with thyroid cancer. PMID:16887886

  12. The Super Elongation Complex Family of RNA Polymerase II Elongation Factors: Gene Target Specificity and Transcriptional Output

    PubMed Central

    Luo, Zhuojuan; Lin, Chengqi; Guest, Erin; Garrett, Alexander S.; Mohaghegh, Nima; Swanson, Selene; Marshall, Stacy; Florens, Laurence; Washburn, Michael P.

    2012-01-01

    The elongation stage of transcription is highly regulated in metazoans. We previously purified the AFF1- and AFF4-containing super elongation complex (SEC) as a major regulator of development and cancer pathogenesis. Here, we report the biochemical isolation of SEC-like 2 (SEC-L2) and SEC-like 3 (SEC-L3) containing AFF2 and AFF3 in association with P-TEFb, ENL/MLLT1, and AF9/MLLT3. The SEC family members demonstrate high levels of polymerase II (Pol II) C-terminal domain kinase activity; however, only SEC is required for the proper induction of the HSP70 gene upon stress. Genome-wide mRNA-Seq analyses demonstrated that SEC-L2 and SEC-L3 control the expression of different subsets of genes, while AFF4/SEC plays a more dominant role in rapid transcriptional induction in cells. MYC is one of the direct targets of AFF4/SEC, and SEC recruitment to the MYC gene regulates its expression in different cancer cells, including those in acute myeloid or lymphoid leukemia. These findings suggest that AFF4/SEC could be a potential therapeutic target for the treatment of leukemia or other cancers associated with MYC overexpression. PMID:22547686

  13. GSTT2, a phase II gene induced by apple polyphenols, protects colon epithelial cells against genotoxic damage.

    PubMed

    Petermann, Astrid; Miene, Claudia; Schulz-Raffelt, Gabriele; Palige, Katja; Hölzer, Jana; Glei, Michael; Böhmer, Frank-D

    2009-10-01

    The potential protective effect of a polyphenol-rich diet for colon carcinogenesis is of great scientific and medical interest. Apples are a main source of polyphenols, and apple juice has been shown to attenuate chemically induced colon carcinogenesis in animal models. In addition to an antioxidant and antiproliferative activity, apple polyphenols have been shown to elevate expression of the phase II gene glutathione S-transferase T2 (GSTT2) in colon epithelial cells. We hypothesized that apple polyphenols may thereby provide protection against oxidant-induced DNA damage. Using GSTT2 promoter constructs and luciferase reporter assays, we found that polyphenolic apple extracts (AE) can directly enhance GSTT2 promoter activity. Comet assays demonstrated that the genotoxicity of the GSTT2 substrate cumene hydroperoxide (CumOOH) was significantly reduced when HT29 colon epithelial cells were pretreated with AE. Overexpression of GSTT2 in HT29 cells significantly reduced CumOOH induced DNA damage, whereas shRNA mediated knockdown of GSTT2 gene expression resulted in higher damage. Our results causally link GSTT2 levels with protection from genotoxic stress, and provide evidence that the antigenotoxic effects of apple polyphenols in vitro are at least in part due to an induction of GSTT2 expression. Induction of phase II genes may contribute to primary chemoprevention of colon cancer by apple polyphenols. PMID:19753610

  14. A tissue-specific DNase I-hypersensitive site in a class II A alpha gene is under trans-regulatory control.

    PubMed Central

    Liou, H C; Polla, B S; Aragnol, D; Leserman, L D; Griffith, I J; Glimcher, L H

    1988-01-01

    Class II major histocompatibility complex molecules are integral membrane glycoproteins whose distribution is limited to certain tissues. To identify the molecular basis for such specificity, the chromatin configuration of the class II A alpha gene was examined in intact nuclei from various cell types. We show that there are three DNase I-hypersensitive sites in the A alpha gene. One of these sites, located near the promoter region, is specific to cells that normally express class II molecules at some stage of differentiation. Furthermore, this tissue-specific site appears to be under trans-regulatory control. Images PMID:3258662

  15. Genomics and Polymorphism of Agph-DAB1, an Mhc Class II B Gene in Red-Winged Blackbirds (Agelaius phoeniceus)

    Microsoft Academic Search

    Scott V. Edwards; Joe Gasper; Mariam March

    To further our understanding of the evolution of avian Mhc genes at the genomic level, we screened a cosmid library made from a red-winged blackbird (Agelaius phoeniceus) with a blackbird cDNA probe and subcloned from one of the Mhc-containing cosmids a gene which we designate Agph-DAB1. The structure of the gene is similar to that found for chicken class II

  16. IiSDD1 , a gene responsive to autopolyploidy and environmental factors in Isatis indigotica

    Microsoft Academic Search

    Ying Xiao; Xiaojing Yu; Junfeng Chen; Peng Di; Wansheng Chen; Lei Zhang

    2010-01-01

    In plants, stomata play a pivotal role in the regulation of gas exchange and are distributed throughout the aerial epidermis.\\u000a SDD1, a gene isolated from Arabidopsis thaliana has been demonstrated to specialize in stomatal density and distribution. In our present study, a comprehensive survey of\\u000a global gene expression performed by using an A. thaliana whole genome Affymetrix gene chip revealed

  17. Loss of lager specific genes and subtelomeric regions define two different Saccharomyces cerevisiae lineages for Saccharomyces pastorianus Group I and II strains.

    PubMed

    Monerawela, Chandre; James, Tharappel C; Wolfe, Kenneth H; Bond, Ursula

    2015-03-01

    Lager yeasts, Saccharomyces pastorianus, are interspecies hybrids between S. cerevisiae and S. eubayanus and are classified into Group I and Group II clades. The genome of the Group II strain, Weihenstephan 34/70, contains eight so-called 'lager-specific' genes that are located in subtelomeric regions. We evaluated the origins of these genes through bioinformatic and PCR analyses of Saccharomyces genomes. We determined that four are of cerevisiae origin while four originate from S. eubayanus. The Group I yeasts contain all four S. eubayanus genes but individual strains contain only a subset of the cerevisiae genes. We identified S. cerevisiae strains that contain all four cerevisiae 'lager-specific' genes, and distinct patterns of loss of these genes in other strains. Analysis of the subtelomeric regions uncovered patterns of loss in different S. cerevisiae strains. We identify two classes of S. cerevisiae strains: ale yeasts (Foster O) and stout yeasts with patterns of 'lager-specific' genes and subtelomeric regions identical to Group I and II S. pastorianus yeasts, respectively. These findings lead us to propose that Group I and II S. pastorianus strains originate from separate hybridization events involving different S. cerevisiae lineages. Using the combined bioinformatic and PCR data, we describe a potential classification map for industrial yeasts. PMID:25673756

  18. NK and B cell deficiency in a MPS type II family with novel mutation in the IDS gene.

    PubMed

    Torres, Leuridan Cavalcante; Soares, Diogo Cordeiro de Queiroz; Kulikowski, Leslie Domenici; Franco, Jose Francisco; Kim, Chong Ae

    2014-10-01

    The mucopolysaccharidoses (MPSs) are a group of rare, inherited lysosomal storage disorders that are clinically characterized by abnormalities in multiple organ systems and reduced life expectancy. Whereas the lysosome is essential to the functioning of the immune system, some authors suggest that the MPS patients have abnormalities in the immune system similar to the patients with primary immunodeficiency. In this study, we evaluated 8 male MPS type II patients of the same family with novel mutation in the IDS gene. We found in this MPS family a quantitative deficiency of NK and B cells with normal values of IgG, IgM and IgA serum antibodies and normal response to polysaccharide antigens. Interestingly, abnormalities found in these patients were not observed in other MPS patients, suggesting that the type of mutation found in the IDS gene can be implicated in the immunodeficiency. PMID:25038527

  19. Frequent intragenic deletion of the P gene in Tanzanian patients with type II oculocutaneous albinism (OCA2).

    PubMed Central

    Spritz, R A; Fukai, K; Holmes, S A; Luande, J

    1995-01-01

    Type II oculocutaneous albinism (OCA2) is an autosomal recessive disorder in which the biosynthesis of melanin pigment is reduced in the skin, hair, and eyes. OCA2, which results from mutations of the P gene, is the most frequent type of albinism in African and African-American patients. OCA2 is especially frequent in Tanzania, where it occurs with an incidence of approximately 1/1,400. We have identified abnormalities of the P gene in each of 13 unrelated patients with OCA2 from Tanzania. One of these, a deletion of exon 7, is strongly predominant, accounting for approximately 77% of mutant alleles in this group of patients. Images Figure 1 PMID:7762554

  20. Frequent intragenic deletion of the P gene in Tanzanian patients with Type II oculocutaneous albinism (OCA2)

    SciTech Connect

    Spritz, R.; Fukai, K.; Holmes, S.A. [Univ. of Wisconsin, Madison, WI (United States)] [and others

    1995-06-01

    Type II oculocutaneous albinism (OCA2) is an autosomal recessive disorder in which the biosynthesis of melanin pigment is reduced in the skin, hair, and eyes. OCA2, which results from mutations of the P gene, is the most frequent type of albinism in African and African-American patients. OCA2 is especially frequent in Tanzania, where it occurs with an incidence of {approximately}1/1,400. We have identified abnormalities of the P gene in each of 13 unrelated patients with OCA2 from Tanzania. One of these, a deletion of exon 7, is strongly predominant, accounting for {approximately}77% of mutant alleles in this group of patients. 20 refs., 2 figs.

  1. Rate of elongation by RNA polymerase II is associated with specific gene features and epigenetic modifications

    PubMed Central

    Veloso, Artur; Kirkconnell, Killeen S.; Magnuson, Brian; Biewen, Benjamin; Paulsen, Michelle T.; Wilson, Thomas E.; Ljungman, Mats

    2014-01-01

    The rate of transcription elongation plays an important role in the timing of expression of full-length transcripts as well as in the regulation of alternative splicing. In this study, we coupled Bru-seq technology with 5,6-dichlorobenzimidazole 1-?-D-ribofuranoside (DRB) to estimate the elongation rates of over 2000 individual genes in human cells. This technique, BruDRB-seq, revealed gene-specific differences in elongation rates with a median rate of around 1.5 kb/min. We found that genes with rapid elongation rates showed higher densities of H3K79me2 and H4K20me1 histone marks compared to slower elongating genes. Furthermore, high elongation rates had a positive correlation with gene length, low complexity DNA sequence, and distance from the nearest active transcription unit. Features that negatively correlated with elongation rate included the density of exons, long terminal repeats, GC content of the gene, and DNA methylation density in the bodies of genes. Our results suggest that some static gene features influence transcription elongation rates and that cells may alter elongation rates by epigenetic regulation. The BruDRB-seq technique offers new opportunities to interrogate mechanisms of regulation of transcription elongation. PMID:24714810

  2. Individual letters of the RNA polymerase II CTD code govern distinct gene expression programs in fission yeast

    PubMed Central

    Schwer, Beate; Bitton, Danny Asher; Sanchez, Ana M.; Bähler, Jürg; Shuman, Stewart

    2014-01-01

    The primary structure and phosphorylation pattern of the tandem Y1S2P3T4S5P6S7 repeats of the RNA polymerase II carboxyl-terminal domain (CTD) comprise an informational code that coordinates transcription, chromatin modification, and RNA processing. To gauge the contributions of individual CTD coding “letters” to gene expression, we analyzed the poly(A)+ transcriptomes of fission yeast mutants that lack each of the four inessential CTD phosphoacceptors: Tyr1, Ser2, Thr4, and Ser7. There was a hierarchy of CTD mutational effects with respect to the number of dysregulated protein-coding RNAs, with S2A (n = 227) >> Y1F (n = 71) > S7A (n = 58) >> T4A (n = 7). The majority of the protein-coding RNAs affected in Y1F cells were coordinately affected by S2A, suggesting that Tyr1-Ser2 constitutes a two-letter code “word.” Y1F and S2A elicited increased expression of genes encoding proteins involved in iron uptake (Frp1, Fip1, Fio1, Str3, Str1, Sib1), without affecting the expression of the genes that repress the iron regulon, implying that Tyr1-Ser2 transduces a repressive signal. Y1F and S2A cells had increased levels of ferric reductase activity and were hypersensitive to phleomycin, indicative of elevated intracellular iron. The T4A and S7A mutations had opposing effects on the phosphate response pathway. T4A reduced the expression of two genes encoding proteins involved in phosphate acquisition (the Pho1 acid phosphatase and the phosphate transporter SPBC8E4.01c), without affecting the expression of known genes that regulate the phosphate response pathway, whereas S7A increased pho1+ expression. These results highlight specific cellular gene expression programs that are responsive to distinct CTD cues. PMID:24591591

  3. Genetic variation of the major histocompatibility complex (MHC class II B gene) in the threatened Hume's pheasant, Syrmaticus humiae.

    PubMed

    Chen, Weicai; Bei, Yongjian; Li, Hanhua

    2015-01-01

    Major histocompatibility complex (MHC) genes are the most polymorphic genes in vertebrates and encode molecules that play a crucial role in pathogen resistance. As a result of their diversity, they have received much attention in the fields of evolutionary and conservation biology. Here, we described the genetic variation of MHC class II B (MHCIIB) exon 2 in a wild population of Hume's pheasant (Syrmaticus humiae), which has suffered a dramatic decline in population over the last three decades across its ranges in the face of heavy exploitation and habitat loss. Twenty-four distinct alleles were found in 73 S. humiae specimens. We found seven shared alleles among four geographical groups as well as six rare MHCIIB alleles. Most individuals displayed between one to five alleles, suggesting that there are at least three MHCIIB loci of the Hume's pheasant. The dN ? dS ratio at putative antigen-binding sites (ABS) was significantly greater than one, indicating balancing selection is acting on MHCIIB exon 2. Additionally, recombination and gene conversion contributed to generating MHCIIB diversity in the Hume's pheasant. One to three recombination events and seventy-five significant gene conversion events were observed within the Hume's pheasant MHCIIB loci. The phylogenetic tree and network analysis revealed that the Hume's pheasant alleles do not cluster together, but are scattered through the tree or network indicating a trans-species evolutionary mode. These findings revealed the evolution of the Hume's pheasant MHC after suffering extreme habitat fragmentation. PMID:25629763

  4. MicroTom—a high-throughput model transformation system for functional genomics

    Microsoft Academic Search

    Yinghui Dan; Hua Yan; Tichafa Munyikwa; Jimmy Dong; Yanling Zhang; Charles L. Armstrong

    2006-01-01

    We have developed a high-throughput Agrobacterium-mediated transformation model system using both nptII and the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4) based selections in MicroTom, a miniature rapid-cycling cherry tomato variety. With the NPTII selection system, transformation\\u000a frequency calculated as independent transgenic events per inoculated explant ranged from 24 to 80% with an average of 56%,\\u000a in industrial

  5. Expanding our Understanding of Sequence-Function Relationships of Type II Polyketide Biosynthetic Gene Clusters: Bioinformatics-Guided Identification of Frankiamicin A from Frankia sp. EAN1pec

    PubMed Central

    Ogasawara, Yasushi; Yackley, Benjamin J.; Greenberg, Jacob A.; Rogelj, Snezna; Melançon, Charles E.

    2015-01-01

    A large and rapidly increasing number of unstudied “orphan” natural product biosynthetic gene clusters are being uncovered in sequenced microbial genomes. An important goal of modern natural products research is to be able to accurately predict natural product structures and biosynthetic pathways from these gene cluster sequences. This requires both development of bioinformatic methods for global analysis of these gene clusters and experimental characterization of select products produced by gene clusters with divergent sequence characteristics. Here, we conduct global bioinformatic analysis of all available type II polyketide gene cluster sequences and identify a conserved set of gene clusters with unique ketosynthase ?/? sequence characteristics in the genomes of Frankia species, a group of Actinobacteria with underexploited natural product biosynthetic potential. Through LC-MS profiling of extracts from several Frankia species grown under various conditions, we identified Frankia sp. EAN1pec as producing a compound with spectral characteristics consistent with the type II polyketide produced by this gene cluster. We isolated the compound, a pentangular polyketide which we named frankiamicin A, and elucidated its structure by NMR and labeled precursor feeding. We also propose biosynthetic and regulatory pathways for frankiamicin A based on comparative genomic analysis and literature precedent, and conduct bioactivity assays of the compound. Our findings provide new information linking this set of Frankia gene clusters with the compound they produce, and our approach has implications for accurate functional prediction of the many other type II polyketide clusters present in bacterial genomes. PMID:25837682

  6. RSF1010 and a conjugative plasmid contain sulII, one of two known genes for plasmid-borne sulfonamide resistance dihydropteroate synthase.

    PubMed Central

    Rådström, P; Swedberg, G

    1988-01-01

    The nucleotide sequence of the type II sulfonamide resistance dihydropteroate synthase (sulII) gene was determined. The molecular weight determined by maxicells was 30,000, and the predicted molecular weight for the polypeptide was 28,469. Comparison with the sulI gene encoded by Tn21 showed 57% DNA similarity. The sulII-encoded polypeptide has 138 of 271 amino acids in common with the polypeptide encoded by sulI. The sulII gene is located on various IncQ (broad-host-range) plasmids and other small nonconjugative resistance plasmids. Detailed restriction maps were constructed to compare the different plasmids in which sulII is found. The large conjugative plasmid pGS05 and the IncQ plasmid RSF1010 contained identical nucleotide sequences for the sulII gene. This type of sulfonamide resistance is very frequently found among gram-negative bacteria because of its efficient spread to various plasmids. Images PMID:3075438

  7. Herpes simplex virus 1 activates cdc2 to recruit topoisomerase II alpha for post-DNA synthesis expression of late genes.

    PubMed

    Advani, Sunil J; Weichselbaum, Ralph R; Roizman, Bernard

    2003-04-15

    A subset (gamma(2)) of late herpes simplex virus 1 genes depends on viral DNA synthesis for its expression. For optimal expression, a small number of these genes, exemplified by U(S)11, also requires two viral proteins, the alpha protein infected cell protein (ICP) 22 and the protein kinase U(L)13. Earlier we showed that U(L)13 and ICP22 mediate the stabilization of cdc2 and the replacement of its cellular partner, cyclin B, with the viral DNA polymerase processivity factor U(L)42. Here we report that cdc2 and its new partner, U(L)42, bind a phosphorylated form of topoisomerase II alpha. The posttranslational modification of topoisomerase II alpha and its interaction with cdc2-U(L)42 proteins depend on ICP22 in infected cells. Although topoisomerase II is required for viral DNA synthesis, ICP22 is not, indicating a second function for topoisomerase II alpha. The intricate manner in which the virus recruits topoisomerase II alpha for post-DNA synthesis expression of viral genes suggests that topoisomerase II alpha also is required for untangling concatemeric DNA progeny for optimal transcription of late genes. PMID:12665617

  8. IGF-I, IGF-II, and Insulin Stimulate Different Gene Expression Responses through Binding to the IGF-I Receptor

    PubMed Central

    Versteyhe, Soetkin; Klaproth, Birgit; Borup, Rehannah; Palsgaard, Jane; Jensen, Maja; Gray, Steven G.; De Meyts, Pierre

    2013-01-01

    Insulin and the insulin-like growth factors (IGF)-I and -II are closely related peptides important for regulation of metabolism, growth, differentiation, and development. The IGFs exert their main effects through the IGF-I receptor. Although the insulin receptor is the main physiological receptor for insulin, this peptide hormone can also bind at higher concentrations to the IGF-I receptor and exert effects through it. We used microarray gene expression profiling to investigate the gene expression regulated by IGF-I, IGF-II, and insulin after stimulation of the IGF-I receptor. Fibroblasts from mice, knockout for IGF-II and the IGF-II/cation-independent mannose-6-phosphate receptor, and expressing functional IGF-I but no insulin receptors, were stimulated for 4?h with equipotent saturating concentrations of insulin, IGF-I, and IGF-II. Each ligand specifically regulated a group of transcripts that was not regulated by the other two ligands. Many of the functions and pathways these regulated genes were involved in, were consistent with the known biological effects of these ligands. The differences in gene expression might therefore account for some of the different biological effects of insulin, IGF-I, and IGF-II. This work adds to the evidence that not only the affinity of a ligand determines its biological response, but also its nature, even through the same receptor. PMID:23950756

  9. Carbonic anhydrase II deficiency syndrome in a Belgian family is caused by a point mutation at an invariant histidine residue (107 His----Tyr): complete structure of the normal human CA II gene.

    PubMed Central

    Venta, P J; Welty, R J; Johnson, T M; Sly, W S; Tashian, R E

    1991-01-01

    Carbonic anhydrase II (CA II), which has the highest turnover number and widest tissue distribution of any of the seven CA isozymes known in humans, is absent from the red blood cells and probably from other tissues of patients with CA II deficiency syndrome. We have sequenced the CA II gene in a patient from a consanguinous marriage in a Belgian family and identified the mutation that is probably the cause of the CA II deficiency in that family. The change is a C-to-T transition which results in the substitution of Tyr (TAT) for His (CAT) at position 107. This histidine is invariant in all amniotic CA isozymes sequenced to date, as well as the CAs from elasmobranch and algal sources and in a viral CA-related protein. His-107 appears to have a stabilizing function in the structure of all CA molecules, and its substitution by Tyr apparently disrupts the critical hydrogen bonding of His-107 to two other similarly invariant residues, Glu-117 and Tyr-194, resulting in an unstable CA II molecule. We have also completed the intron-exon structure of the normal human CA II gene, which has allowed us to prepare PCR primers for all exons. These primers will facilitate the determination of the mutations in other inherited CA II deficiencies. Images Figure 2 Figure 3 Figure 4 PMID:1928091

  10. Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter

    SciTech Connect

    Kwon, Deug-Nam; Park, Mi-Ryung; Park, Jong-Yi; Cho, Ssang-Goo; Park, Chankyu [Department of Animal Biotechnology, Konkuk University, Seoul 143-701 (Korea, Republic of)] [Department of Animal Biotechnology, Konkuk University, Seoul 143-701 (Korea, Republic of); Oh, Jae-Wook [Division of Animal Life Science, College of Animal Bioscience and Technology, Konkuk University, Seoul 143-701 (Korea, Republic of)] [Division of Animal Life Science, College of Animal Bioscience and Technology, Konkuk University, Seoul 143-701 (Korea, Republic of); Song, Hyuk [Department of Animal Science, College of Natural Science, Konkuk University, Chung-ju 380-701 (Korea, Republic of)] [Department of Animal Science, College of Natural Science, Konkuk University, Chung-ju 380-701 (Korea, Republic of); Kim, Jae-Hwan [College of Life Science, CHA University, Seongnam, Gyeonggi-do 463-836 (Korea, Republic of)] [College of Life Science, CHA University, Seongnam, Gyeonggi-do 463-836 (Korea, Republic of); Kim, Jin-Hoi, E-mail: jhkim541@konkuk.ac.kr [Department of Animal Biotechnology, Konkuk University, Seoul 143-701 (Korea, Republic of)] [Department of Animal Biotechnology, Konkuk University, Seoul 143-701 (Korea, Republic of)

    2011-07-01

    Highlights: {yields} The sequences of -604 to -84 bp of the pUPII promoter contained the region of a putative negative cis-regulatory element. {yields} The core promoter was located in the 5F-1. {yields} Transcription factor HNF4 can directly bind in the pUPII core promoter region, which plays a critical role in controlling promoter activity. {yields} These features of the pUPII promoter are fundamental to development of a target-specific vector. -- Abstract: Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells. We also identified a new core promoter region and putative negative cis-regulatory element within a minimal promoter region. In addition, we showed that hepatocyte nuclear factor 4 (HNF4) can directly bind in the pUPII core promoter (5F-1) region, which plays a critical role in controlling promoter activity. Transient cotransfection experiments showed that HNF4 positively regulates pUPII gene promoter activity. Thus, the binding element and its binding protein, HNF4 transcription factor, may be involved in the mechanism that specifically regulates pUPII gene transcription.

  11. Mediator, TATA-binding protein, and RNA polymerase II contribute to low histone occupancy at active gene promoters in yeast.

    PubMed

    Ansari, Suraiya A; Paul, Emily; Sommer, Sebastian; Lieleg, Corinna; He, Qiye; Daly, Alexandre Z; Rode, Kara A; Barber, Wesley T; Ellis, Laura C; LaPorta, Erika; Orzechowski, Amanda M; Taylor, Emily; Reeb, Tanner; Wong, Jason; Korber, Philipp; Morse, Randall H

    2014-05-23

    Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction. PMID:24727477

  12. Multiple Sites of Type II Site Ligand (Luteolin and BMHPC) Regulation of Gene Expression in PC-3 Cells

    PubMed Central

    Markaverich, Barry M.; Vijjeswarapu, Mary

    2012-01-01

    Type II [3H]estradiol binding site ligands including luteolin (a naturally occurring bioflavonoid) and synthetic compounds such as 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)cyclohexanone (BMHPC) inhibit normal and malignant prostate cell (PC-3, LNCaP, DU-145) proliferation in vitro and in vivo. Type II sites represent a binding domain on histone H4 possibly involved in an epigenetic mechanism for controlling gene transcription. Treatment of PC-3 human prostate cancer cells with luteolin or BMHPC modulated the expression of a number of genes in the epidermal growth factor receptor signaling pathway (EGFRSP) and cell cycle pathway (CCP). Pronounced stimulation (400-2000% of control) of c-FOS and p21 RNA expression was observed, suggesting that these were primary sites of action. Both compounds also caused irreversible G2/M arrest (p<0.001). siRNA’s for c-FOS or p21 reduced the RNA expression of their respective targets by 85-95%, with minimal effects on cell proliferation. Furthermore, neither siRNA alone (single knockdown), or in combination (double knockdown), blocked luteolin or BMHPC inhibition of PC-3 cell proliferation. Thus, although c-FOS and p21 are known to modulate the expression of genes in the ESGRSP (EGFR, SOS, GRB2, JNK1, MKK4, RasGAP) and CCP (CCNA2, CCNE2, CDC25A, CDKN1A, CDKN1B, p27, PLK1) involved in the regulation of cell proliferation by luteolin and BMHPC, the c-FOS and p21 siRNA knockdown studies reported here suggest that c-FOS and p21 may be secondary bystanders in the overall response to these ligands in the regulation of PC-3 cell proliferation. PMID:23675277

  13. Stable integration and expression of wasabi defensin gene in "Egusi" melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot.

    PubMed

    Ntui, Valentine Otang; Thirukkumaran, Gunaratnam; Azadi, Pejman; Khan, Raham Sher; Nakamura, Ikuo; Mii, Masahiro

    2010-09-01

    Production of "Egusi" melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer "Egusi" resistant to these diseases, cotyledonary explants of two "Egusi" genotypes, 'Ejagham' and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mg l(-l) kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and 'Ejagham', respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype 'Ejagham' were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1-5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants. PMID:20552202

  14. Cotransformation and differential expression of introduced genes into potato (Solanum tuberosum L.) cv Bintje.

    PubMed

    Ottaviani, M P; Hänisch Ten Cate, C H

    1991-06-01

    The Dutch potato cultivar Bintje has been transformed by Agrobacterium strain LBA1060KG, which contains two plasmids carrying three different DNAs (TL- and TR-DNA on the Agrobacterium rhizogenes plasmid and TKG-DNA on the pBI121 plasmid). Several transformed root clones were obtained after transformation of leaf, stem, and tuber segments, and plants were then regenerated from these root clones. The expression of the various marker genes [rol, opine, ?-glucuronidase (GUS), and neomycin phosphotransferase (NPTII)] was determined in several root clones and in regenerated plants. The selection of vigorously growing root clones was as efficient as selection for kanamycin resistance. In spite of the location of NPTII and GUS genes on the same T-DNA, 17% of the root clones did not show GUS activity. Nevertheless, Southern blot analysis showed that these root clones contained at least three copies of the GUS gene. Sixty-four per cent of the root clones contained opines. The expression of these genes, however, was negatively correlated with plant regeneration capacity and normal plant development. The differential expression of the marker genes in the transgenic potato tissues is discussed. PMID:24221438

  15. A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes

    PubMed Central

    Diesch, Jeannine; Lesmana, Analia; Poortinga, Gretchen; Hein, Nadine; Lidgerwood, Grace; Cameron, Donald P.; Ellul, Jason; Goodall, Gregory J.; Wong, Lee H.; Dhillon, Amardeep S.; Hamdane, Nourdine; Rothblum, Lawrence I.; Pearson, Richard B.; Haviv, Izhak; Moss, Tom

    2015-01-01

    Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity). PMID:25452314

  16. Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus).

    PubMed

    Gasper, J S; Shiina, T; Inoko, H; Edwards, S V

    2001-07-01

    Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences. PMID:11472064

  17. The genes encoding the type II gonadotropin-releasing hormone receptor and the ribonucleoprotein RBM8A in humans overlap in two genomic loci.

    PubMed

    Faurholm, B; Millar, R P; Katz, A A

    2001-11-01

    We have cloned and characterized two genomic loci encoding the human type II gonadotropin-releasing hormone (GnRH) receptor and RNA-binding motif protein-8 (RBM8A). In both loci the genes overlap and are in antisense orientation to each other. The locus on chromosome 1 encompasses the type II GnRH receptor gene (GNRHR2), which is composed of three exons. We found transcripts from this gene in a wide range of tissues, but they lacked a methionine initiation codon and had a stop codon in exon 2. In the antisense orientation, this locus contains RBM8A, which consists of six exons and directs the synthesis of an RBM8A protein of 173 or 174 amino acids by alternative splicing. A second locus on chromosome 14 contains pseudogenes of RBM8A and the type II GnRH receptor and probably originated from the chromosome 1 locus by retrotransposition. PMID:11707068

  18. Topoisomerase II? binding protein 1 c.*229C>T (rs115160714) gene polymorphism and endometrial cancer risk.

    PubMed

    Forma, Ewa; Wójcik-Krowiranda, Katarzyna; Jó?wiak, Pawe?; Szymczyk, Agnieszka; Bie?kiewicz, Andrzej; Bry?, Magdalena; Krze?lak, Anna

    2014-07-01

    TopBP1 (topoisomerase II? binding protein 1) protein is involved in DNA replication, DNA damage checkpoint response and transcriptional regulation. In this study we investigated whether alterations in the TopBP1 gene can influence the risk of endometrial cancer. We examined the association between five single nucleotide polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 3'UTR region of the TopBP1 gene and endometrial cancer risk as well as allele-specific gene expression. One hundred twenty-one endometrial cancer patients were genotyped for these SNPs. Allele-specific TopBP1 mRNA and protein expressions were determined by real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with endometrial cancer. Compared to homozygous common allele carriers, heterozygous for the T variant had significantly increased risk of endometrial cancer [adjusted odds ratio (OR)?=?5.59, 95 % confidence interval (CI): 1.96-15.91, p?=?0.0003]. Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT genotype. There was a significant association between the rs115160714 and tumor grade and FIGO classification. Most carriers of minor allele had a high grade tumors (G3) classified as FIGO III/IV. The results of our study raise a possibility that a genetic variation of TopBP1 may be implicated in the etiology of endometrial cancer. PMID:24346708

  19. Molecular cloning of cDNA of mammalian and chicken II gonadotropin-releasing hormones (mGnRHs and cGnRH-II) in the beluga ( Huso huso ) and the disruptive effect of methylmercury on gene expression

    Microsoft Academic Search

    Ahmad Gharaei; Fereidoun Mahboudi; Abbas Esmaili-Sari; Rozita Edalat; Ahmad Adeli; Saeed Keyvanshokooh

    2010-01-01

    Two gonadotropin-releasing hormone (GnRH) isoforms were identified in the beluga (Huso huso) brain by cDNA sequencing: prepro-mammalian GnRH (mGnRH) and prepro-chicken GnRH-II (cGnRH-II). The nucleotide sequences\\u000a of the beluga mGnRH and cGnRH-II precursors are 273 and 258 base pairs (bp) long, encoding peptides of 91 and 86 amino acids,\\u000a respectively. To investigate the effect of methylmercury (MeHg) on GnRH gene

  20. Transcriptional up-regulation of antioxidant genes by PPAR{delta} inhibits angiotensin II-induced premature senescence in vascular smooth muscle cells

    SciTech Connect

    Kim, Hyo Jung; Ham, Sun Ah [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of)] [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of); Paek, Kyung Shin [Department of Nursing, Semyung University, Jechon (Korea, Republic of)] [Department of Nursing, Semyung University, Jechon (Korea, Republic of); Hwang, Jung Seok; Jung, Si Young; Kim, Min Young; Jin, Hanna; Kang, Eun Sil; Woo, Im Sun; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of)] [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of); Han, Chang Woo [Department of Internal Medicine, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of)] [Department of Internal Medicine, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of); Seo, Han Geuk, E-mail: hgseo@gnu.ac.kr [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of)

    2011-03-25

    Research highlights: {yields} Activation of PPAR{delta} by GW501516 significantly inhibited Ang II-induced premature senescence in hVSMCs. {yields} Agonist-activated PPAR{delta} suppressed generation of Ang II-triggered ROS with a concomitant reduction in DNA damage. {yields} GW501516 up-regulated expression of antioxidant genes, such as GPx1, Trx1, Mn-SOD and HO-1. {yields} Knock-down of these antioxidant genes abolished the effects of GW501516 on ROS production and premature senescence. -- Abstract: This study evaluated peroxisome proliferator-activated receptor (PPAR) {delta} as a potential target for therapeutic intervention in Ang II-induced senescence in human vascular smooth muscle cells (hVSMCs). Activation of PPAR{delta} by GW501516, a specific agonist of PPAR{delta}, significantly inhibited the Ang II-induced premature senescence of hVSMCs. Agonist-activated PPAR{delta} suppressed the generation of Ang II-triggered reactive oxygen species (ROS) with a concomitant reduction in DNA damage. Notably, GW501516 up-regulated the expression of antioxidant genes, such as glutathione peroxidase 1, thioredoxin 1, manganese superoxide dismutase and heme oxygenase 1. siRNA-mediated down-regulation of these antioxidant genes almost completely abolished the effects of GW501516 on ROS production and premature senescence in hVSMCs treated with Ang II. Taken together, the enhanced transcription of antioxidant genes is responsible for the PPAR{delta}-mediated inhibition of premature senescence through sequestration of ROS in hVSMCs treated with Ang II.

  1. EVIDENCE OF MINOR GENES IN CHICKPEA 'SPANISH WHITE' FOR RESISTANCE TO ASCOCHYTA RABIEI PATHOTYPE II

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND and OBJECTIVES Isolates from the United States of Ascochyta rabiei, the causal agent of chickpea Ascochyta blight, were divided into two pathotypes (I and II). Cultivar ‘Spanish White’ is susceptible to both pathotypes and ‘Dwelley’ is resistant to pathotype I but susceptible to pathotyp...

  2. Jumping gene adaptations of NSGA-II and their use in the multi-objective optimal design of shell and tube heat exchangers

    Microsoft Academic Search

    Aaditya Agarwal; Santosh K. Gupta

    2008-01-01

    Two new jumping gene (JG) adaptations of the binary-coded, elitist non-dominated sorting genetic algorithm, NSGA-II are developed. Three benchmark problems are first solved to compare the performance of these adaptations with the earlier JG adaptations of NSGA-II. Single- and multi-objective optimal design of a shell and tube heat exchanger is then carried out using the new sJG (specific JG) adaptation

  3. Loss of Timp3 Gene Leads to Abdominal Aortic Aneurysm Formation in Response to Angiotensin II*

    PubMed Central

    Basu, Ratnadeep; Fan, Dong; Kandalam, Vijay; Lee, Jiwon; Das, Subhash K.; Wang, Xiuhua; Baldwin, Troy A.; Oudit, Gavin Y.; Kassiri, Zamaneh

    2012-01-01

    Aortic aneurysm is dilation of the aorta primarily due to degradation of the aortic wall extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs), the proteases that degrade the ECM. Timp3 is the only ECM-bound Timp, and its levels are altered in the aorta from patients with abdominal aortic aneurysm (AAA). We investigated the causal role of Timp3 in AAA formation. Infusion of angiotensin II (Ang II) using micro-osmotic (Alzet) pumps in Timp3?/? male mice, but not in wild type control mice, led to adverse remodeling of the abdominal aorta, reduced collagen and elastin proteins but not mRNA, and elevated proteolytic activities, suggesting excess protein degradation within 2 weeks that led to formation of AAA by 4 weeks. Intriguingly, despite early up-regulation of MMP2 in Timp3?/?Ang II aortas, additional deletion of Mmp2 in these mice (Timp3?/?/Mmp2?/?) resulted in exacerbated AAA, compromised survival due to aortic rupture, and inflammation in the abdominal aorta. Reconstitution of WT bone marrow in Timp3?/?/Mmp2?/? mice reduced inflammation and prevented AAA in these animals following Ang II infusion. Treatment with a broad spectrum MMP inhibitor (PD166793) prevented the Ang II-induced AAA in Timp3?/? and Timp3?/?/Mmp2?/? mice. Our study demonstrates that the regulatory function of TIMP3 is critical in preventing adverse vascular remodeling and AAA. Hence, replenishing TIMP3, a physiological inhibitor of a number of metalloproteinases, could serve as a therapeutic approach in limiting AAA development or expansion. PMID:23144462

  4. USF and NF-E2 Cooperate to Regulate the Recruitment and Activity of RNA Polymerase II in the ?-Globin Gene Locus*

    PubMed Central

    Zhou, Zhuo; Li, Xingguo; Deng, Changwang; Ney, Paul A.; Huang, Suming; Bungert, Jörg

    2010-01-01

    The human ?-globin gene is expressed at high levels in erythroid cells and regulated by proximal and distal cis-acting DNA elements, including promoter, enhancer, and a locus control region (LCR). Transcription complexes are recruited not only to the globin gene promoters but also to the LCR. Previous studies have implicated the ubiquitously expressed transcription factor USF and the tissue-restricted activator NF-E2 in the recruitment of transcription complexes to the ?-globin gene locus. Here we demonstrate that although USF is required for the efficient association of RNA polymerase II (Pol II) with immobilized LCR templates, USF and NF-E2 together regulate the association of Pol II with the adult ?-globin gene promoter. Recruitment of Pol II to the LCR occurs in undifferentiated murine erythroleukemia cells, but phosphorylation of LCR-associated Pol II at serine 5 of the C-terminal domain is mediated by erythroid differentiation and requires the activity of NF-E2. Furthermore, we provide evidence showing that USF interacts with NF-E2 in erythroid cells. The data provide mechanistic insight into how ubiquitous and tissue-restricted transcription factors cooperate to regulate the recruitment and activity of transcription complexes in a tissue-specific chromatin domain. PMID:20236933

  5. Genomic Study of RNA Polymerase II and III SNAPc-Bound Promoters Reveals a Gene Transcribed by Both Enzymes and a Broad Use of Common Activators

    PubMed Central

    Praz, Viviane; Michaud, Joëlle; Romascano, David; Hernandez, Nouria

    2012-01-01

    SNAPc is one of a few basal transcription factors used by both RNA polymerase (pol) II and pol III. To define the set of active SNAPc-dependent promoters in human cells, we have localized genome-wide four SNAPc subunits, GTF2B (TFIIB), BRF2, pol II, and pol III. Among some seventy loci occupied by SNAPc and other factors, including pol II snRNA genes, pol III genes with type 3 promoters, and a few un-annotated loci, most are primarily occupied by either pol II and GTF2B, or pol III and BRF2. A notable exception is the RPPH1 gene, which is occupied by significant amounts of both polymerases. We show that the large majority of SNAPc-dependent promoters recruit POU2F1 and/or ZNF143 on their enhancer region, and a subset also recruits GABP, a factor newly implicated in SNAPc-dependent transcription. These activators associate with pol II and III promoters in G1 slightly before the polymerase, and ZNF143 is required for efficient transcription initiation complex assembly. The results characterize a set of genes with unique properties and establish that polymerase specificity is not absolute in vivo. PMID:23166507

  6. Two HlyIIR dimers bind to a long perfect inverted repeat in the operator of the hemolysin II gene from Bacillus cereus.

    PubMed

    Rodikova, Ekaterina A; Kovalevskiy, Oleg V; Mayorov, Sergey G; Budarina, Zhanna I; Marchenkov, Victor V; Melnik, Bogdan S; Leech, Andrew P; Nikitin, Dmitri V; Shlyapnikov, Michael G; Solonin, Alexander S

    2007-03-20

    HlyIIR is a negative transcriptional regulator of hemolysin II gene from B. cereus. It binds to a long DNA perfect inverted repeat (44bp) located upstream the hlyII gene. Here we show that HlyIIR is dimeric in solution and in bacterial cells. No protein-protein interactions between dimers and no significant modification of target DNA conformation upon complex formation were observed. Two HlyIIR dimers were found to bind to native operator independently with Kd level in the nanomolar range. The minimal HlyIIR binding site was identified as a half of the long DNA perfect inverted repeat. PMID:17346714

  7. Ribosomal RNA genes in species of the Cynareae tribe ( Compositae ). II

    Microsoft Academic Search

    F. Maggini; G. F. Tucci; M. T. Gelati

    1988-01-01

    Summary The structure of the ribosomal DNA has been analyzed in three species of theCynareae tribe using Southern blot hybridization.Silybum marianum possesses two types of ribosomal genes 12.3 and 13.4 kb long;Cirsium vulgare has at least four types of rDNA repeats 10.6, 10.5, 11.7 and 11.9 kb long;Carlina acaulis only one type of ribosomal genes 9.7 kb long. The rRNA

  8. Molecular characterization of major histocompatibility complex class II gene expression and demonstration of antigen-specific T cell response indicate a new phenotype in class II-deficient patients

    PubMed Central

    1995-01-01

    Major histocompatibility complex (MHC) class II deficiency is an inherited autosomal recessive combined immunodeficiency. The disease is known as bare lymphocyte syndrome (BLS). BLS is characterized by a lack of constitutive MHC class II expression on macrophages and B cells as well as a lack of induced MHC class II expression on cells other than professional antigen-presenting cells (APCs) due to the absence of mRNA and protein of the human leukocyte antigen (HLA) class II molecules, designated HLA-DR, -DQ, and -DP. The defect in gene expression is located at the transcriptional level and affects all class II genes simultaneously. Here we have analyzed transcription and protein expression of class II antigens in Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines and mononuclear cells (MNCs) of twin brothers. Whereas flow cytometric analysis failed to detect class II antigens on the cell surface of the patients' EBV-B cells and MNCs, examination of the genes coding for HLA-DR, -DQ, -DP, and the invariant chain (Ii) by reverse transcriptase-polymerase chain reaction amplification resulted in an unusual mRNA pattern in the B cell lines of the patients (HLA-DR alpha +, -DR beta, -DQ alpha +, -DQ beta -, -DP alpha -; -DP beta +, Ii+). In accordance with these findings no HLA-DR beta-specific protein was detected by immunoblotting, whereas low levels of HLA-DR alpha and normal levels of Ii were present. In contrast to EBV-B cells, the MNCs of both patients displayed a residual HLA-DR beta, -DQ beta, and -DP alpha mRNA signal. Furthermore, HLA-DR beta-specific protein was found in addition to HLA-DR alpha by immunoblotting of cell lysates, even though it was clearly decreased as compared with controls. Our results indicate that the defect in class II antigen expression is not necessarily present to the same extent in B cells and cells of other lineages. mRNA levels of HLA-DR beta were found to be enriched in adherent cells within the MNC fraction. Further investigations indicated that the MHC class II expressed is functional in antigen presentation, as the two boys' CD4+ T cells became activated and expressed interleukin-2R after stimulation of peripheral blood mononuclear cell cultures with recall antigen (tetanus toxoid). Furthermore, T cells tested in one of the two patients responded to both MHC class I and II allostimulation, and this response was inhibited by monoclonal antibodies of the respective specificity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7699327

  9. Giant panda genomic data provide insight into the birth-and-death process of mammalian major histocompatibility complex class II genes.

    PubMed

    Wan, Qiu-Hong; Zeng, Chang-Jun; Ni, Xiao-Wei; Pan, Hui-Juan; Fang, Sheng-Guo

    2009-01-01

    To gain an understanding of the genomic structure and evolutionary history of the giant panda major histocompatibility complex (MHC) genes, we determined a 636,503-bp nucleotide sequence spanning the MHC class II region. Analysis revealed that the MHC class II region from this rare species contained 26 loci (17 predicted to be expressed), of which 10 are classical class II genes (1 DRA, 2 DRB, 2 DQA, 3 DQB, 1 DYB, 1 DPA, and 2 DPB) and 4 are non-classical class II genes (1 DOA, 1 DOB, 1 DMA, and 1 DMB). The presence of DYB, a gene specific to ruminants, prompted a comparison of the giant panda class II sequence with those of humans, cats, dogs, cattle, pigs, and mice. The results indicated that birth and death events within the DQ and DRB-DY regions led to major lineage differences, with absence of these regions in the cat and in humans and mice respectively. The phylogenetic trees constructed using all expressed alpha and beta genes from marsupials and placental mammals showed that: (1) because marsupials carry loci corresponding to DR, DP, DO and DM genes, those subregions most likely developed before the divergence of marsupials and placental mammals, approximately 150 million years ago (MYA); (2) conversely, the DQ and DY regions must have evolved later, but before the radiation of placental mammals (100 MYA). As a result, the typical genomic structure of MHC class II genes for the giant panda is similar to that of the other placental mammals and corresponds to BTNL2 approximately DR1 approximately DQ approximately DR2 approximately DY approximately DO_box approximately DP approximately COL11A2. Over the past 100 million years, there has been birth and death of mammalian DR, DQ, DY, and DP genes, an evolutionary process that has brought about the current species-specific genomic structure of the MHC class II region. Furthermore, facing certain similar pathogens, mammals have adopted intra-subregion (DR and DQ) and inter-subregion (between DQ and DP) convergent evolutionary strategies for their alpha and beta genes, respectively. PMID:19127303

  10. Non-neutral evolution and reciprocal monophyly of two expressed Mhc class II B genes in Leach's storm-petrel.

    PubMed

    Dearborn, Donald C; Gager, Andrea B; Gilmour, Morgan E; McArthur, Andrew G; Hinerfeld, Douglas A; Mauck, Robert A

    2015-02-01

    The major histocompatibility complex (Mhc) is subject to pathogen-mediated balancing selection and can link natural selection with mate choice. We characterized two Mhc class II B loci in Leach's storm-petrel, Oceanodroma leucorhoa, focusing on exon 2 which encodes the portion of the protein that binds pathogen peptides. We amplified and sequenced exon 2 with locus-specific nested PCR and Illumina MiSeq using individually barcoded primers. Repeat genotyping of 78 single-locus genotypes produced identical results in 77 cases (98.7%). Sequencing of messenger RNA (mRNA) from three birds confirmed expression of both loci, consistent with the observed absence of stop codons or frameshifts in all alleles. In 48 birds, we found 9 and 12 alleles at the two loci, respectively, and all 21 alleles translated to unique amino acid sequences. Unlike many studies of duplicated Mhc genes, alleles of the two loci clustered into monophyletic groups. Consistent with this phylogenetic result, interlocus gene conversion appears to have affected only two short fragments of the exon. As predicted under a paradigm of pathogen-mediated selection, comparison of synonymous and non-synonymous substitution rates found evidence of a history of positive selection at putative peptide binding sites. Overall, the results suggest that the gene duplication event leading to these two loci is not recent and that point mutations and positive selection on the peptide binding sites may be the predominant forces acting on these genes. Characterization of these loci sets the stage for population-level work on the evolutionary ecology of Mhc in this species. PMID:25416539

  11. Novel mutations in the long isoform of the USH2A gene in patients with Usher syndrome type II or non-syndromic retinitis pigmentosa

    Microsoft Academic Search

    Terri L McGee; Babak Jian Seyedahmadi; Meredith O Sweeney; Thaddeus P Dryja; Eliot L Berson

    2010-01-01

    BackgroundUsher syndrome type II (USH2) is an autosomal recessive disorder characterised by retinitis pigmentosa (RP) and mild to moderate sensorineural hearing loss. Mutations in the USH2A gene are the most common cause of USH2 and are also a cause of some forms of RP without hearing loss (ie, non-syndromic RP). The USH2A gene was initially identified as a transcript comprised

  12. A Transforming GrowthFactor @J ReceptorTypeII GeneMutationCommonin Colon and Gastric but Rare in Endometrial Cancers with Microsatellite Instability1

    Microsoft Academic Search

    Lois L. Myeroff; Ramon Parsons; Seong-Jin Kim; Lora Hedrick; Kathleen R. Cho; Kim Orth; Michael Mathis; Kenneth W. Kinzler; James Lutterbaugh; Yung-Jue Bang; Henry T. Lynch; Anita B. Roberts; Sanford D. Markowitz

    We have recently demonstrated that mutation of the transforming growth factor43 (TGF-@) receptor type II (RI!) gene is characteristic of colon cancers exhibiting microsatellite instability or replication errors (RER+). Moreover, we have shown that RI! mutations in these RER+ colon cancers are characteristicallyframeshiftmutationswithin a 1O.bp polyadenine repeat present in the RJJ.coding region. We now show that RI! gene mutations in

  13. Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445

    Microsoft Academic Search

    Jean-Frederic Dubern; Eric R. Coppoolse; Willem J. Stiekema; Guido V. Bloemberg

    2008-01-01

    Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene cluster (pso) and its surrounding region were isolated, sequenced and characterized. Three genes, termed psoA, psoB and psoC, were identified and

  14. Selective gamma-chain T-cell receptor gene rearrangements in a patient with Omenn's syndrome: absence of V-II subgroup (V gamma 9) transcripts.

    PubMed Central

    Mathioudakis, G; Good, R A; Chernajovsky, Y; Day, N K; Platsoucas, C D

    1996-01-01

    Only gamma-chain T-cell receptor transcripts utilizing V-1 subgroup gene segments were found in peripheral blood lymphocytes from a patient with Omenn's syndrome. gamma-Chain T-cell receptor transcripts utilizing the V gamma 9 (V-II subgroup) gene segment were absent in peripheral blood lymphocytes from this patient. V gamma 9 J gamma 1.2 C gamma 1 rearrangements are those primarily found in peripheral blood lymphocytes (70 to 85%) from normal donors. PMID:8877149

  15. Molecular phylogeny of Fusarium inferred from partial RNA polymerase II gene sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Currently there are no robust phylogenetic hypotheses for Fusarium based on large-scale sampling across the breadth of this important group of mycotoxigenic phytopathogens. Nucleotide variation within the second largest RNA polymerase subunit (RPB2) protein-coding gene, however, has clearly demonst...

  16. SIX MAJOR CLADES OF AGARICALES INFERRED FROM PNA POLYMERASE II AND NUCLEAR RIBOSOMAL RNA GENES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many taxonomic families of agarics are not monophyletic and require re-evaluation by molecular phylogenetic methods. Using over 5600 nucleotide characters from rpb1, rpb1-intron 2, rpb2 and 18S, 25S, and 5.8S ribosomal RNA genes, we recover six major clades of Agaricales with Bayesian and parsimony ...

  17. Dissection of immune gene networks in primary melanoma tumors critical for antitumor surveillance of patients with stage II-III resectable disease.

    PubMed

    Sivendran, Shanthi; Chang, Rui; Pham, Lisa; Phelps, Robert G; Harcharik, Sara T; Hall, Lawrence D; Bernardo, Sebastian G; Moskalenko, Marina M; Sivendran, Meera; Fu, Yichun; de Moll, Ellen H; Pan, Michael; Moon, Jee Young; Arora, Sonali; Cohain, Ariella; DiFeo, Analisa; Ferringer, Tammie C; Tismenetsky, Mikhail; Tsui, Cindy L; Friedlander, Philip A; Parides, Michael K; Banchereau, Jacques; Chaussabel, Damien; Lebwohl, Mark G; Wolchok, Jedd D; Bhardwaj, Nina; Burakoff, Steven J; Oh, William K; Palucka, Karolina; Merad, Miriam; Schadt, Eric E; Saenger, Yvonne M

    2014-08-01

    Patients with resected stage II-III cutaneous melanomas remain at high risk for metastasis and death. Biomarker development has been limited by the challenge of isolating high-quality RNA for transcriptome-wide profiling from formalin-fixed and paraffin-embedded (FFPE) primary tumor specimens. Using NanoString technology, RNA from 40 stage II-III FFPE primary melanomas was analyzed and a 53-immune-gene panel predictive of non-progression (area under the curve (AUC)=0.920) was defined. The signature predicted disease-specific survival (DSS P<0.001) and recurrence-free survival (RFS P<0.001). CD2, the most differentially expressed gene in the training set, also predicted non-progression (P<0.001). Using publicly available microarray data from 46 primary human melanomas (GSE15605), a coexpression module enriched for the 53-gene panel was then identified using unbiased methods. A Bayesian network of signaling pathways based on this data identified driver genes. Finally, the proposed 53-gene panel was confirmed in an independent test population of 48 patients (AUC=0.787). The gene signature was an independent predictor of non-progression (P<0.001), RFS (P<0.001), and DSS (P=0.024) in the test population. The identified driver genes are potential therapeutic targets, and the 53-gene panel should be tested for clinical application using a larger data set annotated on the basis of prospectively gathered data. PMID:24522433

  18. Analysis of DNA flanking the treA gene of Bacillus subtilis reveals genes encoding a putative specific enzyme II Tre and a potential regulator of the trehalose operon

    Microsoft Academic Search

    Frieder Schöck; Michael K. Dahl

    1996-01-01

    Nucleotide sequencing revealed the genes treP encoding a putative specific enzyme IITre upstream from treA and treR encoding a potential regulator downstream from treA of Bacillus subtilis 168. The treP gene encodes a 470-amino acid (aa) protein (50 kDa) showing high similarities to several different specific enzymes II of phosphoenolpyruvate-dependent phosphotransferase systems. treR encodes a 238-aa protein (28 kDa) with

  19. Cloning of the cDNA Encoding the Urotensin II Precursor in Frog and Human Reveals Intense Expression of the Urotensin II Gene in Motoneurons of the Spinal Cord

    Microsoft Academic Search

    Yolaine Coulouarn; Isabelle Lihrmann; Sylvie Jegou; Youssef Anouar; Her Ve Tostivint; Jean Claude Beauvillain; J. Michael Conlon; Howard A. Bern; Hubert Vaudry

    1998-01-01

    Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish. Subsequently, UII has been characterized from a frog brain extract, indicating that a gene encoding a UII precursor is also present in the genome of a tetrapod. Here, we report the characterization of the cDNAs encoding frog and human UII precursors and the

  20. Epigenetic modification of DRG neuronal gene expression subsequent to nerve injury: etiological contribution to complex regional pain syndromes (Part II).

    PubMed

    Wang, Fuzhou; Stefano, George B; Kream, Richard M

    2014-01-01

    Cumulating evidence indicated that nerve injury-associated cellular and molecular changes play an essential role in contributing to the development of pathological pain, and more recent findings implicated the critical role of epigenetic mechanisms in pain-related sensitization in the DRG subsequent to nerve injury. In this part of the dyad review (Part II), we reviewed and paid special attention on the etiological contribution of DGR gene expression modulated by epigenetic mechanisms of CRPS. As essential effectors to different molecular activation, we first discussed the activation of various signaling pathways that subsequently from nerve injury, and in further illustrated the fundamental and functional underpinnings of nerve injury-induced pain, in which we argued for the potential epigenetic mechanisms in response to sensitizing stimuli or injury. Therefore, understanding the specific mediating factors that influence individual epigenetic differences contributing to pain sensitivity and responsiveness to analgesics possesses crucial clinical implications. PMID:25027291

  1. Epigenetic modification of DRG neuronal gene expression subsequent to nerve injury: Etiological contribution to complex regional pain syndromes (Part II)

    PubMed Central

    Wang, Fuzhou; Stefano, George B.; Kream, Richard M.

    2014-01-01

    Cumulating evidence indicated that nerve injury-associated cellular and molecular changes play an essential role in contributing to the development of pathological pain, and more recent findings implicated the critical role of epigenetic mechanisms in pain-related sensitization in the DRG subsequent to nerve injury. In this part of the dyad review (Part II), we reviewed and paid special attention on the etiological contribution of DGR gene expression modulated by epigenetic mechanisms of CRPS. As essential effectors to different molecular activation, we first discussed the activation of various signaling pathways that subsequently from nerve injury, and in further illustrated the fundamental and functional underpinnings of nerve injury-induced pain, in which we argued for the potential epigenetic mechanisms in response to sensitizing stimuli or injury. Therefore, understanding the specific mediating factors that influence individual epigenetic differences contributing to pain sensitivity and responsiveness to analgesics possesses crucial clinical implications. PMID:25027291

  2. Suppression of the {alpha}-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death

    SciTech Connect

    Kang, Shinhae [Technology Innovation Center, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Song, Jihoon [Department of Life Science, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Kang, Jihoon [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Kang, Heekyoung [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Lee, Daeho [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Lee, Youngki [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of); Park, Deokbae [Department of Medicine, Cheju National University, Jeju, Jeju 690-756 (Korea, Republic of)]. E-mail: parkdb@cheju.ac.kr

    2005-04-01

    Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms ({alpha}, {beta}, {gamma}). Although the {alpha}-isoform (PI3K-C2{alpha}) is considered ubiquitous and preferentially activated by insulin rather than the {beta}-isoform, the physiological significance of PI3K-C2{alpha} is poorly understood. The present study aimed to determine whether PI3K-C2{alpha} is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2{alpha} gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2{alpha} whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2{alpha} gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2{alpha} mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2{alpha} mRNA levels. This finding strongly suggests that PI3K-C2{alpha} is a crucial survival factor.

  3. The effect of a nuclear localization sequence on transfection efficacy of genes delivered by cobalt(II)-polybenzimidazole complexes.

    PubMed

    Yin, Jun; Meng, Xianggao; Zhang, Shibing; Zhang, Dan; Wang, Li; Liu, Changlin

    2012-11-01

    We have demonstrated that the metal complexes of polybenzimidazoles are emerging likely as a new type of gene-delivery systems based on their strong DNA-condensing ability. However, the in vitro transfection efficacy of the DNA condensates formed with the metal complexes was relatively low. The positively charged peptides, such as cell-penetrating peptides and nuclear localization sequences (NLSs), have been reported to be capable of enhancing expression of the transgenes, likely as they promote entrance of their electrostatic complexes with DNA into the nuclear through nuclear pores. Here, we explored expression of the genes transferred by a series of Co(II) complexes in the presence of NLS (PKKKRKV) in normal and cancer cell lines. The results showed that the Co(II) complexes lead to the more pronounced DNA condensation in the presence of NLS than that in the absence of NLS. The binding of NLS prior to addition of the Co complexes can significantly reduce both the size and the population of the condensates at the given Co complexes/DNA ratios, compared with the NLS-free condensates. Meanwhile, the binding of NLS can considerably increase surface positive charges on the DNA nanoparticles. The suitable sizes and high surface positive charges facilitate the entrance of the nanoparticles into cells. Luciferase activity assay indicated that the transfection efficacy of the NLS-bound condensates was five-fold of that of the NLS-free ones in different cell lines, and comparable to that of the condensate formed with the commercially available carrier PEI. Moreover, cell viability assay of the NLS-bound condensates showed lower cytotoxicity than the NLS-free ones. Thus, the combination of NLS and cationic metal complexes might offer a new type of ternary delivery systems. PMID:22840232

  4. Spread of recombinant DNA by roots and pollen of transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp.

    PubMed

    de Vries, Johann; Heine, Martin; Harms, Klaus; Wackernagel, Wilfried

    2003-08-01

    Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII'; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII' and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment. PMID:12902229

  5. Gene expression of herpes simplex virus. II. Uv radiological analysis of viral transcription units

    SciTech Connect

    Millette, R. L.; Klaiber, R.

    1980-06-01

    The transcriptional organization of the genome of herpes simplex virus type 1 was analyzed by measuring the sensitivity of viral polypeptide synthesis to uv irradiation of the infecting virus. Herpes simplex virus type 1 was irradiated with various doses of uv light and used to infect xeroderma pigmentosum fibroblasts. Immediate early transcription units were analyzed by having cycloheximide present throughout the period of infection, removing the drug at 8 h postinfection, and pulse-labeling proteins with (355)methionine. Delayed early transcription units were analyzed in similar studies by having 9-beta-D-arabinofuranosyladenine present during the experiment to block replication of the input irradiated genome. The results indicate that none of the immediate early genes analyzed can be cotranscribed, whereas some of the delayed early genes might be cotranscribed. No evidence was found for the existence of large, multigene transcription units.

  6. Resistance to wilt in chickpea. II. Further evidence for two genes for resistance to race 1

    Microsoft Academic Search

    H. D. Upadhyaya; J. B. Smithson; M. P. Haware; J. Kumar

    1983-01-01

    Tests of parents and F1, F2 and F3 generations of crosses of JG-62 (early-rilting) and C-104 (late-wilting) with resistant cultivars provide further evidence that resistance in chickpea (Cicer arietinum L.) to Race 1 of Fusarium oxysporum f.sp. ciceris is controlled by at least two genes, both of which must be present in homozygous recessive form for complete resistance. Singly, one

  7. 894. A Novel Mechanism between Type II Diabetes Mellitus and Procalcitonin Gene Expression

    Microsoft Academic Search

    Mehment Ali Soylemez; Oktay Seyment; Gunnur Yigit

    2005-01-01

    Objective: Procalcitonin (PCT) was originally described in 1984 as a 116-aminoacid protein with a molecular weight of 14,5 kDa. The PCT gene, referred to as Calc-1, is located on chromosome 11p15.4 and was sequenced in 1989. The promoter has sites for basal transcription factors but more interestingly, also has sites for NF?? (Nuclear factor ??) and AP-1(Activator protein-1), factors induced

  8. Patterns of evolution of MHC class II genes of crows (Corvus) suggest trans-species polymorphism

    PubMed Central

    Townsend, Andrea K.; Sepil, Irem; Nishiumi, Isao; Satta, Yoko

    2015-01-01

    A distinguishing characteristic of genes that code for the major histocompatibility complex (MHC) is that alleles often share more similarity between, rather than within species. There are two likely mechanisms that can explain this pattern: convergent evolution and trans-species polymorphism (TSP), in which ancient allelic lineages are maintained by balancing selection and retained by descendant species. Distinguishing between these two mechanisms has major implications in how we view adaptation of immune genes. In this study we analyzed exon 2 of the MHC class IIB in three passerine bird species in the genus Corvus: jungle crows (Corvus macrorhynchos japonensis) American crows (C. brachyrhynchos) and carrion crows (C. corone orientalis). Carrion crows and American crows are recently diverged, but allopatric, sister species, whereas carrion crows and jungle crows are more distantly related but sympatric species, and possibly share pathogens linked to MHC IIB polymorphisms. These patterns of evolutionary divergence and current geographic ranges enabled us to test for trans-species polymorphism and convergent evolution of the MHC IIB in crows. Phylogenetic reconstructions of MHC IIB sequences revealed several well supported interspecific clusters containing all three species, and there was no biased clustering of variants among the sympatric carrion crows and jungle crows. The topologies of phylogenetic trees constructed from putatively selected sites were remarkably different than those constructed from putatively neutral sites. In addition, trees constructed using non-synonymous substitutions from a continuous fragment of exon 2 had more, and generally more inclusive, supported interspecific MHC IIB variant clusters than those constructed from the same fragment using synonymous substitutions. These phylogenetic patterns suggest that recombination, especially gene conversion, has partially erased the signal of allelic ancestry in these species. While clustering of positively selected amino acids by supertyping revealed a single supertype shared by only jungle and carrion crows, a pattern consistent with convergence, the overall phylogenetic patterns we observed suggest that TSP, rather than convergence, explains the interspecific allelic similarity of MHC IIB genes in these species of crows. PMID:25802816

  9. Patterns of evolution of MHC class II genes of crows (Corvus) suggest trans-species polymorphism.

    PubMed

    Eimes, John A; Townsend, Andrea K; Sepil, Irem; Nishiumi, Isao; Satta, Yoko

    2015-01-01

    A distinguishing characteristic of genes that code for the major histocompatibility complex (MHC) is that alleles often share more similarity between, rather than within species. There are two likely mechanisms that can explain this pattern: convergent evolution and trans-species polymorphism (TSP), in which ancient allelic lineages are maintained by balancing selection and retained by descendant species. Distinguishing between these two mechanisms has major implications in how we view adaptation of immune genes. In this study we analyzed exon 2 of the MHC class IIB in three passerine bird species in the genus Corvus: jungle crows (Corvus macrorhynchos japonensis) American crows (C. brachyrhynchos) and carrion crows (C. corone orientalis). Carrion crows and American crows are recently diverged, but allopatric, sister species, whereas carrion crows and jungle crows are more distantly related but sympatric species, and possibly share pathogens linked to MHC IIB polymorphisms. These patterns of evolutionary divergence and current geographic ranges enabled us to test for trans-species polymorphism and convergent evolution of the MHC IIB in crows. Phylogenetic reconstructions of MHC IIB sequences revealed several well supported interspecific clusters containing all three species, and there was no biased clustering of variants among the sympatric carrion crows and jungle crows. The topologies of phylogenetic trees constructed from putatively selected sites were remarkably different than those constructed from putatively neutral sites. In addition, trees constructed using non-synonymous substitutions from a continuous fragment of exon 2 had more, and generally more inclusive, supported interspecific MHC IIB variant clusters than those constructed from the same fragment using synonymous substitutions. These phylogenetic patterns suggest that recombination, especially gene conversion, has partially erased the signal of allelic ancestry in these species. While clustering of positively selected amino acids by supertyping revealed a single supertype shared by only jungle and carrion crows, a pattern consistent with convergence, the overall phylogenetic patterns we observed suggest that TSP, rather than convergence, explains the interspecific allelic similarity of MHC IIB genes in these species of crows. PMID:25802816

  10. The Glucocorticoid Receptor Type II Complex is a Target of the HIV1 vpr Gene Product

    Microsoft Academic Search

    Yosef Refaeli; David N. Levy; David B. Weiner

    1995-01-01

    The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 15-kDa virion-associated protein that functions as a regulator of cellular processes linked to the HIV life cycle. We report the interaction of a 41-kDa cytosolic viral protein R interacting protein 1 (Rip-1) with Vpr in vitro. Rip-1 displays a wide tissue distribution, including relevant targets of HIV infection.

  11. p13 from group II baculoviruses is a killing-associated gene

    PubMed Central

    Lu, Nan; Du, Enqi; Liu, Yangkun; Qiao, Hong; Yao, Lunguang; Pan, Zishu; Lu, Songya; Qi, Yipeng

    2012-01-01

    p13 gene was first described in Leucania separata multinuclear polyhedrosis virus (Ls-p13) several years ago, but the function of P13 protein has not been experimentally investigated to date. In this article, we indicated that the expression of p13 from Heliothis armigera single nucleocapsid nucleopolyhedrovirus (Ha-p13) was regulated by both early and late promoter. Luciferase assay demonstrated that the activity of Ha-p13 promoter with hr4 enhancer was more than 100 times in heterologous Sf9 cells than that in nature host Hz-AM1 cells. Both Ls-P13 and Ha-P13 are transmembrane proteins. Confocal microscopic analysis showed that both mainly located in the cytoplasm membrane at 48 h. Results of RNA interference indicated that Ha-p13 was a killing-associated gene for host insects H. armigera. The AcMNPV acquired the mentioned killing activity and markedly accelerate the killing rate when expressing Ls-p13. In conclusion, p13 is a killing associated gene in both homologous and heterologous nucleopolyhedrovirus. [BMB Reports 2012; 45(12): 730-735] PMID:23261060

  12. Mutations in the HLA class II genes leading to loss of expression of HLA-DR and HLA-DQ in diffuse large B-cell lymphoma.

    PubMed

    Jordanova, Ekaterina S; Philippo, Katja; Giphart, Marius J; Schuuring, Ed; Kluin, Philip M

    2003-07-01

    Loss of expression of human leukocyte antigen (HLA) class II molecules on tumor cells affects the onset and modulation of the immune response through lack of activation of CD4+ T lymphocytes. Previously, we showed that the frequent loss of expression of HLA class II in diffuse large B-cell lymphoma (DLBCL) of the testis and the central nervous system (CNS) is mainly due to homozygous deletions in the HLA region on chromosome band 6p21.3. A minority of cases showed hemizygous deletions or mitotic recombination, implying that mutation of the remaining copy of the class II genes might be involved. Here, we studied three DLBCLs with loss of HLA-DQ expression for mutations in the DQB1 and DQA1 genes and three tumors with loss of HLA-DR expression for mutations in the DRB1 and DRA genes. In one case, a point mutation in exon 2 of the DQB1 gene, leading to the formation of a stop codon, was detected at position 47. In a second case, a stop codon was found at position 11 due to a deletion of 19 bp in exon 1 of the DRA gene. No mutations were found in the promoter sequences of the DRA, DQA1 and DQB1 genes. We conclude that both homozygous deletions and hemizygous deletions or mitotic recombination with mutations of the remaining allele may lead to loss of expression of the HLA class II genes, which is comparable to the mechanisms affecting HLA class I expression in solid cancers. PMID:12756506

  13. Analysis of HLA class II genes in Hashimoto's thyroiditis reveals differences compared to Graves’ disease

    Microsoft Academic Search

    A A Zeitlin; J M Heward; P R Newby; J D Carr-Smith; J A Franklyn; S C L Gough; M J Simmonds

    2008-01-01

    Graves’ disease (GD) and Hashimoto's thyroiditis (HT) represent the commonest forms of autoimmune thyroid disease (AITD) each presenting with distinct clinical features. Progress has been made in determining association of HLA class II DRB1, DQB1 and DQA1 loci with GD demonstrating a predisposing effect for DR3 (DRB1*03-DQB1*02-DQA1*05) and a protective effect for DR7 (DRB1*07-DQB1*02-DQA1*02). Small data sets have hindered progress

  14. A Novel Frameshift Mutation of the USH2A Gene in a Korean Patient with Usher Syndrome Type II.

    PubMed

    Boo, Sung Hyun; Song, Min-Jung; Kim, Hee-Jin; Cho, Yang-Sun; Chu, Hosuk; Ko, Moon-Hee; Chung, Won-Ho; Kim, Jong-Won; Hong, Sung Hwa

    2013-03-01

    Usher syndrome type II (USH2) is the most common form of Usher syndrome, characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa. It has been shown that mutations in the USH2A gene are responsible for USH2. The authors herein describe a 34-year-old Korean woman with the typical clinical manifestation of USH2; she had bilateral hearing disturbance and progressive visual deterioration, without vestibular dysfunction. Molecular genetic study of the USH2A gene revealed a novel frameshift mutation (c.2310delA; Glu771LysfsX17). She was heterozygous for this mutation, and no other mutation was found in USH2A, suggesting the possibility of an intronic or large genomic rearrangement mutation. To the best of our knowledge, this is the first report of a genetically confirmed case of USH2 in Korea. More investigations are needed to delineate genotype-phenotype correlations and ethnicity-specific genetic background of Usher syndrome. PMID:23526569

  15. Mammary gland-specific hypomethylation of Hpa II sites flanking the bovine alpha S1-casein gene.

    PubMed

    Platenburg, G J; Vollebregt, E J; Karatzas, C N; Kootwijk, E P; De Boer, H A; Strijker, R

    1996-11-01

    In the lactating cow, mammary gland-specific hypomethylation occurs at two Hpa II sites in the 5'-flanking region of the alpha S1-casein gene, and one in the 3'-region. These sites, A, B and C, are at nucleotide position -1388, -774 and +18034, respectively, relative to the major transcription start site. Site B was hypomethylated when the alpha S1-casein gene was expressed, and methylated when not expressed. In transgenic mice containing the bovine alpha S1-casein 5' and 3' regulatory elements fused to the human lactoferrin (hLF) cDNA, in some cases similar methylation patterns of sites A and B, as compared to the situation in the cow, were observed. In five mouse lines (out of the seven analysed) expressing the transgene in the milk, site B was hypomethylated in the mammary gland, while it was methylated in liver. In the two other mouse lines, no correlation was found between transgene expression and mammary gland-specific hypomethylation of site B. One of the five mouse lines with transgene expression and showing mammary-gland-specific hypomethylation of site B was studied in detail. In this mouse line, induction of transgene expression preceded hypomethylation of site B. PMID:8840525

  16. Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters

    PubMed Central

    Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak; Ilgu, Muslum; Beasley, Jonathan; Bendickson, Lee; Mehanovic, Samir; Kraus, George A.; Nilsen-Hamilton, Marit

    2014-01-01

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time. PMID:24753407

  17. Resemblance and Dissemblance of Arabidopsis Type II Peroxiredoxins: Similar Sequences for Divergent Gene Expression, Protein Localization, and Activity1

    PubMed Central

    Bréhélin, Claire; Meyer, Etienne H.; de Souris, Jean-Paul; Bonnard, Géraldine; Meyer, Yves

    2003-01-01

    The Arabidopsis type II peroxiredoxin (PRXII) family is composed of six different genes, five of which are expressed. On the basis of the nucleotide and protein sequences, we were able to define three subgroups among the PRXII family. The first subgroup is composed of AtPRXII-B, -C, and -D, which are highly similar and localized in the cytosol. AtPRXII-B is ubiquitously expressed. More striking is the specific expression of AtPRXII-C and AtPRXII-D localized in pollen. The second subgroup comprises the mitochondrial AtPRXII-F, the corresponding gene of which is expressed constitutively. We show that AtPRXII-E, belonging to the last subgroup, is expressed mostly in reproductive tissues and that its product is addressed to the plastid. By in vitro enzymatic experiments, we demonstrate that glutaredoxin is the electron donor of recombinant AtPRXII-B for peroxidase reaction, but the donors of AtPRXII-E and AtPRXII-F have still to be identified. PMID:12913160

  18. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    SciTech Connect

    Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  19. Gene expression in the DpnI and DpnII restriction enzyme systems of Streptococcus pneumoniae

    SciTech Connect

    Lacks, S.A.; Sabelnikov, A.G.; Chen, Jau-Der; Greenberg, B.

    1992-12-31

    Although a number of bacterial species are naturally transformable, that is, their cells are able to take up external DNA in substantial amounts and integrate it into the chromosome without artificial manipulation of the cell surface, Streptococcus pneumoniae, the first species in which this phenomenon was detected, remains a prototype of such transformation. Cells of S. pneumonias also contain potent restriction endonucleases able to severely restrict DNA introduced during viral infection. Our current understanding of the genetic basis of the complementary DpnI and DpnII restriction systems and of the biochemistry of their component enzymes are briefly reviewed. The manner in which these enzymes impinge on the transfer of chromosomal genes and of plasmeds will be examined in detail. It will be seen that far from acting against foreign DNA in general, the restriction systems seem to be designed to exclude only infecting viral DNA The presence of complementary restriction systems in different cells of S. pneumonias enhances their effectiveness in blocking viral infection and promoting species survival. This enhanced effectiveness requires the expression of alternative restriction systems. Therefore, the ability of the cells to transfer the restriction enzyme genes and to regulate their expression are important for survival of the species.

  20. Atrazine resistance in the grass Poa annua is due to a single base change in the chloroplast gene for the D1 protein of photosystem II

    Microsoft Academic Search

    M. D. C. Barros; T. A. Dyer

    1988-01-01

    We report here the first molecular characterization of maternally inherited atrazine resistance in a monocot. As has been found in dicots, resistance in the grass Poa annua is correlated with a decrease in the ability of herbicides to bind to thylakoids and with an alteration in the gene for the D1 protein of photosystem II which would result in a

  1. Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia

    SciTech Connect

    Vandenberg, P.; Khillan, J.S.; Prockop, D.J.; Helminen, H.; Kontusaari, S.; Ala-Kokko, L. (Thomas Jefferson Univ., Philadelphia, PA (United States))

    1991-09-01

    A minigene version of the human gene for type II procollagen (COL2AI) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro{alpha} chains that associate with normal pro{alpha} chains and thereby cause degradation of the shortened and normal pro{alpha} chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing the minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro{alpha}1(II) chains that were disulfide-linked to normal mouse pro{alpha}1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.

  2. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22

    SciTech Connect

    Tsai-Pflugfelder, M.; Liu, L.F.; Liu, A.A.; Tewey, K.M.; Whang-Peng, J.; Knutsen, T.; Huebner, K.; Croce, C.M.; Wang, J.C. (Harvard Univ., Cambridge, MA (USA))

    1988-10-01

    Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage {lambda} was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a {lambda}gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.

  3. Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts

    PubMed Central

    1991-01-01

    The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo. PMID:1918153

  4. Genes

    NSDL National Science Digital Library

    BEGIN:VCARD VERSION:2.1 FN:Access Excellence N:Excellence; Access REV:2005-03-12 END:VCARD

    2005-03-12

    Illustration of the placement of genes in a chromosome. A gene can be defined as a region of DNA that controls a hereditary characteristic. It usually corresponds to a sequence used in the production of a specific protein or RNA. A gene carries biological information in a form that must be copied and transmitted from each cell to all its progeny. This includes the entire functional unit: coding DNA sequences, non-coding regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as several hundred thousand base pairs. It can even be carried by more than one chromosome. The estimate for the number of genes in humans has decreased as our knowledge has increased. As of 2001, humans are thought to have between 30,000 and 40,000 genes.

  5. Alteration of Topoisomerase II–Alpha Gene in Human Breast Cancer: Association With Responsiveness to Anthracycline-Based Chemotherapy

    PubMed Central

    Press, Michael F.; Sauter, Guido; Buyse, Marc; Bernstein, Leslie; Guzman, Roberta; Santiago, Angela; Villalobos, Ivonne E.; Eiermann, Wolfgang; Pienkowski, Tadeusz; Martin, Miguel; Robert, Nicholas; Crown, John; Bee, Valerie; Taupin, Henry; Flom, Kerry J.; Tabah-Fisch, Isabelle; Pauletti, Giovanni; Lindsay, Mary-Ann; Riva, Alessandro; Slamon, Dennis J.

    2011-01-01

    Purpose Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A) gene encoding an enzyme that is a major target of anthracyclines. This study was designed to evaluate whether TOP2A gene alterations may predict incremental responsiveness to anthracyclines in some breast cancers. Methods A total of 4,943 breast cancers were analyzed for alterations in TOP2A and HER2. Primary tumor tissues from patients with metastatic breast cancer treated in a trial of chemotherapy plus/minus trastuzumab were studied for amplification/deletion of TOP2A and HER2 as a test set followed by evaluation of malignancies from two separate, large trials for changes in these same genes as a validation set. Association between these alterations and clinical outcomes was determined. Results Test set cases containing HER2 amplification treated with doxorubicin and cyclophosphamide (AC) plus trastuzumab, demonstrated longer progression-free survival compared to those treated with AC alone (P = .0002). However, patients treated with AC alone whose tumors contain HER2/TOP2A coamplification experienced a similar improvement in survival (P = .004). Conversely, for patients treated with paclitaxel, HER2/TOP2A coamplification was not associated with improved outcomes. These observations were confirmed in a larger validation set, where HER2/TOP2A coamplification was again associated with longer survival when only anthracycline-containing chemotherapy was used for treatment compared with outcome in HER2-positive cancers lacking TOP2A coamplification. Conclusion In a study involving nearly 5,000 breast malignancies, both test set and validation set demonstrate that TOP2A coamplification, not HER2 amplification, is the clinically useful predictive marker of an incremental response to anthracycline-based chemotherapy. Absence of HER2/TOP2A coamplification may indicate a more restricted efficacy advantage for breast cancers than previously thought. PMID:21189395

  6. Comparative genomic analysis reveals independent expansion of a lineage-specific gene family in vertebrates: The class II cytokine receptors and their ligands in mammals and fish

    PubMed Central

    Lutfalla, Georges; Crollius, Hugues Roest; Stange-thomann, Nicole; Jaillon, Olivier; Mogensen, Knud; Monneron, Danièle

    2003-01-01

    Background The high degree of sequence conservation between coding regions in fish and mammals can be exploited to identify genes in mammalian genomes by comparison with the sequence of similar genes in fish. Conversely, experimentally characterized mammalian genes may be used to annotate fish genomes. However, gene families that escape this principle include the rapidly diverging cytokines that regulate the immune system, and their receptors. A classic example is the class II helical cytokines (HCII) including type I, type II and lambda interferons, IL10 related cytokines (IL10, IL19, IL20, IL22, IL24 and IL26) and their receptors (HCRII). Despite the report of a near complete pufferfish (Takifugu rubripes) genome sequence, these genes remain undescribed in fish. Results We have used an original strategy based both on conserved amino acid sequence and gene structure to identify HCII and HCRII in the genome of another pufferfish, Tetraodon nigroviridis that is amenable to laboratory experiments. The 15 genes that were identified are highly divergent and include a single interferon molecule, three IL10 related cytokines and their potential receptors together with two Tissue Factor (TF). Some of these genes form tandem clusters on the Tetraodon genome. Their expression pattern was determined in different tissues. Most importantly, Tetraodon interferon was identified and we show that the recombinant protein can induce antiviral MX gene expression in Tetraodon primary kidney cells. Similar results were obtained in Zebrafish which has 7 MX genes. Conclusion We propose a scheme for the evolution of HCII and their receptors during the radiation of bony vertebrates and suggest that the diversification that played an important role in the fine-tuning of the ancestral mechanism for host defense against infections probably followed different pathways in amniotes and fish. PMID:12869211

  7. Role of protein kinase C and tyrosine kinase activity in IFN-gamma-induced expression of the class II MHC gene.

    PubMed

    Lee, Y J; Panek, R B; Huston, M; Benveniste, E N

    1995-01-01

    Astrocytes are induced by interferon-gamma (IFN-gamma) to express class II major histocompatibility complex (MHC) antigens. Our previous studies demonstrated that IFN-gamma-initiated signaling events important for class II expression include activation of protein kinase C (PKC) and the Na+/H+ antiporter. We have extended these studies and found that protein tyrosine kinase (PTK) activity is also required for class II expression. Treatment of astrocytes with inhibitors specific for PKC and PTK blocked INF-gamma-induced class II gene transcription, mRNA expression, and protein expression. Immunoblotting and immunoprecipitation experiments demonstrated that IFN-gamma induced tyrosine phosphorylation of the p91 component of ISGF3, which is blocked by preincubation of cells with PTK inhibitors. Treatment of astrocytes with IFN-gamma and either PKC and PTK inhibitors changed the mobility and intensity of a nuclear factor, IFN-gamma-enhanced factor X, which binds to the X box of the class II MHC promoter. Taken together, these data provide evidence that activation of both PTK and PKC is required for IFN-gamma-induced expression of the class II gene. PMID:7840140

  8. Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma). Genetic linkage to chromosome 12q in the region of the type II keratin gene cluster.

    PubMed Central

    Pulkkinen, L; Christiano, A M; Knowlton, R G; Uitto, J

    1993-01-01

    Epidermolytic hyperkeratosis (EHK) is an autosomal dominant genodermatosis characterized by hyperkeratosis and blistering of the skin. Histopathology demonstrates suprabasilar blister formation with aggregation of tonofilaments. In this study, we tested the hypothesis that the EHK phenotype is linked to one of the suprabasilar keratins (KRT10 or KRT1) present in the types I and II keratin gene clusters in chromosomes 17q and 12q, respectively. For this purpose, Southern hybridizations were performed with DNA from a large kindred with EHK, consisting of 11 affected individuals in three generations. Segregation analysis with markers flanking the keratin gene clusters demonstrated linkage (Z = 3.61 at theta = 0) to a locus on 12q, while markers on 17q were excluded. These data implicate KRT1, the type II keratin expressed in suprabasilar keratinocytes, as a candidate gene in this family with EHK. Images PMID:7678607

  9. Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics.

    PubMed

    Tollenaere, C; Bryja, J; Galan, M; Cadet, P; Deter, J; Chaval, Y; Berthier, K; Ribas Salvador, A; Voutilainen, L; Laakkonen, J; Henttonen, H; Cosson, J-F; Charbonnel, N

    2008-09-01

    We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system. PMID:18624885

  10. Tetranuclear ruthenium(ii) complexes with oligo-oxyethylene linkers as one- and two-photon luminescent tracking non-viral gene vectors.

    PubMed

    Qiu, Kangqiang; Yu, Bole; Huang, Huaiyi; Zhang, Pingyu; Ji, Liangnian; Chao, Hui

    2015-04-01

    To prolong the observation time, increase the penetration depth and decrease self-absorption and phototoxicity, two-photon luminescent vectors have emerged as promising tools for tracking gene delivery in living cells. Herein, we report four new tetranuclear Ru(ii) complexes based on oligo-oxyethylene and polybenzimidazole as one- and two- photon luminescent tracking non-viral gene vectors. In such a molecular design, the oligo-oxyethylene, polybenzimidazole and Ru(ii) polypyridyl complexes were expected to render the vectors with increased cell permeability, biocompatibility, proton buffering capacity and one- and two-photon luminescence. Corresponding DNA interaction studies showed that the ability of the complexes to condense DNA decreased with increasing oligo-oxyethylene lengths. Additionally, all complexes protected DNA. The complexes were investigated as one- and two-photon tracking non-viral gene vectors in living cells and showed proper cellular uptake, good luciferase expression and low cytotoxicity. PMID:25785372

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

    PubMed

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

    2008-01-01

    The expression of inulinolytic genes in Aspergillus niger is co-regulated and induced by inulin and sucrose. We have identified a positive acting transcription factor InuR, which is required for the induced expression of inulinolytic genes. InuR is a member of the fungal specific class of transcription factors of the Zn(II)2Cys6 type. Involvement of InuR in inulin and sucrose metabolism was suspected because of the clustering of inuR gene with sucB, which encodes an intracellular invertase with transfructosylation activity and a putative sugar transporter encoding gene (An15g00310). Deletion of the inuR gene resulted in a strain displaying a severe reduction in growth on inulin and sucrose medium. Northern analysis revealed that expression of inulinolytic and sucrolytic genes, e.g., inuE, inuA, sucA, as well as the putative sugar transporter gene (An15g00310) is dependent on InuR. Genome-wide expression analysis revealed, three additional putative sugar transporters encoding genes (An15g04060, An15g03940 and An17g01710), which were strongly induced by sucrose in an InuR dependent way. In silico analysis of the promoter sequences of strongly InuR regulated genes suggests that InuR might bind as dimer to two CGG triplets, which are separated by eight nucleotides. PMID:17917744

  12. The expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats

    SciTech Connect

    Katayama, Seiichi [Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Kamisu, Ibaraki 314-0255 (Japan) and Science of Bioresource Production, United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065 (Japan)]. E-mail: katayama@ankaken.co.jp; Ashizawa, Koji [Laboratory of Animal Reproduction, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192 (Japan); Gohma, Hiroshi [Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Kamisu, Ibaraki 314-0255 (Japan); Fukuhara, Tadahiro [Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Kamisu, Ibaraki 314-0255 (Japan); Narumi, Kazunori [Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Kamisu, Ibaraki 314-0255 (Japan); Tsuzuki, Yasuhiro [Laboratory of Animal Reproduction, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192 (Japan); Tatemoto, Hideki [Department of Bioproduction, Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213 (Japan); Nakada, Tadashi [Department of Bioproduction, Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213 (Japan); Nagai, Kenji [Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Kamisu, Ibaraki 314-0255 (Japan)

    2006-12-15

    The objective of this study was to investigate the effects of estrogen receptor (ER) agonists and an ER antagonist on the expression of Hedgehog genes (Indian hedgehog: Ihh; Desert hedgehog: Dhh) and Hedgehog target genes (Patched 1: Ptc1; glioma-associated oncogene homolog 1: Gli1; chicken ovalbumin upstream promoter transcription factor II: Coup-TfII) in the rat uterus. Immature female rats were administered once with 17{alpha}-ethynyl estradiol (EE, an ER agonist), propyl pyrazole triole (PPT, an ER{alpha}-selective agonist), diarylpropionitrile (DPN, an ER{beta}-selective agonist), or ICI 182,780 (an ER antagonist). Expression of mRNA for Ihh, Dhh, and Ptc1 was dose-dependently downregulated by EE in the uterus of immature rats, mediated by ER as confirmed by coadministration of ICI 182,780. The mRNA expression levels of Ptc1, Gli1, and Coup-TfII were simultaneously downregulated during the period in which the mRNA expression levels of Ihh and Dhh were downregulated in the uterus after administration of EE. PPT downregulated the transcription of Ihh, Dhh, Ptc1, Gli1, and Coup-TfII, indicating that expression of these genes was regulated by the ER{alpha}-dependent pathway. DPN also downregulated the transcription of Ihh and Dhh, although the effect was weaker than that of PPT, indicating that the regulation of uterine Ihh and Dhh transcription was also affected by the ER{beta}-dependent pathway. These results suggest that the expression of Hedgehog genes (Ihh, Dhh) and Hedgehog target genes (Ptc1, Gli1, Coup-TfII) is affected by estrogenic stimuli in the uterus of immature female rats.

  13. Characterization of the dual start motif of a class II holin gene

    E-print Network

    Barenboim, Maxim

    2001-01-01

    . Possible membrane topologies of X and 21 holins. . . . . . . . . . . . Construction of plasmids and A. hy21kan phages. . . . . 30 A. In vitro translation products of P' mRNA. B. Western blot analysis of 8" gene products in membranes. C. Western blot...(SR)kan Plasmids pBR322 pMTB100 pMTB102 pMTB103 supE44 relA I lac /F'proA B tact Zll&MI 5::Tn10 (tet')] l c185 7 hy(QSRRzRz1) A, c1857 hy(QSRRzRzI) ' bor::kan A, c1857 hy(gzt(SR)RzRzl) ' bor::kan amp' tet' IncColE rop p TZ18R:;(SRRzRz1) pMTB100 S '68...

  14. MHC II-? chain gene expression studies define the regional organization of the thymus in the developing bony fish Dicentrarchus labrax (L.).

    PubMed

    Picchietti, S; Abelli, L; Guerra, L; Randelli, E; Proietti Serafini, F; Belardinelli, M C; Buonocore, F; Bernini, C; Fausto, A M; Scapigliati, G

    2015-02-01

    MHC II-? chain gene transcripts were quantified by real-time PCR and localised by in situ hybridization in the developing thymus of the teleost Dicentrarchus labrax, regarding the specialization of the thymic compartments. MHC II-? expression significantly rose when the first lymphoid colonization of the thymus occurred, thereafter increased further when the organ progressively developed cortex and medulla regions. The evolving patterns of MHC II-? expression provided anatomical insights into some mechanisms of thymocyte selection. Among the stromal cells transcribing MHC II-?, scattered cortical epithelial cells appeared likely involved in the positive selection, while those abundant in the cortico-medullary border and medulla in the negative selection. These latter most represent dendritic cells, based on typical localization and phenotype. These findings provide further proofs that efficient mechanisms leading to maturation of naïve T cells are operative in teleosts, strongly reminiscent of the models conserved in more evolved gnathostomes. PMID:25475077

  15. Sugar-mediated transcriptional regulation of the Gap gene system and concerted photosystem II functional modulation in the microalga Scenedesmus vacuolatus.

    PubMed

    Valverde, Federico; Ortega, José M; Losada, Manuel; Serrano, Aurelio

    2005-08-01

    Partial cDNAs corresponding to the GapA, GapC and GapN genes that encode the three different glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) of the green microalga Scenedesmus vacuolatus SAG 211-8b have been cloned and characterized. Northern blot experiments, as well as immunoblots and activity measurements, demonstrate a differential regulation by sugars of the components of the algal Gap gene system. Addition of glucose or other metabolizable sugars to photoautotrophic cultures promoted a drastic repression of the GapA gene and depletion to negligible levels of the corresponding GAPDHA, a chloroplastic protein involved in photosynthetic CO2 assimilation. By contrast, expression of the GapC and GapN genes encoding their cytosolic counterparts involved in glycolysis was enhanced. However, no down-regulation of the GapA gene by glucose took place in the dark, indicating that the observed effect is associated with sugar assimilation in the light. Likewise, glucose promoted in illuminated algal cultures a severe decrease of photosystem II functionality, estimated by O2 evolution activity, thermoluminescence emission and D1 protein level, while again, no effect was observed in the dark. On the basis of the correlation found between photosystem II performance and sugar transcriptional regulation of the GapA gene, a scenario of sugar-mediated regulation of photosynthetic metabolism in microalgae is proposed that will help to explain the so-called glucose bleaching effect in photosynthetic eukaryotes. PMID:15830207

  16. Towards the simplification of MHC typing protocols: targeting classical MHC class II genes in a passerine, the pied flycatcher Ficedula hypoleuca

    PubMed Central

    2010-01-01

    Background Major Histocompatibility Complex (MHC) has drawn the attention of evolutionary biologists due to its importance in crucial biological processes, such as sexual selection and immune response in jawed vertebrates. However, the characterization of classical MHC genes subjected to the effects of natural selection still remains elusive in many vertebrate groups. Here, we have tested the suitability of flanking intron sequences to guide the selective exploration of classical MHC genes driving the co-evolutionary dynamics between pathogens and their passerine (Aves, Order Passeriformes) hosts. Findings Intronic sequences flanking the usually polymorphic exon 2 were isolated from different species using primers sitting on conserved coding regions of MHC class II genes (? chain). Taking the pied flycatcher Ficedula hypoleuca as an example, we demonstrate that careful primer design can evade non-classical MHC gene and pseudogene amplification. At least four polymorphic and expressed loci were co-replicated using a single pair of primers in five non-related individuals (N = 28 alleles). The cross-amplification and preliminary inspection of similar MHC fragments in eight unrelated songbird taxa suggests that similar approaches can also be applied to other species. Conclusions Intron sequences flanking the usually polymorphic exon 2 may assist the specific investigation of classical MHC class II B genes in species characterized by extensive gene duplication and pseudogenization. Importantly, the evasion of non-classical MHC genes with a more specific function and non-functional pseudogenes may accelerate data collection and diminish lab costs. Comprehensive knowledge of gene structure, polymorphism and expression profiles may be useful not only for the selective examination of evolutionarily relevant genes but also to restrict chimera formation by minimizing the number of co-amplifying loci. PMID:20815923

  17. Angiotensin II modulates interleukin-1{beta}-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-{kappa}B crosstalk

    SciTech Connect

    Xu, Shanqin [Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA (United States)] [Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA (United States); Zhi, Hui [Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA (United States)] [Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA (United States); Hou, Xiuyun [Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA (United States)] [Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA (United States); Jiang, Bingbing, E-mail: bjiang1@rics.bwh.harvard.edu [Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA (United States) [Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA (United States); Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA (United States)

    2011-07-08

    Highlights: {yields} We examine how angiotensin II modulates ERK-NF-{kappa}B crosstalk and gene expression. {yields} Angiotensin II suppresses IL-1{beta}-induced prolonged ERK and NF-{kappa}B activation. {yields} ERK-RSK1 signaling is required for IL-1{beta}-induced prolonged NF-{kappa}B activation. {yields} Angiotensin II modulates NF-{kappa}B responsive genes via regulating ERK-NF-{kappa}B crosstalk. {yields} ERK-NF-{kappa}B crosstalk is a novel mechanism regulating inflammatory gene expression. -- Abstract: Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. In cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1{beta}-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-{kappa}B, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1{beta}-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1{beta}, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE{sup -/-}) mice. VCAM-1 and iNOS expression were higher in ApoE{sup -/-} than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE{sup -/-} mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin II can differentially modulate inflammatory gene expression in aortic smooth muscle cells through influencing ERK-NF-{kappa}B crosstalk, which may contribute to angiotensin II-induced inflammatory disorders related to cardiovascular diseases.

  18. Cork Oak Trees (Quercus suber L.).

    PubMed

    Alvarez, Rubén; Toribio, Mariano; Cortizo, Millán; Ordás Fernández, Ricardo-Javier

    2006-01-01

    A transformation system for selected mature Quercus suber L. trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses are inoculated with AGL1 strain harbouring the plasmid pBINUbiGUSint, which carries the nptII and uidA genes. Evidence of stable transgene integration is obtained by polymerase chain reaction for nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos are germinated and successfully transferred to soil. PMID:17033056

  19. TaqMan probe-based real-time PCR assay for detection and discrimination of class I, II, and III tfdA genes in soils treated with phenoxy acid herbicides.

    PubMed

    Baelum, Jacob; Jacobsen, Carsten S

    2009-05-01

    Separate quantification of three classes of tfdA genes was performed using TaqMan quantitative real-time PCR for 13 different soils subsequent to mineralization of three phenoxy acids. Class III tfdA genes were found to be involved in mineralization more often than class I and II tfdA genes. PMID:19251892

  20. Schisandra Chinensis Baillon regulates the gene expression of phase II antioxidant/detoxifying enzymes in hepatic damage induced rats

    PubMed Central

    Jang, Han I; Do, Gyeong-Min; Lee, Hye Min; Ok, Hyang Mok; Shin, Jae-Ho

    2014-01-01

    BACKGROUND/OBJECTIVES This study investigated the antioxidant activities and hepatoprotective effects of Schisandra chinensis Baillon extract (SCE) against tert-butyl hydroperoxide (t-BHP)-induced oxidative hepatic damage in rats. MATERIALS/METHODS Sprague-Dawley (SD) rats were pretreated with SCE (300, 600, and 1,200 mg/kg BW) or saline once daily for 14 consecutive days. On day 14, each animal, except those belonging to the normal control group, were injected with t-BHP (0.8 mmol/kg BW/i.p.), and all of the rats were sacrificed 16 h after t-BHP injection. RESULTS Although no significant differences in AST and ALT levels were observed among the TC and SCE groups, the high-dose SCE group showed a decreasing tendency compared to the TC group. However, erythrocyte SOD activity showed a significant increase in the low-dose SCE group compared with the TC group. On the other hand, no significant differences in hepatic total glutathione (GSH) level, glutathione reductase (GR), and glutathione peroxidase (GSH-Px) activities were observed among the TC and SCE groups. Hepatic histopathological evaluation revealed that pretreatment with SCE resulted in reduced t-BHP-induced incidence of lesions, such as neutrophil infiltration, swelling of liver cells, and necrosis. In particular, treatment with a high dose of SCE resulted in induction of phase II antioxidant/detoxifying enzyme expression, such as glutathione S-transferase (GST) and glutamate-cysteine ligase catalytic subunit (GCLC). CONCLUSIONS Based on these results, we conclude that SCE exerts protective effects against t-BHP induced oxidative hepatic damage through the reduction of neutrophil infiltration, swelling of liver cells, and necrosis. In addition, SCE regulates the gene expression of phase II antioxidant/detoxifying enzymes independent of hepatic antioxidant enzyme activity. PMID:24944771

  1. Absence of MHC class II gene expression in a patient with a single amino acid substitution in the class II transactivator protein CIITA

    Microsoft Academic Search

    Virginia Quan; Michael Towey; Steven Sacks; Adrian P. Kelly

    1999-01-01

    We investigated the underlying genetic defect in an immunodeficient patient who presented with recurrent bacterial infections\\u000a in his late twenties and demonstrated a transcriptional defect in major histocompatibility complex (MHC) class II regulation.\\u000a Transient heterokaryon analysis implicated functional loss of CIITA, the MHC class II transactivator protein, and in support\\u000a of this MHC class II antigen expression was restored by

  2. Molecular analysis of HLA Class I and Class II genes in four indigenous Malaysian populations.

    PubMed

    Jinam, T A; Saitou, N; Edo, J; Mahmood, A; Phipps, M E

    2010-02-01

    This is the first report of high-resolution human leukocyte antigen (HLA) typing in four indigenous groups in Malaysia. A total of 99 normal, healthy participants representing the Negrito (Jehai and Kensiu), Proto-Malay (Temuan) and a native group of Borneo (Bidayuh) were typed for HLA-A, -B, -DRB1 and -DQB1 genes using sequence-based typing. Eleven HLA-A, 26 HLA-B, 16 HLA-DRB1 and 14 HLA-DQB1 alleles were detected, including a new allele, HLA-B*3589 in the Jehai. Highly frequent alleles were A*2407, B*1513, B*1801, DRB1*0901, DRB1*1202, DRB1*1502, DQB1*0303 and DQB1*0502. Principal component analysis based on high-resolution HLA-A, -B and -DRB1 allele frequencies showed close affinities among all four groups, including the Negritos, with other Southeast Asian populations. These results showed the scope of HLA diversity in these indigenous minority groups and may prove beneficial for future disease association, anthropological and forensic studies. PMID:20003135

  3. Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis

    SciTech Connect

    Gonzalez-Gay, M.A.; Zanelli, E.; Krco, C.J. [Mayo Clinic and Mayo Graduate School of Medicine, Rochester, MN (United States)] [and others

    1995-05-01

    Collagen-induced arthritis (CIA) is an animal model of auto immune polyarthritis, sharing similarities with rheumatoid arthritis (RA). Paradoxally, susceptibility to mouse CIA is controlled by the H2A loci (DQ homologous) while RA is linked to HLA.DR genes (H2E homologous). We recently showed that the E{beta}{sup d} molecule prevents CIA development in susceptible H2{sup q} mice. We addressed the question of whether H2Eb polymorphism will influence CIA incidence as HLA.DRB1 polymorphism does in RA. In F{sub 1} mice, only H2Eb{sup d} and H2Eb{sup s} molecules showed protection. Using recombinant B10.RDD (Eb{sup d/b}) mice, we found that CIA protection was mediated by the first domain of the E{beta}{sup d} molecule. Using peptides covering the third hypervariable region of the E{beta} chain, we found a perfect correlation between presentation of E{beta} peptides by the H2A{sup q} molecule and protection on CIA. Therefore, the mechanism by which H2Eb protects against CIA seems to rely on the affinity of E{beta} peptides for the H2A{sup q} molecule. 35 refs., 2 figs., 3 tabs.

  4. (?)-Xanthatin up-regulation of the GADD45? tumor suppressor gene in MDA-MB-231 breast cancer cells: Role of topoisomerase II? inhibition and reactive oxygen species

    PubMed Central

    Takeda, Shuso; Noguchi, Momoko; Matsuo, Kazumasa; Yamaguchi, Yasuhiro; Kudo, Taichi; Nishimura, Hajime; Okamoto, Yoshiko; Amamoto, Toshiaki; Shindo, Mitsuru; Omiecinski, Curtis J.; Aramaki, Hironori

    2014-01-01

    Previously, we reported that (?)-xanthatin, a naturally occurring xanthanolide present in the Cocklebur plant, exhibits potent anti-proliferative effects on human breast cancer cells, accompanied by an induction of the growth arrest and DNA damage-inducible gene 45? (GADD45?), recognized recently as a novel tumor suppressor gene. However, the mechanisms mediating this activation were unknown. Topoisomerase II? (Topo II?) inhibition has been reported to produce a cell death response accompanied by an atypical DNA laddering fragmentation profile, similar to that noted previously for (–)-xanthatin. Therefore we hypothesized that (?)-xanthatin’s GADD45? activation was mediated through the Topo II? pathway. Here, we identify that (?)-xanthatin does function as a catalytic inhibitor of Topo II?, promoting DNA damage. In addition, reactive oxygen species (ROS) were elevated in cells treated with this agent. Mechanistically, it was determined that the induced levels of GADD45? mRNA resulting from (?)-xanthatin exposures were stabilized by coordinately produced ROS, and that the consequent induction of GADD45? mRNA, GADD45? protein and ROS generation were abrogated by co-treatment with N-acetyl-l-cysteine. Taken together, the data support the concept that Topo II? inhibition by (?)-xanthatin is a trigger that stimulates expression of DNA damage-inducible GADD45? mRNA and that concomitantly produced ROS act downstream to further enhance the GADD45? mRNA/GADD45? protein induction process, resulting in breast cancer cell death. PMID:23313378

  5. Expression of type II iodothyronine deiodinase gene in the brain of a tropical spinefoot, Siganus guttatus.

    PubMed

    Wambiji, Nina; Park, Yong-Ju; Kim, Se-Jae; Hur, Sung-Pyo; Takeuchi, Yuki; Takemura, Akihiro

    2011-12-01

    Type II iodothyronine deiodinase (D2) converts 3,5,3',5'-tetraiodothyronine to 3,5,3'-triiodothyronine and is involved in regulating thyroid hormone-dependent processes in various tissues. D2 mRNA expression in the mediobasal hypothalamus is affected by photoperiod, which influences reproductive processes in temperate birds and mammals. We examined whether D2 mRNA is expressed in the hypothalamus (located in the forebrain within the diencephalon area) and whether its abundance is affected by day length, temperature, or food availability in the tropical spinefoot, Siganus guttatus, which is endemic to tropical monsoon areas. The reverse transcription-polymerase chain reaction (RT-PCR) revealed that D2 mRNA is expressed in various brain regions. The abundance of hypothalamic D2 mRNA was higher at 12.00h than at 06.00h or 24.00h. Rearing fish under constant dark conditions resulted in a decrease in D2 mRNA abundance during the subjective night. A single injection of melatonin lowered D2 mRNA abundance within 3h. Collectively, it appears that hypothalamic D2 mRNA abundance is regulated by the circadian system and/or melatonin. No differences in D2 mRNA abundance were observed, when fish were reared at 20, 25, and 30°C. However, food deprivation stimulated D2 mRNA expression during the daytime. These results suggest that photoperiodic and nutritive conditions affect hypothalamic D2 mRNA expression in S. guttatus. PMID:21463701

  6. Cell-Type-Specific Profiling of Gene Expression and Chromatin Binding without Cell Isolation: Assaying RNA Pol II Occupancy in Neural Stem Cells

    PubMed Central

    Southall, Tony D.; Gold, Katrina S.; Egger, Boris; Davidson, Catherine M.; Caygill, Elizabeth E.; Marshall, Owen J.; Brand, Andrea H.

    2013-01-01

    Summary Cell-type-specific transcriptional profiling often requires the isolation of specific cell types from complex tissues. We have developed “TaDa,” a technique that enables cell-specific profiling without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation, or affinity purification. The method is simple, sensitive, highly reproducible, and transferable to any model system. We show that TaDa can be used to identify transcribed genes in a cell-type-specific manner with considerable temporal precision, enabling the identification of differential gene expression between neuroblasts and the neuroepithelial cells from which they derive. We profile the genome-wide binding of RNA polymerase II in these adjacent, clonally related stem cells within intact Drosophila brains. Our data reveal expression of specific metabolic genes in neuroepithelial cells, but not in neuroblasts, and highlight gene regulatory networks that may pattern neural stem cell fates. PMID:23792147

  7. Genetic transformation and gene expression in white pine (pinus strobus)

    SciTech Connect

    Minocha, R.

    1987-10-01

    The objectives of the study were: (1) to develop protocols for transformation of white pine (Pinus strobus) embryonic tissue; and (2) to analyze the regulation of foreign gene expression in Pinus strobus. A number of Agrobacterium tumefaciens strains containing chimeric genes for neomycin phosphotransferase (NPTII for kanamycin resistance) and chloramphenicol acetyl transferase (CAT) under the control of either a constitutive promoter (NOS-nopaline synthase) or light-inducible promoters (RuBisCO small subunit and chlorophyll a/b binding protein) were used. A variety of tissues from white pine seedlings and mature trees was used. The techniques for transformation were modified from those used for tobacco transformation. The results show that white pine tissue from young seedlings is high suitable for transformation by A. tumefaciens. Whereas the normal tissues are very sensitive to kanamycin, transformed callus was quite resistant to this antibiotic.

  8. Nucleotide sequence of a multiple-copy gene for the B protein of photosystem II of a cyanobacterium.

    PubMed

    Mulligan, B; Schultes, N; Chen, L; Bogorad, L

    1984-05-01

    Chloroplast photogene 32 codes for the 32-kilodalton triazine herbicide-binding protein at the B site of electron transport in photosystem II of the photosynthetic apparatus-its product is the B protein and the gene is accordingly designated ps2B here. The cyanobacteria Anacystis nidulans R2, Fremyella diplosiphon, and Nostoc sp. MAC each contain several copies of ps2B. The sequence of one copy of ps2B from Fremyella, ps2B-1, has been determined. The longest open reading frame would code for a protein of 360 amino acids. Although the deduced amino acid sequence of ps2B-1 is highly homologous overall to that of the corresponding spinach protein [Zurawski, G., Bohnert, H. J., Whitfeld, P. R. & Bottomley, W. (1982) Proc. Natl. Acad. Sci. USA 79, 7699-7703] and, excluding neutral substitutions, the homology is 95% for an internal segment of 309 amino acids, there are a number of nonneutral amino acid substitutions. Most of the differences in net charge and polarity occur in the first 20 amino acids at the amino terminus and in the amino acid composition at the carboxyl terminus. The nucleotide sequences are 76% homologous overall. Conserved sequences resembling prokaryotic "-10" and "-35" regions are found at remarkably similar positions in the spinach and F. diplosiphon sequences although the surrounding sequences show only occasional homologies. PMID:16578775

  9. Polycomb Associates Genome-wide with a Specific RNA Polymerase II Variant, and Regulates Metabolic Genes in ESCs

    PubMed Central

    Brookes, Emily; de Santiago, Inês; Hebenstreit, Daniel; Morris, Kelly J.; Carroll, Tom; Xie, Sheila Q.; Stock, Julie K.; Heidemann, Martin; Eick, Dirk; Nozaki, Naohito; Kimura, Hiroshi; Ragoussis, Jiannis; Teichmann, Sarah A.; Pombo, Ana

    2012-01-01

    Summary Polycomb repressor complexes (PRCs) are important chromatin modifiers fundamentally implicated in pluripotency and cancer. Polycomb silencing in embryonic stem cells (ESCs) can be accompanied by active chromatin and primed RNA polymerase II (RNAPII), but the relationship between PRCs and RNAPII remains unclear genome-wide. We mapped PRC repression markers and four RNAPII states in ESCs using ChIP-seq, and found that PRC targets exhibit a range of RNAPII variants. First, developmental PRC targets are bound by unproductive RNAPII (S5p+S7p?S2p?) genome-wide. Sequential ChIP, Ring1B depletion, and genome-wide correlations show that PRCs and RNAPII-S5p physically bind to the same chromatin and functionally synergize. Second, we identify a cohort of genes marked by PRC and elongating RNAPII (S5p+S7p+S2p+); they produce mRNA and protein, and their expression increases upon PRC1 knockdown. We show that this group of PRC targets switches between active and PRC-repressed states within the ESC population, and that many have roles in metabolism. PMID:22305566

  10. The Us9 Gene Product of Pseudorabies Virus, an Alphaherpesvirus, Is a Phosphorylated, Tail-Anchored Type II Membrane Protein

    PubMed Central

    Brideau, A. D.; Banfield, Bruce W.; Enquist, L. W.

    1998-01-01

    The Us9 gene is highly conserved among the alphaherpesviruses sequenced to date, yet its function remains unknown. In this report, we demonstrate that the pseudorabies virus (PRV) Us9 protein is present in infected cell lysates as several phosphorylated polypeptides ranging from 17 to 20 kDa. Synthesis is first detected at 6 h postinfection and is sensitive to the DNA synthesis inhibitor phosphonoacetic acid. Unlike the herpes simplex virus type 1 Us9 homolog, which was reported to be associated with nucleocapsids in the nuclei of infected cells (M. C. Frame, D. J. McGeoch, F. J. Rixon, A. C. Orr, and H. S. Marsden, Virology 150:321–332, 1986), PRV Us9 localizes to the secretory pathway (predominately to the Golgi apparatus) and not to the nucleus. By fusing the enhanced green fluorescent protein (EGFP) reporter molecule to the carboxy terminus of Us9, we demonstrated that Us9 not only is capable of targeting a Us9-EGFP fusion protein to the Golgi compartment but also is able to direct efficient incorporation of such chimeric molecules into infectious viral particles. Moreover, through protease digestion experiments with Us9-EGFP-containing viral particles, we demonstrated that the Us9 protein is inserted into the viral envelope as a type II, tail-anchored membrane protein. PMID:9573219

  11. Ectopic expression of the agouti gene in transgenic mice causes obesity, features of type II diabetes, and yellow fur

    SciTech Connect

    Klebig, M.L.; Woychik, R.P. [Oak Ridge National Laboratory, Oak Ridge, TN (United States); Wilkinson, J.E. [Univ. of Tennessee, Knoxville, TN (United States); Geisler, J.G. [Oak Ridge National Laboratory, Oak Ridge, TN (United States)]|[Univ. of Tennessee, Knoxville, TN (United States)

    1995-05-23

    Mice that carry the lethal yellow (A{sup y}) or viable yellow (A{sup vy}) mutation, two dominant mutations of the agouti (a) gene in mouse chromosome 2, exhibit a phenotype that includes yellow fur, marked obesity, a form of type II diabetes associated with insulin resistance, and an increased susceptibility to tumor development. Molecular analyses of these and several other dominant {open_quotes}obese yellow{close_quotes} a-locus mutations suggested that ectopic expression of the normal agouti protein gives rise to this complex pleiotropic phenotype. We have now tested this hypothesis directly by generating transgenic mice that ectopically express an agouti cDNA clone encoding the normal agouti protein in all tissues examined. Transgenic mice of both sexes have yellow fur, become obese, and develop hyperinsulinemia. In addition, male transgenic mice develop hyperglycemia by 12-20 weeks of age. These results demonstrate conclusively that the ectopic agouti expression is responsible for most, if not all, of the phenotypic traits of the dominant, obese yellow mutants. 42 refs., 5 figs.

  12. Autosomal dominant familial neurohypophyseal diabetes insipidus caused by a mutation in the arginine-vasopressin II gene in four generations of a Korean family

    PubMed Central

    Kim, Myo-Jing; Kim, Young-Eun; Ki, Chang-Seok

    2014-01-01

    Autosomal dominant neurohypophyseal diabetes insipidus is a rare form of central diabetes insipidus that is caused by mutations in the vasopressin-neurophysin II (AVP-NPII) gene. It is characterized by persistent polydipsia and polyuria induced by deficient or absent secretion of arginine vasopressin (AVP). Here we report a case of familial neurohypophyseal diabetes insipidus in four generations of a Korean family, caused by heterozygous missense mutation in exon 2 of the AVP-NPII gene (c.286G>T). This is the first report of such a case in Korea. PMID:25654069

  13. A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells

    SciTech Connect

    Singer-Sam, J.; Dai, A.; Riggs, A.D. (Beckman Research Inst., Duarte, CA (United States)); Goldstein, L.; Gartler, S.M. (Univ. of Washington, Seattle (United States))

    1992-02-15

    Hpa II site H8 is in the CpG-rich 5{prime} untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGK1). It is the only Hpa II site in the CpG island' whose methylation pattern is perfectly correlated with transcriptional silence of this gene. The authors measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. They conclude that site H8 is the unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.

  14. Mutations in the MGAT2 gene controlling complex N-glycan synthesis cause carbohydrate-deficient glycoprotein syndrome type II, an autosomal recessive disease with defective brain development

    SciTech Connect

    Tan, J.; Schachter, H.; Dunn, J. [Univ. of Toronto (Canada)] [and others

    1996-10-01

    Carbohydrate-deficient glycoprotein syndrome (CDGS) type II is a multisystemic congenital disease with severe involvement of the nervous system. Two unrelated CDGS type II patients are shown to have point mutations (one patient having Ser{r_arrow}Phe and the other having His{r_arrow}Arg) in the catalytic domain of the gene MGAT2, encoding UDP-GlcNAc:{alpha}-6-D-mannoside {Beta}-1,2-N-ace-tylglucosaminyltransferase II (GnT II), an enzyme essential for biosynthesis of complex Asn-linked glycans. Both mutations caused both decreased expression of enzyme protein in a baculovirus/insect cell system and inactivation of enzyme activity. Restriction-endonuclease analysis of DNA from 23 blood relatives of one of these patients showed that 13 donors were heterozygotes; the other relatives and 21 unrelated donors were normal homozygotes. All heterozygotes showed a significant reduction (33%-68%) in mononuclear-cell GnT II activity. The data indicate that CDGS type II is an autosomal recessive disease and that complex Asn-linked glycans are essential for normal neurological development. 38 refs., 4 figs., 1 tab.

  15. Songbird Genomics: Analysis of 45 kb Upstream of a Polymorphic Mhc Class II Gene in Red-Winged Blackbirds ( Agelaius phoeniceus)

    Microsoft Academic Search

    Joe S. Gasper; Takashi Shiina; Hidetoshi Inoko; Scott V. Edwards

    2001-01-01

    Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent

  16. Cyclin D1, p16 and retinoblastoma gene product expression as a predictor for prognosis in non-small cell lung cancer at stages I and II

    Microsoft Academic Search

    Mulan Jin; Shoichi Inoue; Tomohiro Umemura; Jun Moriya; Mikio Arakawa; Kazuo Nagashima; Hiroyuki Kato

    2001-01-01

    The association of the immunohistochemical expressions of cyclin D1, p16 and the retinoblastoma gene product (pRB) with the prognoses of 106 patients with non-small cell lung cancer (NSCLC) at stages I and II after a complete resection was investigated. We used antibodies recognizing nuclear and cytoplasmic cyclin D1, p16 and pRB. In 106 tumors, the positive rates of cyclin D1,

  17. Gene polymorphisms of angiotensin II type 1 receptor and angiotensin-converting enzyme in two ethnic groups living in Zhejiang Province, China

    Microsoft Academic Search

    Chunlan Yan; Jinbiao Zhan; Weihong Feng

    2005-01-01

    Keywords: Angiotensin receptor, Angiotensin- converting enzyme, Gene polymorphism, Chinese population Abstract Polymorphisms of ACE insertion\\/deletion (I\\/D) and angiotensin II type 1 receptor (AT1R) 1166A-C have been associated with many diseases, and distributions of their genotypes vary in different races and populations. The aim of this study was to investigate distributions of angiotensin-converting enzyme (ACE) and AT1R genotypes in Han and

  18. Identification of two major histocompatibility (MH) class II A genes and their association to Vibrio anguillarum infection in half-smooth tongue sole ( Cynoglossus semilaevis)

    NASA Astrophysics Data System (ADS)

    Li, Chunmei; Wang, Xubo; Zhang, Quanqi; Wang, Zhigang; Qi, Jie; Yi, Qilin; Liu, Zhipeng; Wang, Yanan; Yu, Haiyang

    2012-03-01

    Major histocompatibility complex class II antigens are important in vertebrate immune system. In the present study, the full cDNA sequence of class II A gene was synthesized by RACE-PCR from half-smooth tongue sole ( Cynoglossus semilaevis), and its open reading frame (ORF) polymorphism was studied. The whole cDNA sequence was 992 bp in length, including the ORF with 717 bp. Twenty-five alleles were identified and clustered into two distinct groups according to the specific nucleotides/ amino acids in specific positions. Eleven alleles belonged to Cyse-DAA while fourteen alleles belonged to Cyse-DBA. Four Cyse-DAA alleles were observed in one individual, and three to five Cyse-DBA alleles were observed in each of the three detected individuals, which indicated that at least two loci existed in each gene. Moreover, in order to study the function of the alleles in resistance to infection, 200 individuals were intraperitoneally injected with Vibrio anguillarum and the first 20 dead individuals and 20 surviving ones were selected for genotype analysis. Fifty-six alleles were identified among the 40 individuals. Twenty-nine alleles belonged to Cyse-DAA and the other 27 alleles belonged to Cyse-DBA. Eighteen alleles were selected for studying their function in resistance to infection. Alleles Cyse-DAA*0201, Cyse-DAA*1101, Cyse-DBA*0401, Cyse-DBA*1102, Cyse-DBA*1801 and Cyse-DBA*2201 were identified only in surviving individuals, while alleles Cyse- DAA*0901, Cyse-DBA*1101 and Cyse-DBA*1401 occurred more frequently in dead individuals. This study confirmed the existence and polymorphism of two class II A genes as well as the relationship between alleles of class II A genes and disease susceptibility/ resistance in half-smooth tongue sole.

  19. Genomics and polymorphism of Agph-DAB1, an Mhc class II B gene in red-winged blackbirds (Agelaius phoeniceus).

    PubMed

    Edwards, S V; Gasper, J; March, M

    1998-03-01

    To further our understanding of the evolution of avian Mhc genes at the genomic level, we screened a cosmid library made from a red-winged blackbird (Agelaius phoeniceus) with a blackbird cDNA probe and subcloned from one of the Mhc-containing cosmids a gene which we designate Agph-DAB1. The structure of the gene is similar to that found for chicken class II B genes, except that the introns are surprisingly large, ranging from 98 to over 600 bp, making this the longest avian class II B gene to date. Using primers targeted toward the introns flanking the peptide-binding region (PBR), we amplified the entirety of the second exon and determined nucleotide sequences of 41 PCR products from eight individual blackbirds. The 10 sequence types found, among which were two probable pseudogene sequences, exhibit the classic hallmarks for evolution of PBRs, namely, an excess of nonsynonymous over synonymous substitutions and evidence of gene conversion events in polymorphic subdomains. Despite these patterns and our use of intron primers, the distribution of sequences among individuals suggests that more than one locus was amplified in most individuals, and the bushlike tree of sequences provides little information as to locus-specific clusters. These results imply a complex history of gene conversion, recent duplication, or possibly, concerted evolution among multiple loci, although Agph-DAB1, the first genomic Mhc sequence from a bird other than chicken, provides important clues in the quest for locus-specific Mhc primers in birds. PMID:9501491

  20. Light-Intensity-Dependent Expression of Lhc Gene Family Encoding Light-Harvesting Chlorophyll-a/b Proteins of Photosystem II in Chlamydomonas reinhardtii1

    PubMed Central

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

    2002-01-01

    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 CO2 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 CO2 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. PMID:12226512

  1. Molecular cloning, transcriptional, and expression analysis of the first cellulase gene ( cbh2), encoding cellobiohydrolase II, from the moderately thermophilic fungus Talaromyces emersonii and structure prediction of the gene product

    Microsoft Academic Search

    P. G. Murray; C. M. Collins; A. Grassick; M. G. Tuohy

    2003-01-01

    A gene (cbh2) encoding cellobiohydrolase II was isolated from the fungus Talaromyces emersonii by rapid amplification of cDNA ends techniques and the equivalent genomic sequence was subsequently cloned. This represents the first report of a key component of the cellulase regulon from this organism. DNA sequencing revealed that cbh2 has an open reading frame of 1377bp, which encodes a putative

  2. Immature transformed rat islet beta-cells differentially express C-peptides derived from the genes coding for insulin I and II as well as a transfected human insulin gene.

    PubMed

    Blume, N; Petersen, J S; Andersen, L C; Kofod, H; Dyrberg, T; Michelsen, B K; Serup, P; Madsen, O D

    1992-02-01

    Synthetic peptides representing unique sequences in rat proinsulin C-peptide I and II were used to generate highly specific antisera, which, when applied on sections of normal rat pancreas, confirm a homogeneous coexpression of the two C-peptides in all islet beta-cells. Insulin gene expression is induced in the transformed heterogeneous rat islet cell clone, NHI-6F, by transient in vivo passage. During this process a transfected human insulin gene is coactivated with the endogenous nonallelic rat insulin I and II genes. Newly established cultures from NHI-6F insulinomas having a high frequency of insulin-producing cells showed highly differential expression at the cellular level of the three proinsulin C-peptide immunoreactivities, as follows: C-peptide I greater than human C-peptide greater than C-peptide II. The fractions of cells expressing human C-peptide and C-peptide II decreased in time and were absent after more than 50 successive passages, while a C-peptide I-producing population was still present. Double-labeling experiments revealed a heterogeneous distribution of the three different C-peptides. Surprisingly, in the early passages a large fraction of cells would express only a single species of proinsulin-C-peptide immunoreactivity but still at high levels. However, rat C-peptide II and human C-peptide were often colocalized, even in later passages. In situ hybridization studies combined with the immunocytochemical data suggest that the differential expression occurs at the level of transcription.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1569972

  3. Association of Angiotensin II Type 1 Receptor (A1166C) Gene Polymorphism and Its Increased Expression in Essential Hypertension: A Case-Control Study

    PubMed Central

    Chandra, Sudhir; Narang, Rajiv; Sreenivas, Vishnubhatla; Bhatia, Jagriti; Saluja, Daman; Srivastava, Kamna

    2014-01-01

    Objectives Hypertension is one of the major cardiovascular diseases. It affects nearly 1.56 billion people worldwide. The present study is about a particular genetic polymorphism (A1166C), gene expression and protein expression of the angiotensin II type I receptor (AT1R) (SNP ID: rs5186) and its association with essential hypertension in a Northern Indian population. Methods We analyzed the A1166C polymorphism and expression of AT1R gene in 250 patients with essential hypertension and 250 normal healthy controls. Results A significant association was found in the AT1R genotypes (AC+CC) with essential hypertension (?2?=?22.48, p?=?0.0001). Individuals with CC genotypes were at 2.4 times higher odds (p?=?0.0001) to develop essential hypertension than individuals with AC and AA genotypes. The statistically significant intergenotypic variation in the systolic blood pressure was found higher in the patients with CC (169.4±36.3 mmHg) as compared to that of AA (143.5±28.1 mmHg) and AC (153.9±30.5 mmHg) genotypes (p?=?0.0001). We found a significant difference in the average delta-CT value (p?=?0.0001) wherein an upregulated gene expression (approximately 16 fold) was observed in case of patients as compared to controls. Furthermore, higher expression of AT1R gene was observed in patients with CC genotype than with AC and AA genotypes. A significant difference (p?=?0.0001) in the protein expression of angiotensin II Type 1 receptor was also observed in the plasma of patients (1.49±0.27) as compared to controls (0.80±0.24). Conclusion Our findings suggest that C allele of A1166C polymorphism in the angiotensin II type 1 receptor gene is associated with essential hypertension and its upregulation could play an important role in essential hypertension. PMID:24992666

  4. Knockdown of mineralocorticoid or angiotensin II type 1 receptor gene expression in the paraventricular nucleus prevents angiotensin II hypertension in rats.

    PubMed

    Chen, Aidong; Huang, Bing S; Wang, Hong-Wei; Ahmad, Monir; Leenen, Frans H H

    2014-08-15

    Circulating Ang II activates an aldosterone-mineralocorticoid receptor (MR) - angiotensin II (Ang II) - angiotensin type 1 receptor (AT1R) pathway in the hypothalamus. To obtain insights into the actual neuronal projections involved, adeno-associated virus carrying small interfering RNA against either AT1aR (AAV-AT1aR-siRNA) or MR (AAV-MR-siRNA) were infused into the paraventricular nucleus (PVN) in Wistar rats. Intra-PVN infusion of AAV-AT1aR-siRNA or AAV-MR-siRNA decreased AT1R or MR expression in the PVN but not in the subfornical organ (SFO) or supraoptic nucleus (SON). Subcutaneous infusion of Ang II at 500 ng kg(-1) min(-1) for 2 weeks increased mean arterial pressure by 60-70 mmHg, and increased AT1R and MR expression in the SFO, SON and PVN. Intra-PVN AT1aR-siRNA prevented the Ang II-induced increase in AT1R but not MR expression in the PVN, and MR-siRNA prevented MR but not AT1R expression in the PVN. The increases in AT1R and MR expression in both the SFO and the SON were not changed by the two AAV-siRNAs. Specific knockdown of AT1R or MR in the PVN by AAV-siRNA each prevented most of the Ang II-induced hypertension. Prevention of the subcutaneous Ang II-induced increase in MR but not the increase in AT1R by knockdown of MR and vice versa suggests an independent regulation of MR and AT1R expression in the PVN. Both AT1R and MR activation in the PVN play a critical role in Ang II-induced hypertension in rats. PMID:24973408

  5. Phase I/II enzyme gene polymorphisms and esophageal cancer risk: A meta-analysis of the literature

    PubMed Central

    Yang, Chun-Xia; Matsuo, Keitaro; Wang, Zhi-Ming; Tajima, Kazuo

    2005-01-01

    AIM: Phase I/II enzymes metabolize environmental carcin-ogens and several functional polymorphisms have been reported in their encoding genes. Although their significance with regard to esophageal carcinogenicity has been examined epidemiologically, it remains controversial. The present systematic review of the literature was performed to clarify associations. METHODS: Eligible studies were case-control or cohort studies published until September 2004 that were written in any language. From PubMed and a manual review of refe-rence lists in relevant review articles, we obtained 16 studies related to the CYP1A1 Ile-Val substitution in exon 7, CYP1A1 MspI polymorphisms, CYP2E1 RsaI polymorphisms, GSTM1 null type, GSTT1 null type and GSTP1 Ile104Val. All were of case-control design. Summary statistics were odds ratios (ORs) comparing heterozygous-, homozygous-non-wild type or these two in combination with the homozygous wild type, or the null type with the non-null type for GSTM1 and GSTT1. A random effect model was used to estimate the summary ORs. A meta-regression analysis was applied to explore sources of heterogeneity. RESULTS: Individuals with the Ile-Val substitution in CYP1A1 exon 7 had increased esophageal cancer risk, with ORs (95%CI) compared with Ile/Ile of 1.37 (1.09-1.71), 2.52 (1.62-3.91) and 1.44 (1.17-1.78) for Ile-Val, Val/Val genotype and the combined group. No significant association was found between esophageal cancer risk and the other genetic parameters. CONCLUSION: A significant association exists between the CYP1A1 Ile-Val polymorphism and risk of esophageal cancer. Polymorphisms that increase the internal exposure to activated carcinogens may increase the risk of esophageal cancer. PMID:15849806

  6. Brassica napus responses to short-term excessive copper treatment with decrease of photosynthetic pigments, differential expression of heavy metal homeostasis genes including activation of gene NRAMP4 involved in photosystem II stabilization.

    PubMed

    Zlobin, I E; Kholodova, V P; Rakhmankulova, Z F; Kuznetsov, Vl V

    2014-11-01

    In the present study, the influence of 50 and 100 µM CuSO4 was investigated starting from 3 h till 72 h treatment of 4-weeks Brassica napus plants. High CuSO4 concentrations in nutrient medium resulted in the rapid copper accumulation in plants, especially in roots, much slower and to lower degree in leaves. Copper excess induced early decrease in the leaf water content and temporary leaf wilting. The decrease in content of photosynthetic pigments became significant to 24 h of excessive copper treatments and reached 35 % decrease to 72 h, but there were no significant changes in maximum quantum efficiency of photosystem II photochemistry. The copper excess affected the expression of ten genes involved in heavy metal homeostasis and copper detoxification. The results showed the differential and organ-specific expression of most genes. The potential roles of copper-activated genes encoding heavy metal transporters (ZIP5, NRAMP4, YSL2, and MRP1), metallothioneins (MT1a and MT2b), low-molecular chelator synthesis enzymes (PCS1 and NAS2), and metallochaperones (CCS and HIPP06) in heavy metal homeostasis and copper ion detoxification were discussed. The highest increase in gene expression was shown for NRAMP4 in leaves in spite of relatively moderate Cu accumulation there. The opinion was advanced that the NRAMP4 activation can be considered among the early reactions in the defense of the photosystem II against copper excess. PMID:25361533

  7. Biologic Determinants of Tumor Recurrence in Stage II Colon Cancer: Validation Study of the 12-Gene Recurrence Score in Cancer and Leukemia Group B (CALGB) 9581

    PubMed Central

    Venook, Alan P.; Niedzwiecki, Donna; Lopatin, Margarita; Ye, Xing; Lee, Mark; Friedman, Paula N.; Frankel, Wendy; Clark-Langone, Kim; Millward, Carl; Shak, Steven; Goldberg, Richard M.; Mahmoud, Najjia N.; Warren, Robert S.; Schilsky, Richard L.; Bertagnolli, Monica M.

    2013-01-01

    Purpose A greater understanding of the biology of tumor recurrence should improve adjuvant treatment decision making. We conducted a validation study of the 12-gene recurrence score (RS), a quantitative assay integrating stromal response and cell cycle gene expression, in tumor specimens from patients enrolled onto Cancer and Leukemia Group B (CALGB) 9581. Patients and Methods CALGB 9581 randomly assigned 1,713 patients with stage II colon cancer to treatment with edrecolomab or observation and found no survival difference. The analysis reported here included all patients with available tissue and recurrence (n = 162) and a random (approximately 1:3) selection of nonrecurring patients. RS was assessed in 690 formalin-fixed paraffin-embedded tumor samples with quantitative reverse transcriptase polymerase chain reaction by using prespecified genes and a previously validated algorithm. Association of RS and recurrence was analyzed by weighted Cox proportional hazards regression. Results Continuous RS was significantly associated with risk of recurrence (P = .013) as was mismatch repair (MMR) gene deficiency (P = .044). In multivariate analyses, RS was the strongest predictor of recurrence (P = .004), independent of T stage, MMR, number of nodes examined, grade, and lymphovascular invasion. In T3 MMR-intact (MMR-I) patients, prespecified low and high RS groups had average 5-year recurrence risks of 13% (95% CI, 10% to 16%) and 21% (95% CI, 16% to 26%), respectively. Conclusion The 12-gene RS predicts recurrence in stage II colon cancer in CALGB 9581. This is consistent with the importance of stromal response and cell cycle gene expression in colon tumor recurrence. RS appears to be most discerning for patients with T3 MMR-I tumors, although markers such as grade and lymphovascular invasion did not add value in this subset of patients. PMID:23530100

  8. HIV associated dementia and HIV encephalitis II: Genes on chromosome 22 expressed in individually microdissected Globus pallidus neurons (Preliminary analysis).

    PubMed

    Shapshak, Paul; Duncan, Robert; Kangueane, Pandajarasamme; Somboonwit, Charurut; Sinnott, John; Commins, Deborah; Singer, Elyse; Levine, Andrew

    2011-01-01

    We analyzed RNA gene expression in neurons from 16 cases in four categories, HIV associated dementia with HIV encephalitis (HAD/HIVE), HAD alone, HIVE alone, and HIV-1-positive (HIV+)with neither HAD nor HIVE. We produced the neurons by laser capture microdissection (LCM) from cryopreserved globus pallidus. Of 55,000 gene fragments analyzed, expression of 197 genes was identified with significance (p = 0.005).We examined each gene for its position in the human genome and found a non-stochastic occurrence for only seven genes, on chromosome 22. Six of the seven genes were identified, CSNK1E (casein kinase 1 epsilon), DGCR8 (Di George syndrome critical region 8), GGA1 (Golgi associated gamma adaptin ear containing ARF binding protein 1), MAPK11 (mitogen activated protein kinase 11), SMCR7L (Smith-Magenis syndrome chromosome region candidate 7-like), andTBC1D22A (TBC1 domain family member 22A). Six genes (CSNK1E, DGCR8, GGA1, MAPK11, SMCR7L, and one unidentified gene) had similar expression profiles across HAD/HIVE, HAD, and HIVE vs. HIV+ whereas one gene (TBC1D22A) had a differing gene expression profile across these patient categories. There are several mental disease-related genes including miRNAs on chromosome 22 and two of the genes (DGCR8 and SMCR7L) identified here are mental disease-related. We speculate that dysregulation of gene expression may occur through mechanisms involving chromatin damage and remodeling. We conclude that the pathogenesis of NeuroAIDS involves dysregulation of expression of mental disease-related genes on chromosome 22 as well as additional genes on other chromosomes. The involvement of these genes as well as miRNA requires additional investigation since numerous genes appear to be involved. PMID:21738310

  9. Terminal Oxidase Diversity and Function in “Metallosphaera yellowstonensis”: Gene Expression and Protein Modeling Suggest Mechanisms of Fe(II) Oxidation in the Sulfolobales? †

    PubMed Central

    Kozubal, M. A.; Dlaki?, M.; Macur, R. E.; Inskeep, W. P.

    2011-01-01

    “Metallosphaera yellowstonensis” is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowstonensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEFGHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b558/566, several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that the fox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance of fox genes (e.g., foxA and foxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores. PMID:21239558

  10. Phase I/II and Phase II Studies of Targeted Gene Delivery In Vivo: Intravenous Rexin-G for Chemotherapy-resistant Sarcoma and Osteosarcoma

    PubMed Central

    Chawla, Sant P; Chua, Victoria S; Fernandez, Lita; Quon, Doris; Saralou, Andreh; Blackwelder, William C; Hall, Frederick L; Gordon, Erlinda M

    2009-01-01

    Rexin-G, a pathotropic nanoparticle bearing a cytocidal cyclin G1 construct was tested in a phase I/II study for chemotherapy-resistant sarcomas and a phase II study for chemotherapy-resistant osteosarcoma. Twenty sarcoma patients and 22 osteosarcoma patients received escalating doses of Rexin-G intravenously from 8 × 1011 to 24 × 1011 colony forming units (cfu)/cycle. Treatment was continued if there was ? grade 1 toxicity. No dose-limiting toxicity (DLT) was observed, and no vector DNA integration, replication-competent retrovirus (RCR) or vector-neutralizing antibodies were noted. In the phase I/II study, 3/6 patients had stable disease (SD) at the lowest dose; median progression-free survival (PFS) was 1.2 months, and overall survival (OS), 3.3 months. At higher doses, 10/14 patients had SD; median PFS was 3.7 months and median OS, 7.8 months. In this phase I/II study, a dose–response relationship with Rexin-G dosage was observed for progression-free and OS times (P = 0.02 and 0.005, respectively). In the phase II study, 10/17 evaluable patients had SD, median PFS was ?3 months and median OS, 6.9 months. These studies suggest that Rexin-G is safe, may help control tumor growth, and may possibly improve survival in chemotherapy-resistant sarcoma and osteosarcoma. PMID:19532136

  11. Phase I/II and phase II studies of targeted gene delivery in vivo: intravenous Rexin-G for chemotherapy-resistant sarcoma and osteosarcoma.

    PubMed

    Chawla, Sant P; Chua, Victoria S; Fernandez, Lita; Quon, Doris; Saralou, Andreh; Blackwelder, William C; Hall, Frederick L; Gordon, Erlinda M

    2009-09-01

    Rexin-G, a pathotropic nanoparticle bearing a cytocidal cyclin G1 construct was tested in a phase I/II study for chemotherapy-resistant sarcomas and a phase II study for chemotherapy-resistant osteosarcoma. Twenty sarcoma patients and 22 osteosarcoma patients received escalating doses of Rexin-G intravenously from 8 x 10(11) to 24 x 10(11) colony forming units (cfu)/cycle. Treatment was continued if there was II study, 3/6 patients had stable disease (SD) at the lowest dose; median progression-free survival (PFS) was 1.2 months, and overall survival (OS), 3.3 months. At higher doses, 10/14 patients had SD; median PFS was 3.7 months and median OS, 7.8 months. In this phase I/II study, a dose-response relationship with Rexin-G dosage was observed for progression-free and OS times (P = 0.02 and 0.005, respectively). In the phase II study, 10/17 evaluable patients had SD, median PFS was >or=3 months and median OS, 6.9 months. These studies suggest that Rexin-G is safe, may help control tumor growth, and may possibly improve survival in chemotherapy-resistant sarcoma and osteosarcoma. PMID:19532136

  12. A novel point mutation in the translation initiation codon of the pre-pro-vasopressin-neurophysin II gene: Cosegregation with morphological abnormalities and clinical symptoms in autosomal dominant neurohypophyseal diabetes insipidus

    Microsoft Academic Search

    J. Rutishauser; M. Boeni-Schnetzler; E. R. Froesch; W. Wichmann; T. Huisman

    1996-01-01

    Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a rare variant of idiopathic central diabetes insipidus. Several different mutations in the human vasopressin-neurophysin II (AVP-NP II) gene have been described. We studied nine family members from three generations of an ADNDI pedigree at the clinical, morphological, and molecular levels. AVP concentrations were measured during diagnostic fluid restriction tests. Coronal and sagittal

  13. Natural selection coupled with intragenic recombination shapes diversity patterns in the major histocompatibility complex class II genes of the giant panda.

    PubMed

    Chen, Yi-Yan; Zhang, Ying-Ying; Zhang, He-Min; Ge, Yun-Fa; Wan, Qiu-Hong; Fang, Sheng-Guo

    2010-05-15

    Ample variations of the major histocompatibility complex (MHC) genes are essential for vertebrates to adapt to various environmental conditions. In this study, we investigated the genetic variations and evolutionary patterns of seven functional MHC class II genes (one DRA, two DRB, two DQA, and two DQB) of the giant panda. The results showed the presence of two monomorphic loci (DRA and DQB2) and five polymorphic loci with different numbers of alleles (seven at DRB1, six at DRB3, seven at DQA1, four at DQA2, six at DQB1). The presence of balancing selection in the giant panda was supported by the following pieces of evidence: (1) The observed heterozygosity was higher than expected. (2) Amino acid heterozygosity was significantly higher at antigen-binding sites (ABS) compared with non-ABS sequences. (3) The selection parameter omega (d(N)/d(S)) was significantly higher at ABS compared with non-ABS sequences. (4) Approximately 95.45% of the positively selected codons (P>0.95) were located at or adjacent to an ABS. Furthermore, this study showed that (1) The Qinling subspecies exhibited high omega values across each locus (all >1), supporting its extensive positive selection. (2) The Sichuan subspecies displayed small omega at DRB1 (omega<0.72) and DQA2 (omega<0.48), suggesting that these sites underwent strong purifying selection. (3) Intragenic recombination was detected in DRB1, DQA1, and DQB1. The molecular diversity in classic Aime-MHC class II genes implies that the giant panda had evolved relatively abundant variations in its adaptive immunity along the history of host-pathogen co-evolution. Collectively, these findings indicate that natural selection accompanied by recombination drives the contrasting diversity patterns of the MHC class II genes between the two studied subspecies of giant panda. PMID:19950128

  14. Distribution of genes associated with yield potential and water-saving in Chinese Zone II wheat detected by developed functional markers.

    PubMed

    Gao, Zhenxian; Shi, Zhanliang; Zhang, Aimin; Guo, Jinkao

    2015-03-01

    Functional markers (FMs) developed from sequence polymorphisms are present in allelic variants of a functional gene at a locus and are directly associated with phenotypic variations. In this study, FM linked to Rht-B1, Rht-D1, TaCwi-A1, TaSus2-2B, TaGW2-6A and Dreb-B1 genes conferring to yield potential and water-saving were selected to analyse the distribution in 102 wheat varieties, most of which were authorized in the past decade and adapted to grow in Zone II of China. First, the semidwarfing genes Rht-B1b and Rht-D1b (mutant alleles) conferring to grain yield were analysed. The frequencies of favourable alleles Rht-B1b and Rht-D1b were 32.4 and 58.8%, respectively. Comparing with the previous report, the frequency of Rht-B1b among cultivars in this study is similar to the frequency among cultivars released in the 1990s, while the frequency of Rht-D1b is slightly lower than the previous report 63.9%. Twelve (11.8%) cultivars neither contained Rht-B1b nor Rht-D1b, while only Yumai 66 contained both semidwarfing genes. Linyuan8 and Xinong 928 are heterozygous at RhtB1 locus and Zhengmai 9023 is heterozygous at both RhtB1 and Rht-D1 loci. Second, the TaCwi-A1, TaSus2-2B and TaGW2-6A genes considered as candidate genes related to grain weight were detected. We found that the frequencies of the favourable alleles were 76.5, 56.9 and 69.6%, respectively. Among the 102 wheat varieties, 30 contained all the three favourable genes, 45 contained two of the three favourable genes and 27 contained only one. There are eight wheat varieties (7.8%) in hybrid state at the TaCWI-A1 locus. Third, the designed FM linked to water-saving gene Dreb-B1 were validated on 102 wheat varieties. The results showed that the haplotypes of 47 wheat varieties at the Dreb-B1 locus were same as that of Opata 85, and 55 wheat varieties showed the signal expected for W7984 (Opata 85 and W7984 are parents of the ITMI mapping population). This information will be useful for the wheat breeding programmes aiming at improving yield and water use efficiency in Shijiazhuang located in China Zone II. PMID:25846875

  15. Effects of alien and intraspecies cytoplasms on manifestation of nuclear genes for wheat resistance to brown rust: II. Specificity of cytoplasm influence on different Lr genes

    SciTech Connect

    Voluevich, E.A.; Buloichik, A.A.; Palilova, A.N. [Institute of Genetics and Cytology, Minsk (Belarus)

    1995-04-01

    Specificity of expression of the major nuclear genes Lr to two brown rust clones in hybrids with the same maternal cytoplasm was analyzed. It was evaluated by a resistant: susceptible ratio in the F{sub 2}. Reciprocal hybrids were obtained from the cross between the progeny of homozygous susceptible plants of the cultivar Penjamo 62 and its alloplasmatic lines carrying cytoplasms of Triticum dicoccoides var. fulvovillosum, Aegilops squarrosa var. typical, Agropyron trichophorum, and isogenic lines of the cultivar Thatcher (Th) with the Lr1, Lr9, Lr15, and Lr19 genes. It was shown that the effect of the Lr1 gene in the cytoplasm of cultivar Thatcher and in eu-, and alloplasmatic forms of Penjamo 62 was less expressed than that of other Lr genes. Cytoplasm of the alloplasmatic line (dicoccoides)-Penjamo 62 was the only exception: in the F{sub 2}, hybrids with Th (Lr1) had a higher yield of resistant forms than those with Th (Lr15). In the hybrid combinations studied, expression and/or transmission of the Lr19 gene was more significant than that of other genes. This gene had no advantages over Lr15 and Lr19 only in cytoplasm of the alloplasmatic line (squarrosa)-Penjamo 62. In certain hybrid cytoplasms, the display of the Lr1, Lr15, and Lr19 genes, in contrast to Lr9, varied with the virulence of the pathogen clones. 15 refs., 5 tabs.

  16. Transfer of recombinant plasmids containing the gene for DpnII DNA methylase into strains of Streptococcus pneumoniae that produce DpnI or DpnII restriction endonucleases

    SciTech Connect

    Lacks, S.A.; Springhorn, S.S.

    1984-06-01

    Plasmid transfer via the transformation pathway of Streptococcus pneumoniae was weakly restricted by the DpnI or DpnII restriction endonuclease, either of which gave a reduction only to 0.4, compared with phage infection, which was restricted to 10/sup -5/. The greater sensitivity of plasmid transfer compared with chromosomal transformation, which was not at all restricted, can be attributed to partially double-stranded intermediates formed from two complementary donor fragments. However, clustering of potential restriction sites in the plasmids increased the probability of escape from restriction. The recombinant plasmid pMP10, in which the gene for the DpnII DNA methylase was cloned, can be transferred to strains that contain neither restriction enzyme or that contain DpnII as readily as can the vector pMP5. Introduction of pMP10 raised the level of methylase by five times the level normally present in DpnII strains. Transfer of pMP10 to DpnI-containing strains was infrequent, presumably owing to the suicidal methylation of DNA which rendered it susceptible to the host endonuclease. The few clones in which pMP10 was established had lost DpnI. Loss of the plasmid after curing of the cell eliminated the methylase but did not restore DpnI. Although this loss of DpnI could result from spontaneous mutations, its relatively high frequency, 0.1% suggested that the loss was due to a regulatory shift.

  17. Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells, fibroblasts, alveolar type II cells, airway epithelial cells and lymphocytes

    PubMed Central

    Sun, Xiaoru; Zheng, Minghuan; Zhang, Miaomiao; Qian, Mengjia; Zheng, Yonghua; Li, Meiyi; Cretoiu, Dragos; Chen, Chengshui; Chen, Luonan; Popescu, Laurentiu M; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC-specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8+ T cells from bronchial lymph nodes (T-BL) and CD8+ T cells from lungs (T-LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up-regulated and 70% down-regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over-expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types. PMID:24826900

  18. Construction of a YAC contig and STS map spanning at least 10 cM in 1q41, the critical region of Usher II gene

    SciTech Connect

    Wang, J.Y.; Zhen, D.K.; Li, B.F. [Univ. of Nebraska, Omaha, NE (United States)] [and others

    1994-09-01

    Usher syndrome is an autosomal recessive disorder causing congenital hearing loss, progressive retinitis pigmentosa and vestibular dysfunction. The Usher syndrome is both clinically and genetically heterogeneous. At least three genetic types of Usher syndrome are know to exist. The Usher II (USH2) syndrome has originally been linked to 1q41 between D1S70 and D1S81. more recently its location was refined and placed between D1S217 and D1S229. We have constructed a YAC contig containing 23 clones and a minimum of 10 Mbp of human DNA. A total of three NotI linking clones, fourteen polymorphic microsatelite markers, eight YAC end clones and twenty lambda and cosmid subclones have been used to order the YACs and assess their integrity. The YAC subclones were used to reassess the location of the USH2 gene. Seven CpG islands have already been identified in the region. Several potential exons have been identified by exon amplification in the cosmid subclones. This map of overlapping clones, the set of densely spaced physical markers and potential exons will promote our understanding of the 1q1 region, its associated genes and eventually the gene mutated in Usher syndrome type II.

  19. Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells

    PubMed Central

    2014-01-01

    Background Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. Methods WT and N0 cells were treated with 5 and 10 ?g/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [NAD(P)H: quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Results Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. Conclusion The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the exception of SOD1 which may not be dependent on this pathway. PMID:24559113

  20. An Nrf2\\/Small Maf Heterodimer Mediates the Induction of Phase II Detoxifying Enzyme Genes through Antioxidant Response Elements

    Microsoft Academic Search

    Ken Itoh; Tomoki Chiba; Satoru Takahashi; Tetsuro Ishii; Kazuhiko Igarashi; Yasutake Katoh; Tatsuya Oyake; Norio Hayashi; Kimihiko Satoh; Ichiro Hatayama; Masayuki Yamamoto; Yo-ichi Nabeshima

    1997-01-01

    The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme

  1. Polymorphisms in type I and II Inosine Monophosphate Dehydrogenase (IMPDH) genes and association with clinical outcome in patients on

    E-print Network

    Boyer, Edmond

    Monitoring. inserm-00494526,version1-23Jun2010 #12;2 Abstract: Background: Type I and II inosine treatment related outcome. We investigated the associations of biopsy proven acute rejection (BPAR, biological and treatment data using multivariate analysis. Results: Many IMPDH II variant alleles referenced

  2. Mapping of the human insulin receptor substrate-2 gene, identification of a linked polymorphic marker and linkage analysis in families with Type II diabetes: no evidence for a major susceptibility role

    Microsoft Academic Search

    K. Kalidas; J. Wasson; B. Glaser; J. M. Meyer; L. J. Duprat; M. F. White; M. Alan Permutt

    1998-01-01

    Summary   Insulin receptor substrate 2 (IRS-2) is a substrate of the insulin receptor and mediates the action of the insulin. Disruption\\u000a of the IRS-2 gene in mice results in peripheral insulin resistance and relative insulin deficiency. It is therefore possible\\u000a that defects in the IRS-2 gene contribute to Type II (non-insulin-dependent) diabetes mellitus. We have examined the gene\\u000a for evidence

  3. I-OmiI and I-OmiII: two intron-encoded homing endonucleases within the Ophiostoma minus rns gene.

    PubMed

    Hafez, Mohamed; Guha, Tuhin Kumar; Hausner, Georg

    2014-08-01

    The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3' overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes. PMID:25110134

  4. A horizontally acquired group II intron in the chloroplast psbA gene of a psychrophilic Chlamydomonas: In vitro self-splicing and genetic evidence for maturase activity

    PubMed Central

    ODOM, OBED W.; SHENKENBERG, DAVID L.; GARCIA, JOSHUA A.; HERRIN, DAVID L.

    2004-01-01

    The majority of known group II introns are from chloroplast genomes, yet the first self-splicing group II intron from a chloroplast gene was reported only recently, from the psbA gene of the euglenoid, Euglena myxocylindracea. Herein, we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas sp. from Antarctica that self-splices accurately in vitro. Remarkably, this intron, which also encodes an ORF with putative reverse transcriptase, maturase, and endonuclease domains, is in the same location, and is related to the E. myxocylindracea intron, as well as to group IIB2 introns from cyanobacteria. In vitro self-splicing of Chs.psbA1 occurred via a lariat, and required Mg2+ (>12 mM) and NH4+. Self-splicing was improved by deleting most of the ORF and by using pre-RNAs directly from transcription reactions, suggestive of a role for folding during transcription. Self-splicing of Chs.psbA1 pre-RNAs showed temperature optima of ~44°C, but with a broad shoulder on the low side of the peak; splicing was nearly absent at 50°C, indicative of thermolability. Splicing of wild-type Chs.psbA1 also occurred in Escherichia coli, but not when the ORF was disrupted by mutations, providing genetic evidence that it has maturase activity. This work provides the first description of a ribozyme from a psychrophilic organism. It also appears to provide a second instance of interkingdom horizontal transfer of this group IIB2 intron (or a close relative) from cyanobacteria to chloroplasts. PMID:15208445

  5. Susceptibility and gene interaction study of the angiotensin II type 1 receptor (AGTR1) gene polymorphisms with non-alcoholic fatty liver disease in a multi-ethnic population.

    PubMed

    Zain, Shamsul Mohd; Mohamed, Zahurin; Mahadeva, Sanjiv; Rampal, Sanjay; Basu, Roma Choudhury; Cheah, Phaik-Leng; Salim, Agus; Mohamed, Rosmawati

    2013-01-01

    Angiotensin II type 1 receptor (AGTR1) has been reported to play a fibrogenic role in non-alcoholic fatty liver disease (NAFLD). In this study, five variants of the AGTR1 gene (rs3772622, rs3772627, rs3772630, rs3772633, and rs2276736) were examined for their association with susceptibility to NAFLD. Subjects made up of 144 biopsy-proven NAFLD patients and 198 controls were genotyped using TaqMan assays. The liver biopsy specimens were histologically graded and scored according to the method of Brunt. Single locus analysis in pooled subjects revealed no association between each of the five variants with susceptibility to NAFLD. In the Indian ethnic group, the rs2276736, rs3772630 and rs3772627 appear to be protective against NAFLD (p = 0.010, p = 0.016 and p = 0.026, respectively). Haplotype ACGCA is shown to be protective against NAFLD for the Indian ethnic subgroup (p = 0.03). Gene-gene interaction between the AGTR1 gene and the patatin-like phospholipase domain-containing 3 (PNPLA3) gene, which we previously reported as associated with NAFLD in this sample, showed a strong interaction between AGTR1 (rs3772627), AGTRI (rs3772630) and PNPLA3 (rs738409) polymorphisms on NAFLD susceptibility (p = 0.007). Further analysis of the NAFLD patients revealed that the G allele of the AGTR1 rs3772622 is associated with increased fibrosis score (p = 0.003). This is the first study that replicates an association between AGTR1 polymorphism and NAFLD, with further details in histological features of NAFLD. There is lack of evidence to suggest an association between any of the five variants of the AGTR1 gene and NAFLD in the Malays and Chinese. In the Indians, the rs2276736, rs3772630 and rs3772627 appear to protect against NAFLD. We report novel findings of an association between the G allele of the rs3772622 with occurrence of fibrosis and of the gene-gene interaction between AGTR1gene and the much-studied PNPLA3 gene. PMID:23484035

  6. Phylogeographic analysis of the firefly, Luciola lateralis, in Japan and Korea based on mitochondrial cytochrome oxidase II gene sequences (Coleoptera: Lampyridae).

    PubMed

    Suzuki, Hirobumi; Sato, Yasushi; Ohba, Nobuyoshi; Bae, Jin-Sik; Jin, Byung-Rae; Sohn, Hung-Dae; Kim, Sam-Eun

    2004-10-01

    Luciola lateralis is widely distributed throughout the Korean Peninsula, northeast China, Sakhalin, and Japan. Two ecological types are recognized in Japan based on flash and hatching time characteristics. The mitochondrial cytochrome oxidase II gene was surveyed by restriction fragment length polymorphism analysis for Japan (46 populations) and Korea (two populations). Eleven haplotypes were detected. Gene trees revealed that haplotypes between Japan and Korea are much more differentiated in nucleotide sequences (8.1%) than those within Japan (0.3-1.4%) and Korea (0.7%). Haplotypes between Honshu and Hokkaido are not separated as clades, and the two ecological types cannot be segregated from each other phylogenetically. We suggest that the Japanese populations of this species may have dispersed within one million years ago and that ecological differences may be the result of physiological adaptation to cold climates. PMID:15524308

  7. The class II HD-ZIP JAIBA gene is involved in meristematic activity and important for gynoecium and fruit development in Arabidopsis

    PubMed Central

    Zúñiga-Mayo, Victor M.; Marsch-Martínez, Nayelli; de Folter, Stefan

    2012-01-01

    Development and patterning of the gynoecium – and later the fruit – must be finely regulated to ensure the survival of the species that produces them. The process that leads to successful fruit formation starts at early stages of floral meristem development and follows a series of chronologically successive events. In a recent work we reported the functional characterization of the class II HD-ZIP JAIBA (JAB) gene. Mutant jab plants showed sporophytic defects in male and female reproductive development, and combined with the mutant crabs claw (crc) caused defects in the floral meristem (FM) determination process and gynoecium medial tissue development. Furthermore, the JAB protein interacted with transcription factors known to regulate meristematic activity, fruit development and FM determinacy. Preliminary results presented here suggest a genetic interaction between JAB and the gene SHOOT MERISTEMLESS (STM). PMID:22951401

  8. Transposon mutagenesis and physiological analysis of strains containing inactivated form I and form II ribulose bisphosphate carboxylase/oxygenase genes in Rhodobacter sphaeroides.

    PubMed Central

    Falcone, D L; Quivey, R G; Tabita, F R

    1988-01-01

    Strains of Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) were constructed such that either the gene encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC-O) or the gene encoding form II RuBPC-O was inactivated. Both strains were capable of photoheterotrophic growth with malate as the electron donor, with only slight differences in growth rate and overall carboxylase specific activity compared with the wild-type strain. Photolithotrophic growth with 1.5% CO2 in hydrogen was also possible for R. sphaeroides strains containing only one of the two RuBPC-O enzyme forms, although the differences in growth rates between wild-type and carboxylase mutant strains were greater under these conditions. These results indicate that the two forms of RuBPC-O are independently regulated. In addition, the regulatory system governing RuBPC-O synthesis may, in some cases, compensate for the lack of the missing enzyme. Images PMID:2826406

  9. Mice with a homozygous deletion of the Mgat2 gene encoding UDP N-acetylglucosamine:?-6- d-mannoside ?1,2- N-acetylglucosaminyltransferase II: a model for congenital disorder of glycosylation type IIa

    Microsoft Academic Search

    Yan Wang; Harry Schachter; Jamey D Marth

    2002-01-01

    Mice homozygous for a deletion of the Mgat2 gene encoding UDP-N-acetylglucosamine:?-6-d-mannoside ?1,2-N-acetylglucosaminyltransferase II (GlcNAcT-II, EC 2.4.1.143) have been reported. GlcNAcT-II is essential for the synthesis of complex N-glycans. The Mgat2-null mice were studied in a comparison with the symptoms of congenital disorder of glycosylation type IIa (CDG-IIa) in humans. Mutant mouse tissues were shown to be deficient in GlcNAcT-II enzyme

  10. RELATIONSHIPS OF HG(II) VOLATILIZATION FROM A FRESHWATER POND TO ABUNDANCE OF MER GENES IN THE GENE POOL OF THE INDIGENOUS MICROBIAL COMMUNITY

    EPA Science Inventory

    The role of biological activities in the reduction and volatilization of Hg(II) from a polluted pond was investigated. lemental mercury was evolved from pond water immediately following spiking with 203 Hg(NO3)2, whereas a lag period of 36 hr was required in control samples colle...

  11. Biological activity of lenalidomide in myelodysplastic syndromes with del5q: results of gene expression profiling from a multicenter phase II study.

    PubMed

    Oliva, Esther Natalie; Cuzzola, Maria; Aloe Spiriti, Maria Antonietta; Poloni, Antonella; Laganà, Carmelo; Rigolino, Carmela; Morabito, Fortunato; Galimberti, Sara; Ghio, Riccardo; Cortelezzi, Agostino; Palumbo, Giuseppe Alberto; Sanpaolo, Grazia; Finelli, Carlo; Ricco, Alessandra; Volpe, Antonio; Rodà, Filippo; Breccia, Massimo; Alimena, Giuliana; Nobile, Francesco; Latagliata, Roberto

    2013-01-01

    In vitro studies suggest that haploinsufficiency is involved in the pathogenesis of myelodysplastic syndromes (MDS). In patients with del5q cytogenetic abnormality, RPS-14 and microRNAs (miRNAs) play a major role. In a multicenter phase II single-arm trial with lenalidomide in anemic primary del5q MDS patients with low- or int-1 risk IPSS, biological changes from baseline were investigated. Gene expression profiling of selected genes was performed (TaqMan® Low Density Array Fluidic card, Applied Biosystems PRISM® 7900HT) and normalized against the expression of the 18S housekeeping gene from a pool of healthy subjects. Thirty-two patients were evaluated at baseline and after 3 and 6 months of treatment. RPS-14, miR-145, and miR-146 were downregulated at baseline and significantly increased during treatment. Nuclear factor kappa B, IL-6, interferon regulatory factor-1, IFN?-R2, IL-2, and many genes in the apoptotic pathways (TNF, IL-1B, and IL-10) were upregulated at baseline and significantly downregulated during lenalidomide treatment, while forkhead box P3, FAS, IFN?, IL-12A, and IL-12B were downregulated at baseline and progressively upregulated during treatment. The crucial role of aberrant immunological pathways and haploinsufficiency in the pathogenesis of del5q MDS is confirmed in the present patient setting. Our results indicate that lenalidomide may act through defined immunological pathways in this condition. PMID:22983750

  12. Evidence for the ‘Good Genes’ Model: Association of MHC Class II DRB Alleles with Ectoparasitism and Reproductive State in the Neotropical Lesser Bulldog Bat, Noctilio albiventris

    PubMed Central

    Schad, Julia; Dechmann, Dina K. N.; Voigt, Christian C.; Sommer, Simone

    2012-01-01

    The adaptive immune system has a major impact on parasite resistance and life history strategies. Immunological defence is costly both in terms of immediate activation and long-term maintenance. The ‘good genes’ model predicts that males with genotypes that promote a good disease resistance have the ability to allocate more resources to reproductive effort which favours the transmission of good alleles into future generations. Our study shows a correlation between immune gene constitution (Major Histocompatibility Complex, MHC class II DRB), ectoparasite loads (ticks and bat flies) and the reproductive state in a neotropical bat, Noctilio albiventris. Infestation rates with ectoparasites were linked to specific Noal-DRB alleles, differed among roosts, increased with body size and co-varied with reproductive state particularly in males. Non-reproductive adult males were more infested with ectoparasites than reproductively active males, and they had more often an allele (Noal-DRB*02) associated with a higher tick infestation than reproductively active males or subadults. We conclude that the individual immune gene constitution affects ectoparasite susceptibility, and contributes to fitness relevant trade-offs in male N. albiventris as suggested by the ‘good genes’ model. PMID:22615910

  13. Human Histone H3K79 Methyltransferase DOT1L Methyltransferase Binds Actively Transcribing RNA Polymerase II to Regulate Gene Expression*

    PubMed Central

    Kim, Seung-Kyoon; Jung, Inkyung; Lee, Hosuk; Kang, Keunsoo; Kim, Mirang; Jeong, Kwiwan; Kwon, Chang Seob; Han, Yong-Mahn; Kim, Yong Sung; Kim, Dongsup; Lee, Daeyoup

    2012-01-01

    Histone-modifying enzymes play a pivotal role in gene expression and repression. In human, DOT1L (Dot1-like) is the only known histone H3 lysine 79 methyltransferase. hDOT1L is associated with transcriptional activation, but the general mechanism connecting hDOT1L to active transcription remains largely unknown. Here, we report that hDOT1L interacts with the phosphorylated C-terminal domain of actively transcribing RNA polymerase II (RNAPII) through a region conserved uniquely in multicellular DOT1 proteins. Genome-wide profiling analyses indicate that the occupancy of hDOT1L largely overlaps with that of RNAPII at actively transcribed genes, especially surrounding transcriptional start sites, in embryonic carcinoma NCCIT cells. We also find that C-terminal domain binding or H3K79 methylations by hDOT1L is important for the expression of target genes such as NANOG and OCT4 and a marker for pluripotency in NCCIT cells. Our results indicate that a functional interaction between hDOT1L and RNAPII targets hDOT1L and subsequent H3K79 methylations to actively transcribed genes. PMID:23012353

  14. Extensive polymorphism and evidence of selection pressure on major histocompatibility complex DLA-DRB1, DQA1 and DQB1 class II genes in Croatian grey wolves.

    PubMed

    Arbanasi?, H; Huber, ?; Kusak, J; Gomer?i?, T; Hrenovi?, J; Galov, A

    2013-01-01

    The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for measuring fitness-related genetic variation in wildlife populations. Because of human persecution and habitat fragmentation, the grey wolf has become extinct from a large part of Western and Central Europe, and remaining populations have become isolated. In Croatia, the grey wolf population, part of the Dinaric-Balkan population, shrank nearly to extinction during the 20th century, and is now legally protected. Using the cloning-sequencing method, we investigated the genetic diversity and evolutionary history of exon 2 of MHC class II DLA-DRB1, DQA1 and DQB1 genes in 77 individuals. We identified 13 DRB1, 7 DQA1 and 11 DQB1 highly divergent alleles, and 13 DLA-DRB1/DQA1/DQB1 haplotypes. Selection analysis comparing the relative rates of non-synonymous to synonymous mutations (d(N)/d(S)) showed evidence of positive selection pressure acting on all three loci. Trans-species polymorphism was found, suggesting the existence of balancing selection. Evolutionary codon models detected considerable difference between alpha and beta chain gene selection patterns: DRB1 and DQB1 appeared to be under stronger selection pressure, while DQA1 showed signs of moderate selection. Our results suggest that, despite the recent contraction of the Croatian wolf population, genetic variability in selectively maintained immune genes has been preserved. PMID:23134500

  15. Evidence for the 'good genes' model: association of MHC class II DRB alleles with ectoparasitism and reproductive state in the neotropical lesser bulldog bat, Noctilio albiventris.

    PubMed

    Schad, Julia; Dechmann, Dina K N; Voigt, Christian C; Sommer, Simone

    2012-01-01

    The adaptive immune system has a major impact on parasite resistance and life history strategies. Immunological defence is costly both in terms of immediate activation and long-term maintenance. The 'good genes' model predicts that males with genotypes that promote a good disease resistance have the ability to allocate more resources to reproductive effort which favours the transmission of good alleles into future generations. Our study shows a correlation between immune gene constitution (Major Histocompatibility Complex, MHC class II DRB), ectoparasite loads (ticks and bat flies) and the reproductive state in a neotropical bat, Noctilio albiventris. Infestation rates with ectoparasites were linked to specific Noal-DRB alleles, differed among roosts, increased with body size and co-varied with reproductive state particularly in males. Non-reproductive adult males were more infested with ectoparasites than reproductively active males, and they had more often an allele (Noal-DRB*02) associated with a higher tick infestation than reproductively active males or subadults. We conclude that the individual immune gene constitution affects ectoparasite susceptibility, and contributes to fitness relevant trade-offs in male N. albiventris as suggested by the 'good genes' model. PMID:22615910

  16. Novel mutations in the RFXANK gene: RFX complex containing in-vitro-generated RFXANK mutant binds the promoter without transactivating MHC II.

    PubMed

    Wiszniewski, Wojciech; Fondaneche, Marie-Claude; Louise-Plence, Pascale; Prochnicka-Chalufour, Ada; Selz, Françoise; Picard, Cappucine; Le Deist, Françoise; Eliaou, Jean-François; Fischer, Alain; Lisowska-Grospierre, Barbara

    2003-02-01

    MHC class II deficiency is a combined immunodeficiency caused by defects in the four regulatory factors, CIITA, RFXANK, RFX5 and RFXAP, that control MHC II expression at the transcriptional level. The RFXANK gene encodes one subunit of the heterotrimeric RFX complex that is involved in the assembly of several transcription factors on MHC II promoters. Seven different RFXANK mutations have previously been reported in 26 unrelated patients. The most frequent mutation, a 26-bp deletion (752delG-25), has been identified in 21 patients. The other mutations are all nonsense or splice-site mutations, leading to proteins lacking all or part of the RFXANK ankyrin repeat region. We report two novel missense mutations, D121V and R212X, resulting in loss of function of the gene. We investigated the in vivo effects of these mutations and of three other point mutations on the expression of the RFXANK RNA and protein. The number of RFXANK transcripts was severely reduced in all patients except one. The RFXANK protein was barely detected in two cases. In addition, guided by a structural model of RFXANK, we investigated experimental mutants of the C-terminal tyrosine 224. Substitution Y224A, but not Y224F, led to the loss of function of RFXANK. Two null mutants, D121V and Y224A, were tested in protein interaction and DNA binding assays. The D121V mutant was unable to form the RFX complex, indicating that D121 is required for RFXAP binding. The Y224A mutant formed an RFX complex that bound normally to the MHC II promoter, but did not lead to MHC class II expression, whereas Y224F RFXANK retained the wild-type function. This indicates that an aromatic ring, but not the phenyl chain of tyrosine, is necessary at position 224 for normal RFXANK function. Studies on the Y224A mutant suggest that, in addition to the RFX subunits and CIITA, another protein is essential for MHC class II expression. This protein appears to interact with the fourth ankyrin repeat of RFXANK. PMID:12618906

  17. Apolipophorin II\\/I, Apolipoprotein B, Vitellogenin, and Microsomal Triglyceride Transfer Protein Genes Are Derived from a Common Ancestor

    Microsoft Academic Search

    Patrick J. Babin; Jan Bogerd; Frank P. Kooiman; Wil J. A. Van Marrewijk; Dick J. Van der Horst

    1999-01-01

    .   Large lipid transfer proteins (LLTP) are nonexchangeable apolipoproteins and intracellular lipid-exchange proteins involved\\u000a in the assembly, secretion, and metabolism of lipoproteins. We have identified contiguous conserved sequence motifs in alignments\\u000a of insect apolipophorin II\\/I precursor (apoLp-II\\/I), human apolipoprotein B (apoB), invertebrate and vertebrate vitellogenins\\u000a (VTG), and the large subunit of mammalian microsomal triglyceride transfer protein (MTP). Conserved motifs present

  18. Trans-species polymorphism of the Mhc class II DRB -like gene in banded penguins (genus Spheniscus )

    Microsoft Academic Search

    Eri F. Kikkawa; Tomi T. Tsuda; Daisuke Sumiyama; Taeko K. Naruse; Michio Fukuda; Masanori Kurita; Rory P. Wilson; Yvon LeMaho; Gary D. Miller; Michio Tsuda; Koichi Murata; Jerzy K. Kulski; Hidetoshi Inoko

    2009-01-01

    The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of\\u000a balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify,\\u000a clone, and sequence overlapping portions of the Mhc class II DRB-like

  19. Regulation of Aldosterone Synthase Gene Expression in the Rat Adrenal Gland and Central Nervous System by Sodium and Angiotensin II

    Microsoft Academic Search

    PING YE; CHRISTOPHER J. KENYON; SCOTT M. MACKENZIE; JONATHAN R. SECKL; ROBERT FRASER; JOHN M. C. CONNELL; ELEANOR DAVIES

    2010-01-01

    We have developed a highly sensitive QRT-PCR method for the measurement of CYP11B1 (11-hydroxylase) and CYP11B2 (aldosterone synthase) mRNAs to study their expression in the rat brain in response to dietary sodium manipulation and angiotensin (Ang)II infusion. Male Wistar Kyoto rats (n 6) were fed normal, high, or low sodium diets for 12 d or were administered AngII or vehicle

  20. Two founder mutations in the SEC23B gene account for the relatively high frequency of CDA II in the Italian population

    PubMed Central

    Russo, Roberta; Gambale, Antonella; Esposito, Maria Rosaria; Serra, Maria Luisa; Troiano, Annaelena; De Maggio, Ilaria; Capasso, Mario; Luzzatto, Lucio; Delaunay, Jean; Tamary, Hannah; Iolascon, Achille

    2011-01-01

    Congenital Dyserythropoietic Anemia type II is an autosomal recessive disorder characterized by unique abnormalities in the differentiation of cells of the erythroid lineage. The vast majority of CDA II cases result from mutations in the SEC23B gene. To date, 53 different causative mutations have been reported in 86 unrelated cases (from the CDA II European Registry), 47 of them Italian. We have now identified SEC23B mutations in 23 additional patients, 17 Italians and 6 non-Italian Europeans. The relative allelic frequency of the mutations was then reassessed in a total of 64 Italian and 45 non-Italian unrelated patients. Two mutations, E109K and R14W, account for over one-half of the cases of CDA II in Italy. Whereas the relative frequency of E109K is similar in Italy and in the rest of Europe (and is also prevalent in Moroccan Jews), the relative frequency of R14W is significantly higher in Italy (26.3% vs. 10.7%). By haplotype analysis we demonstrated that both are founder mutations in the Italian population. By using the DMLE+ program our estimate for the age of the E109K mutation in Italian population is ?2,200 years; whereas for the R14W mutation it is ?3,000 years. We hypothesize that E109K may have originated in the Middle East and may have spread in the heyday of the Roman Empire. Instead, R14W may have originated in Southern Italy. The relatively high frequency of the R14W mutation may account for the known increased prevalence of CDA II in Italy. Am. J. Hematol. 86:727–732, 2011. © 2011 Wiley-Liss, Inc. PMID:21850656

  1. Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane.

    PubMed

    Joyce, Priya; Kuwahata, Melissa; Turner, Nicole; Lakshmanan, Prakash

    2010-02-01

    A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8-4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and beta-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1-3 transgene insertion events that were randomly integrated in the majority of the plants produced. PMID:20041254

  2. The Fusarium verticillioides FUM gene cluster encodes a Zn(II)2Cys6 protein that affects FUM gene expression and fumonisin production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in synthesis of mycotoxins and other secondary met...

  3. Overexpression of the IGF-II/M6P Receptor in Mouse Fibroblast Cell Lines Differentially Alters Expression Profiles of Genes Involved in Alzheimer’s Disease-Related Pathology

    PubMed Central

    Wang, Yanlin; Thinakaran, Gopal; Kar, Satyabrata

    2014-01-01

    Alzheimer’s disease (AD) is the most common type of senile dementia affecting elderly people. The processing of amyloid precursor protein (APP) leading to the generation of ?-amyloid (A?) peptide contributes to neurodegeneration and development of AD pathology. The endocytic trafficking pathway, which comprises of the endosomes and lysosomes, acts as an important site for A? generation, and endocytic dysfunction has been linked to increased A? production and loss of neurons in AD brains. Since insulin-like growth factor-II (IGF-II) receptor plays a critical role in the transport of lysosomal enzymes from the trans-Golgi network to endosomes, it is likely that the receptor may have a role in regulating A? metabolism in AD pathology. However, very little is known on how altered levels of the IGF-II receptor can influence the expression/function of various molecules involved in AD pathology. To address this issue, we evaluated the expression profiles of 87 selected genes related to AD pathology in mouse fibroblast MS cells that are deficient in murine IGF-II receptor and corresponding MS9II cells overexpressing ?500 times the human IGF-II receptors. Our results reveal that an elevation in IGF-II receptor levels alters the expression profiles of a number of genes including APP as well as enzymes regulating A? production, degradation and clearance mechanisms. Additionally, it influences the expression of various lysosomal enzymes and protein kinases that are involved in A? toxicity. IGF-II receptor overexpression also alters expression of several genes involved in intracellular signalling as well as cholesterol metabolism, which play a critical role in AD pathology. The altered gene profiles observed in this study closely match with the corresponding protein levels, with a few exceptions. These results, taken together, suggest that an elevation in IGF-II receptor levels can influence the expression profiles of transcripts as well as proteins that are involved in AD pathogenesis. PMID:24846272

  4. Temperature and length-dependent modulation of the MH class II beta gene expression in brook charr (Salvelinus fontinalis) by a cis-acting minisatellite.

    PubMed

    Croisetière, Sébastien; Bernatchez, Louis; Belhumeur, Pierre

    2010-05-01

    It is widely recognized that the variation in gene regulation is an important factor from which evolutionary changes in diverse aspects of phenotype can be observed in all organisms. Distinctive elements with functional roles on gene regulation have been identified within the non-coding part of the genome, including repeated elements. Major histocompatibility complex (MHC) genes have been the subject of an abundant literature which made them unique candidates for studies of adaptation in natural populations. Yet, the vast majority of studies on MHC genes have dealt with patterns of polymorphism in sequence variation while very few paid attention to the possible implication of differential expression in adaptive responses. In this paper, we report the identification of a polymorphic minisatellite formed of a 32 nucleotides motif (38% G+C) involved in regulation of the major histocompatibility class II beta gene (MHII beta) of brook charr (Salvelinus fontinalis). Our main objectives were: to analyze the variability of this minisatellite found in the second intron of the MHII beta gene and to document its effect to the variation of expression level of this gene under different environmental conditions. Distinctive number of the minisatellite repeats were associated with each different MHII beta alleles identified from exon 2 sequences. Relative expression levels of specific alleles in heterozygous individuals were determined from fish lymphocytes in different genotypes. We found that alleles carrying the longest minisatellite showed a significant 1.67-2.56-fold reduction in the transcript expression relatively to the shortest one. Results obtained in three different genotypes also indicated that the repressive activity associated to the longest minisatellite was more effective at 18 degrees C compared to 6 degrees C. In contrast, no significant difference was observed in transcript levels between alleles with comparable minisatellite length at both temperatures. We also depicted a significant up-regulation of the total MHII beta transcript at 6 degrees C relative to 18 degrees C. These results reveal for the first time that a temperature-sensitive minisatellite could potentially play an important role in the gene regulation of the adaptive immune response in fishes. PMID:20381151

  5. Positive Selection Driving the Evolution of a Gene of Male Reproduction, Acp26Aa, of Drosophila: II. Divergence Versus Polymorphism

    Microsoft Academic Search

    Shun-Chern Tsaur; Chau-Ti Ting; Chung-I Wu

    The evolution of the gene for a male ejaculatory protein, Acp26Aa, has been shown to be driven by positive selection when nonsibling species in the Drosophila melanogaster subgroup are compared. To know if selection has been operating in the recent past and to understand the details of its dynamics, we obtained DNA sequences of Acp26Aa and the nearby Acp26Ab gene

  6. Discrete Gene Loci Regulate Neurodegeneration, Lymphocyte Infiltration, and Major Histocompatibility Complex Class II Expression in the CNS

    Microsoft Academic Search

    Olle Lidman; Maria Swanberg; Linn Horvath; Karl W. Broman; Tomas Olsson; Fredrik Piehl

    Neurodegeneration and inflammation are fundamental aspects of many neurological diseases. A genome-wide scan of the response to ventral root avulsion (VRA) in a rat F2 cross discloses specific gene regions that regulate these processes. Two gene loci displayed linkage to neurodegeneration and T cell infiltration, respectively, and a single locus displayed extreme linkage to VRA-induced major histocom- patibility complex class

  7. Differential regulation of the Sir2 histone deacetylase gene family by inhibitors of class I and II histone deacetylases

    Microsoft Academic Search

    S. Kyrylenko; O. Kyrylenko; T. Suuronen; A. Salminen

    2003-01-01

    The Sir2 histone deacetylase gene family consists of seven mammalian sirtuins (SIRTs) which are NAD-dependent histone\\/protein deacetylases. Sir2 proteins regulate, for instance, genome stability by chromatin silencing in yeast. In mammals, their function is still largely unknown. Due to the NAD + dependency, Sir2 might be the link between metabolic activity and histone\\/protein acetylation. Regulation of gene expression also seems

  8. Characterisation of class II B MHC genes from a ratite bird, the little spotted kiwi ( Apteryx owenii )

    Microsoft Academic Search

    Hilary C. Miller; Gemma Bowker-Wright; Marie Kharkrang; Kristina Ramstad

    2011-01-01

    Major histocompatibility complex (MHC) genes are important for vertebrate immune response and typically display high levels\\u000a of diversity due to balancing selection from exposure to diverse pathogens. An understanding of the structure of the MHC region\\u000a and diversity among functional MHC genes is critical to understanding the evolution of the MHC and species resilience to disease\\u000a exposure. In this study,

  9. The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: evolution of venom group II phospholipase A2's may imply gene duplications.

    PubMed

    Moura-da-Silva, A M; Paine, M J; Diniz, M R; Theakston, R D; Crampton, J M

    1995-08-01

    The sequence coding for a snake venom phospholipase A2 (PLA2), BJUPLA2, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA2's. A striking feature of the cDNA is the high sequence conservation of the 5' and 3' untranslated regions in cDNAs coding for PLA2's from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA2 with group II PLA2's in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA2's. PMID:7666446

  10. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis

    PubMed Central

    2014-01-01

    Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology. PMID:24410776

  11. Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation.

    PubMed Central

    Michel-Reydellet, N; Desnoues, N; Elmerich, C; Kaminski, P A

    1997-01-01

    The nucleotide sequence and transcriptional organization of Azorhizobium caulinodans ORS571 glnA, the structural gene for glutamine synthetase (GS), and glnB, the structural gene for the P(II) protein, have been determined. glnB and glnA are organized as a single operon transcribed from the same start site, under conditions of both nitrogen limitation and nitrogen excess. This start site may be used by two different promoters since the expression of a glnB-lacZ fusion was high in the presence of ammonia and enhanced under conditions of nitrogen limitation in the wild-type strain. The increase was not observed in rpoN or ntrC mutants. In addition, this fusion was overexpressed under both growth conditions, in the glnB mutant strain, suggesting that P(II) negatively regulates its own expression. A DNA motif, similar to a sigma54-dependent promoter consensus, was found in the 5' nontranscribed region. Thus, the glnBA operon seems to be transcribed from a sigma54-dependent promoter that operates under conditions of nitrogen limitation and from another uncharacterized promoter in the presence of ammonia. Both glnB and glnBA mutant strains derepress their nitrogenase in the free-living state, but only the glnBA mutant, auxotrophic for glutamine, does not utilize molecular nitrogen for growth. The level of GS adenylylation is not affected in the glnB mutant as compared to that in the wild type. Under symbiotic conditions, the glnB and glnBA mutant strains induced Fix- nodules on Sesbania rostrata roots. P(II) is the first example in A. caulinodans of a protein required for symbiotic nitrogen fixation but dispensable in bacteria growing in the free-living state. PMID:9171403

  12. Identification of three new mutations in the NADH-cytochrome b5 reductase gene responsible for recessive congenital methemoglobinemia type II

    SciTech Connect

    Mota-Vieira, L.; Kaplan, J.C.; Kahn, A.; Leroux, A. [Universite Rene Descartes-Paris (France)

    1994-09-01

    Recessive congenital methemoglobinemia (RCM; McKusick N{degrees}25800) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases: in type I form, cyanosis is the only symptom and the enzyme is only defective in red blood cells; in type II form, cyanosis is associated with severe mental retardation and neurological impairment and the enzyme defect is systemic. We have identified three new molecular defects in two unrelated patients with type II RCM. A homozygous C{r_arrow}T transition in codon 218 (Arg) was detected in the cDNA of one patient, resulting in a premature stop codon (TGA) in exon 8. Restriction enzyme analysis of genomic DNA confirmed the homozygosity of the propositus and heterozygosity for an identical defect in both parents. The second patient was found to be a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation (T{r_arrow}C) with substitution of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele showed a 3 bp deletion of nucleotides 815-817 of the cDNA. The CTG ATG sequence at position 814-819 in exon 9 coding for Leu-271 and Met-272 was replaced by the CTG triplet, with conservation of the Leu-271 and loss of the Met-272. To our knowledge, these are the first examples of a homozygous nonsense mutation and of a compound heterozygous mutation detected in the cytb5r gene. This finding supports the diversity of genetic defects in the cytb5r gene leading to the severe form of the disease.

  13. ReproducedfromCropScience.PublishedbyCropScienceSocietyofAmerica.Allcopyrightsreserved. Increased Transgene Expression by Breeding and Selection in White Clover

    E-print Network

    Parrott, Wayne

    - transgene at different loci, the genetic background of cating the importance of other genomic factors one transgene should be deployed Plants were transformed with nptII and gusA, and selected on 100 mg to avoid gene silencing between transgenes at different L 1 of kanamycin. Independently transformed plants

  14. The Largest Subunit of RNA Polymerase II as a New Marker Gene to Study Assemblages of Arbuscular Mycorrhizal Fungi in the Field

    PubMed Central

    Stockinger, Herbert; Peyret-Guzzon, Marine; Koegel, Sally; Bouffaud, Marie-Lara; Redecker, Dirk

    2014-01-01

    Due to the potential of arbuscular mycorrhizal fungi (AMF, Glomeromycota) to improve plant growth and soil quality, the influence of agricultural practice on their diversity continues to be an important research question. Up to now studies of community diversity in AMF have exclusively been based on nuclear ribosomal gene regions, which in AMF show high intra-organism polymorphism, seriously complicating interpretation of these data. We designed specific PCR primers for 454 sequencing of a region of the largest subunit of RNA polymerase II gene, and established a new reference dataset comprising all major AMF lineages. This gene is known to be monomorphic within fungal isolates but shows an excellent barcode gap between species. We designed a primer set to amplify all known lineages of AMF and demonstrated its applicability in combination with high-throughput sequencing in a long-term tillage experiment. The PCR primers showed a specificity of 99.94% for glomeromycotan sequences. We found evidence of significant shifts of the AMF communities caused by soil management and showed that tillage effects on different AMF taxa are clearly more complex than previously thought. The high resolving power of high-throughput sequencing highlights the need for quantitative measurements to efficiently detect these effects. PMID:25275381

  15. Mutation of the transforming growth factor-beta type II receptor gene in right-sided colorectal cancer: relationship to clinicopathological features and genetic alterations.

    PubMed

    Iacopetta, B J; Welch, J; Soong, R; House, A K; Zhou, X P; Hamelin, R

    1998-04-01

    The presence of inactivating mutations in the transforming growth factor-beta (TGF-beta) type II receptor (RII) gene in the colon cancer suggests that it may behave like a tumour suppressor gene. RII is mutated in the majority of colon tumours exhibiting widespread microsatellite instability, a characteristic generally referred to as the replication error phenotype (RER+). We investigated the association between RII mutations and various clinicopathological variables and genetic alterations in a large series of sporadic adenocarcinomas arising in the proximal colon. RII mutations were found in 17 per cent (36/210) of right-sided tumours and in 86 per cent (32/37) of those displaying RER+. They were associated with the absence of lymph node invasion (P = 0.04), poor histological differentiation (P = 0.006), and with a trend for improved patient survival. Tumours with an RII mutation also showed non-significant trends for a lower incidence of p53 protein overexpression and of p53, K-ras, and APC gene mutation compared with tumours with normal RII. These results indicate that right-sided colorectal tumours containing RII mutations resemble those with the RER+ phenotype in terms of their clinicopathological features and genetic alterations. PMID:9664904

  16. Analysis of P gene mutations in patients with type II (tyrosinase-positive) oculocutaneous albinism (OCA2)

    SciTech Connect

    Lee, S.T.; Nicholls, R.D.; Schnur, R. [Univ. of Wisconsin, Madison, WI (United States)]|[Case Western Reserve Univ., Cleveland, OH (United States)]|[Children`s Hospital of Philadelphia, PA (United States)] [and others

    1994-09-01

    OCA2 is an autosomal recessive disorder in which the biosynthesis of melanin pigment is greatly reduced in the skin, hair, and eyes. Recently, we showed that OCA2 results from mutations of the P gene, in chromosome segment 15q11-q13. In addition to OCA2, mutations of P account for OCA associated with the Prader-Willi syndrome and some cases of {open_quotes}autosomal recessive ocular albinism{close_quotes} (AROA). We have now studied 38 unrelated patients with various forms of OCA2 or AROA from a variety of different ethnic groups. None of these patients had detectable abnormalities of the tyrosinase (TYR) gene. Among 8 African-American patients with OCA2 we observed apparent locus homogeneity. We detected abnormalities of the P gene in all 8 patients, including 12 different mutations and deletions, most of which are unique to this group and none of which is predominant. In contrast, OCA2 in other populations appears to be genetically heterogeneous. Among 21 Caucasian patients we detected abnormalities of the P gene in only 8, comprising 9 different point mutations and deletions, some of which also occurred among the African-American patients. Among 3 Middle-Eastern, 3 Indo-Pakistani, and 3 Asian patients we detected mutations of the P gene in only one from each group. In a large Indo-Pakistani kindred with OCA2 we have excluded both the TYR and P genes on the basis of genetic linkage. The prevalence of mutations of the P gene thus appears to be much higher among African-Americans with OCA2 than among patients from other ethnic groups. The incidence of OCA2 in some parts of equatorial Africa is extremely high, as frequent as 1 per 1100, and the disease has been linked to P in South African Bantu. The eventual characterization of P gene mutations in Africans will be informative with regard to the origins of P gene mutations in African-American patients.

  17. Retroviral Gene Therapy for X-linked Chronic Granulomatous Disease: Results From Phase I/II Trial

    PubMed Central

    Kang, Hyoung Jin; Bartholomae, Cynthia C; Paruzynski, Anna; Arens, Anne; Kim, Sujeong; Yu, Seung Shin; Hong, Youngtae; Joo, Chang-Wan; Yoon, Nam-Kyung; Rhim, Jung-Woo; Kim, Joong Gon; Von Kalle, Christof; Schmidt, Manfred; Kim, Sunyoung; Ahn, Hyo Seop

    2011-01-01

    X-linked chronic granulomatous disease (CGD) is an inherited immunodeficiency caused by a defect in the gp91phox gene. In an effort to treat X-CGD, we investigated the safety and efficacy of gene therapy using a retroviral vector, MT-gp91. Two X-CGD patients received autologous CD34+ cells transduced with MT-gp91 after a conditioning regimen consisting of fludarabine and busulfan. The level of gene-marked cells was highest at day 21 (8.3 and 11.7% in peripheral blood cells) but decreased to 0.08 and 0.5%, respectively, 3 years after gene transfer. The level of functionally corrected cells, as determined by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assay, reached a peak at day 17 (6.5% patient 1 (P1) and 14.3% patient 2 (P2) of total granulocytes) and declined to 0.05% (P1) and 0.21% (P2), 3 years later. Some retroviral vectors were found to have integrated within or close to the proto-oncogenes MDS1-EVI1, PRDM16, and CCND2; however, no abnormal cell expansion or related hematological malignancy was observed. Overall, the gene transfer procedure did not produce any serious adverse effects and was able to convert a significant fraction of blood cells to biologically functional cells, albeit for a short period of time. PMID:21878903

  18. Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16S rRNA gene, ITS region and glnII, recA, atpD and dnaK genes.

    PubMed

    Menna, Pâmela; Barcellos, Fernando Gomes; Hungria, Mariangela

    2009-12-01

    The genus Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with legumes and non-legumes, and are characterized by physiological and symbiotic versatility and broad geographical distribution. However, despite indications of great genetic variability within the genus, only eight species have been described, mainly because of the highly conserved nature of the 16S rRNA gene. In this study, 169 strains isolated from 43 different legumes were analysed by rep-PCR with the BOX primer, by sequence analysis of the 16S rRNA gene and the 16S-23S rRNA intergenic transcribed spacer (ITS) and by multilocus sequence analysis (MLSA) of four housekeeping genes, glnII, recA, atpD and dnaK. Considering a cut-off at a level of 70 % similarity, 80 rep-PCR profiles were distinguished, which, together with type strains, were clustered at a very low level of similarity (24 %). In both single and concatenated analyses of the 16S rRNA gene and ITS sequences, two large groups were formed, with bootstrap support of 99 % in the concatenated analysis. The first group included the type and/or reference strains of Bradyrhizobium japonicum, B. betae, B. liaoningense, B. canariense and B. yuanmingense and B. japonicum USDA 110, and the second group included strains related to Bradyrhizobium elkanii USDA 76(T), B. pachyrhizi PAC48(T) and B. jicamae PAC68(T). Similar results were obtained with MLSA of glnII, recA, atpD and dnaK. Greatest variability was observed when the atpD gene was amplified, and five strains related to B. elkanii revealed a level of variability never reported before. Another important observation was that a group composed of strains USDA 110, SEMIA 5080 and SEMIA 6059, all isolated from soybean, clustered in all six trees with high bootstrap support and were quite distinct from the clusters that included B. japonicum USDA 6(T). The results confirm that MLSA is a rapid and reliable way of providing information on phylogenetic relationships and of identifying rhizobial strains potentially representative of novel species. PMID:19628593

  19. Deletion of angiotensin II type 1 receptor gene or scavenge of superoxide prevents chronic alcohol-induced aortic damage and remodelling

    PubMed Central

    Bai, Yang; Tan, Yi; Wang, Bo; Miao, Xiao; Chen, Qiang; Zheng, Yang; Cai, Lu

    2012-01-01

    To investigate whether chronic alcohol consumption induces vascular injury via angiotensin II (Ang II) type 1 (AT1) receptor-dependent superoxide generation, male transgenic mice with knockout of AT1 gene (AT1-KO) and age-matched wild-type (WT) C57BL/6 mice were pair-fed a modified Lieber-DeCarli alcohol or isocaloric maltose dextrin control liquid diet for 2 months. Ethanol content (%, W/V) in the diet was 4.8 (34% of total calories) at initiation, and gradually increased up to 5.4 (38% of total calories). For some WT mice with and without alcohol treatment, superoxide dismutase mimetic (MnTMPyP) was given simultaneously by intraperitoneal injection at 5 mg/kg body weight daily for 2 months. At the end of studies, aortas were harvested for histopathological and immunohistochemical examination. Significant increases in the wall thickness and structural disarrangement of aorta were found in alcohol group, along with significant increases in aortic oxidative and/or nitrosative damage, expressions of NADPH oxidases (NOXs), inflammatory response, cell death and proliferation, and remodelling (fibrosis). However, these pathological changes were completely attenuated in alcohol-treated AT1-KO mice or in alcohol-treated WT mice that were also simultaneously treated with MnTMPyP for 2 months. These results suggest that chronic alcohol consumption may activate NOX via Ang II/AT1 receptor, to generate superoxide and associated peroxynitrite that in turn causes aortic nitrosative damage, inflammation, cell death and proliferation, and remodelling. Therefore, blocking Ang II/AT1 system or scavenging superoxide may become a potential preventive and/therapeutic approach to alcoholic vascular damage. PMID:22435601

  20. Generation of transgenic wheat plants producing high levels of the osmoprotectant proline

    Microsoft Academic Search

    Wagdy A. Sawahel; Ali H. Hassan

    2002-01-01

    Plasmid DNA (pBI-P5CS), containing the selectable neomycin phosphotransferase-II `npt II' gene for kanamycin resistance and the reporter ß-glucuronidase `gus' gene as well as the Vigna aconitifolia ?1-pyrroline-5-carboxylate synthetase `P5CS' cDNA that encodes enzymes required for the biosynthesis of proline, was delivered into wheat plants using Agrobacterium-mediated gene transfer via indirect pollen system. Southern, northern and western blot analysis demonstrated that the

  1. Degradation of Xylan to d-Xylose by Recombinant Saccharomyces cerevisiae Coexpressing the Aspergillus niger ?-Xylosidase (xlnD) and the Trichoderma reesei Xylanase II (xyn2) Genes

    PubMed Central

    La Grange, D. C.; Pretorius, I. S.; Claeyssens, M.; van Zyl, W. H.

    2001-01-01

    The ?-xylosidase-encoding xlnD gene of Aspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor ?1 signal peptide (MF?1s) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant ?-xylosidase showed optimum activity at 60°C and pH 3.2 and optimum stability at 50°C. The Ki(app) value for d-xylose and xylobiose for the recombinant ?-xylosidase was determined to be 8.33 and 6.41 mM, respectively. The XLO2 fusion gene and the XYN2 ?-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator. These recombinant S. cerevisiae strains produced 1,577 nkat/ml of ?-xylanase activity when expressing only the ?-xylanase and 860 nkat/ml when coexpressing the ?-xylanase with the ?-xylosidase. The maximum ?-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the ?-xylanase. Coproduction of the ?-xylanase and ?-xylosidase enabled S. cerevisiae to degrade birchwood xylan to d-xylose. PMID:11722900

  2. Sequence Analysis of the Capsid Gene during a Genotype II.4 Dominated Norovirus Season in One University Hospital: Identification of Possible Transmission Routes

    PubMed Central

    Holzknecht, Barbara Juliane; Franck, Kristina Træholt; Nielsen, Rikke Thoft; Böttiger, Blenda; Fischer, Thea Kølsen; Fonager, Jannik

    2015-01-01

    Norovirus (NoV) is a leading cause of gastroenteritis and genotype II.4 (GII.4) is responsible for the majority of nosocomial NoV infections. Our objective was to examine whether sequencing of the capsid gene might be a useful tool for the hospital outbreak investigation to define possible transmission routes. All NoV positive samples submitted from one university hospital during the 2007/8 season were selected. Genotyping of selected samples by partial polymerase gene sequencing had shown that the majority belonged to the GII.4 variant Den Haag 2006b and had identical polymerase sequences. Sequences of the capsid gene (1412 nucleotides) were obtained from the first available sample from 55 patients. From six immunocompromised patients with persistent infections a second sample was also included. As a control for a point-source outbreak, five samples from a foodborne outbreak caused by the same GII.4 variant were analyzed. Forty-seven of the inpatients (85%) were infected with the GII.4 variant Den Haag 2006b. Phylogenetic analysis of the Den Haag 2006b sequences identified four distinct outbreaks in different departments and a fifth outbreak with possible inter-department spread. In addition, a more heterogeneous cluster with evidence of repeated introductions from the community, but also possible inter-department spread was observed. In all six patients with paired sequences, evidence for in vivo evolution of the virus was found. Capsid gene sequencing showed substantial sequence variation among NoV GII.4 variant Den Haag 2006b strains from one single institution during a nine months’ period. This method proved useful to understand the local epidemiology and, when used promptly, has the potential to make infection control measures more targeted. PMID:25590635

  3. An atypical psbA gene encodes a sentinel D1 protein to form a physiologically relevant inactive photosystem II complex in cyanobacteria.

    PubMed

    Wegener, Kimberly M; Nagarajan, Aparna; Pakrasi, Himadri B

    2015-02-01

    Photosystem II, a large membrane-bound enzyme complex in cyanobacteria and chloroplasts, mediates light-induced oxidation of water to molecular oxygen. The D1 protein of PSII, encoded by the psbA gene, provides multiple ligands for cofactors crucial to this enzymatic reaction. Cyanobacteria contain multiple psbA genes that respond to various physiological cues and environmental factors. Certain unicellular cyanobacterial cells, such as Cyanothece sp. ATCC 51142, are capable of nitrogen fixation, a highly oxygen-sensitive process, by separating oxygen evolution from nitrogen fixation using a day-night cycle. We have shown that c-psbA4, one of the five psbA orthologs in this cyanobacterium, is exclusively expressed during nighttime. Remarkably, the corresponding D1 isoform has replacements of a number of amino acids that are essential ligands for the catalytic Mn4CaO5 metal center for water oxidation by PSII. At least 30 cyanobacterial strains, most of which are known to have nitrogen fixing abilities, have similar psbA orthologs. We expressed the c-psbA4 gene from Cyanothece 51142 in a 4E-3 mutant strain of the model non-nitrogen-fixing cyanobacterium Synechocystis sp. PCC 6803, which lacks any psbA gene. The resultant strain could not grow photoautotrophically. Moreover, these Synechocystis 6803 cells were incapable of PSII-mediated oxygen evolution. Based on our findings, we have named this physiologically relevant, unusual D1 isoform sentinel D1. Sentinel D1 represents a new class of D1 protein that, when incorporated in a PSII complex, ensures that PSII cannot mediate water oxidation, thus allowing oxygen-sensitive processes such as nitrogen fixation to occur in cyanobacterial cells. PMID:25525275

  4. Involvement of AlpV, a New Member of the Streptomyces Antibiotic Regulatory Protein Family, in Regulation of the Duplicated Type II Polyketide Synthase alp Gene Cluster in Streptomyces ambofaciens

    Microsoft Academic Search

    Bertrand Aigle; Xiuhua Pang; Bernard Decaris; Pierre Leblond

    2005-01-01

    A type II polyketide synthase gene cluster located in the terminal inverted repeats of Streptomyces ambofaciens ATCC 23877 was shown to be responsible for the production of an orange pigment and alpomycin, a new antibiotic probably belonging to the angucycline\\/angucyclinone class. Remarkably, this alp cluster contains five potential regulatory genes, three of which (alpT, alpU, and alpV) encode proteins with

  5. Extended region of nodulation genes in Rhizobium meliloti 1021. II. Nucleotide sequence, transcription start sites and protein products

    SciTech Connect

    Fisher, R.F.; Swanson, J.A.; Mulligan, J.T.; Long, S.R.

    1987-10-01

    The authors have established the DNA sequence and analyzed the transcription and translation products of a series of putative nodulation (nod) genes in Rhizobium meliloti strain 1021. Four loci have been designated nodF, nodE, nodG and nodH. The correlation of transposon insertion positions with phenotypes and open reading frames was confirmed by sequencing the insertion junctions of the transposons. The protein products of these nod genes were visualized by in vitro expression of cloned DNA segments in a R. meliloti transcription-translation system. In addition, the sequence for nodG was substantiated by creating translational fusions in all three reading frames at several points in the sequence; the resulting fusions were expressed in vitro in both E. coli and R. meliloti transcription-translation systems. A DNA segment bearing several open reading frames downstream of nodG corresponds to the putative nod gene mutated in strain nod-216. The transcription start sites of nodF and nodH were mapped by primer extension of RNA from cells induced with the plant flavone, luteolin. Initiation of transcription occurs approximately 25 bp downstream from the conserved sequence designated the nod box, suggesting that this conserved sequence acts as an upstream regulator of inducible nod gene expression. Its distance from the transcription start site is more suggestive of an activator binding site rather than an RNA polymerase binding site.

  6. A Gene Coding for Tomato Fruit b-Galactosidase II Is Expressed during Fruit Ripening Cloning, Characterization, and Expression Pattern

    Microsoft Academic Search

    David L. Smith; David A. Starrett; Kenneth C. Gross

    b-Galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes characterized by their ability to hydrolyze terminal, non- reducing b-D-galactosyl residues from b-D-galactosides. Several b-galactosidases, sometimes referred to as exo-galactanases, have been purified from plants and shown to possess in vitro activity against extracted cell wall material via the release of galactose from wall polymers containing b(134)-D-galactan. Although b-galactosidase II, a

  7. A new compound heterozygous frameshift mutation in the type II 3{beta}-hydroxysteroid dehydrogenase 3{beta}-HSD gene causes salt-wasting 3{beta}-HSD deficiency congenital adrenal hyperplasia

    SciTech Connect

    Zhang, L.; Sakkal-Alkaddour, S.; Chang, Ying T.; Yang, Xiaojiang; Songya Pang [Univ. of Illinois, Chicago, IL (United States)] [Univ. of Illinois, Chicago, IL (United States)

    1996-01-01

    We report a new compound heterozygous frameshift mutation in the type II 3{Beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) gene in a Pakistanian female child with the salt-wasting form of 3{Beta}-HSD deficiency congenital adrenal hyperplasia. The etiology for her congenital adrenal hyperplasia was not defined. Although the family history suggested possible 3{beta}-HSd deficiency disorder, suppressed adrenal function caused by excess glucocorticoid therapy in this child at 7 yr of age did not allow hormonal diagnosis. To confirm 3{beta}-HSD deficiency, we sequenced the type II 3{beta}-HSD gene in the patient, her family, and the parents of her deceased paternal cousins. The type II 3{beta}-HSD gene region of a putative promotor, exons I, II, III, and IV, and exon-intron boundaries were amplified by PCR and sequenced in all subjects. The DNA sequence of the child revealed a single nucleotide deletion at codon 318 [ACA(Thr){r_arrow}AA] in exon IV in one allele, and two nucleotide deletions at codon 273 [AAA(Lys){r_arrow}A] in exon IV in the other allele. The remaining gene sequences were normal. The codon 318 mutation was found in one allele from the father, brother, and parents of the deceased paternal cousins. The codon 273 mutation was found in one allele of the mother and a sister. These findings confirmed inherited 3{beta}-HSD deficiency in the child caused by the compound heterozygous type II 3{beta}-HSD gene mutation. Both codons at codons 279 and 367, respectively, are predicted to result in an altered and truncated type II 3{beta}-HSD protein, thereby causing salt-wasting 3{beta}-HSD deficiency in the patient. 21 refs., 2 figs., 1 tab.

  8. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) shoot apex with a fungal phytase gene improves phosphorus acquisition.

    PubMed

    Ma, Zhiying; Liu, Jianfeng; Wang, Xingfen

    2013-01-01

    Cotton is an important world economic crop plant. It is considered that cotton is recalcitrant to in vitro proliferation. Somatic embryogenesis and plant regeneration has been successful by using hypocotyl, whereas it is highly genotype dependent. Here, a genotype-independent cotton regeneration protocol from shoot apices is presented. Shoot apices from 3- to 5-day-old seedlings of cotton are infected with an Agrobacterium strain, EHA105, carrying the binary vector pC-KSA contained phytase gene (phyA) and the marker gene neomycin phosphotransferase (NPTII), and directly regenerated as shoots in vitro. Rooted shoots can be obtained within 6-8 weeks. Plants that survived by leaf painting kanamycin (kan) were -further analyzed by DNA and RNA blottings. The transgenic plants with increased the phosphorus (P) acquisition efficiency were obtained following the transformation method. PMID:23143496

  9. Disentangling the effects of recombination, selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois.

    PubMed

    Mona, S; Crestanello, B; Bankhead-Dronnet, S; Pecchioli, E; Ingrosso, S; D'Amelio, S; Rossi, L; Meneguz, P G; Bertorelle, G

    2008-09-01

    The major histocompatibility complex (MHC) harbours some of the most polymorphic loci in vertebrate genomes. MHC genes are thought to be subject to some form of balancing selection, most likely pathogen-mediated selection. Hence, MHC genes are excellent candidates for exploring adaptive processes. In this study, we investigated the genetic variation at exon 2 of the DRB class II MHC locus in 191 alpine chamois (Rupicapra rupicapra) from 10 populations in the eastern Alps of Italy. In particular, we were interested in distinguishing and estimating the relative impact of selective and demographic factors, while taking into account the confounding effect of recombination. The extremely high d(n)/d(s) ratio and the presence of trans-species polymorphisms suggest that a strong long-term balancing selection effect has been operating at this locus throughout the evolutionary history of this species. We analysed patterns of genetic variation within and between populations, and the mitochondrial D-loop polymorphism patterns were analysed to provide a baseline indicator of the effects of demographic processes. These analyses showed that (i) the chamois experienced a demographic decline in the last 5000-30 000 years, most likely related to the postglacial elevation in temperature; (ii) this demographic process can explain the results of neutrality tests applied to MHC variation within populations, but cannot justify the much weaker divergence between populations implied by MHC as opposed to mitochondrial DNA; (iii) similar sets of divergent alleles are probably maintained with similar frequencies by balancing selection in different populations, and this mechanism is also operating in small isolated populations, which are strongly affected by drift. PMID:19238706

  10. IL-21 imposes a type II EBV gene expression on type III and type I B cells by the repression of C- and activation of LMP-1-promoter

    PubMed Central

    Kis, Loránd L.; Salamon, Daniel; Persson, Emma K.; Nagy, Noémi; Scheeren, Ferenc A.; Spits, Hergen; Klein, George; Klein, Eva

    2009-01-01

    Epstein–Barr virus (EBV) is associated with a variety of human tumors. Although the EBV-infected normal B cells in vitro and the EBV-carrying B cell lymphomas in immunodeficient patients express the full set of latent proteins (type III latency), the majority of EBV-associated malignancies express the restricted type I (EBNA-1 only) or type II (EBNA-1 and LMPs) viral program. The mechanisms responsible for these different latent viral gene expression patterns are only partially known. IL-21 is a potent B cell activator and plasma cell differentiation-inducer cytokine produced by CD4+ T cells. We studied its effect on EBV-carrying B cells. In type I Burkitt lymphoma (BL) cell lines and in the conditional lymphoblastoid cell line (LCL) ER/EB2-5, IL-21 potently activated STAT3 and induced the expression of LMP-1, but not EBNA-2. The IL-21-treated type I Jijoye M13 BL line ceased to proliferate, and this was paralleled by the induction of IRF4 and the down-regulation of BCL6 expression. In the type III LCLs and BL lines, IL-21 repressed the C-promoter-derived and LMP-2A mRNAs, whereas it up-regulated the expression of LMP-1 mRNAs. The IL-21-treated type III cells underwent plasma cell differentiation with the induction of Blimp-1, and high levels of Ig and Oct-2. IL-21 might be involved in the EBNA-2-independent expression of LMP-1 in EBV-carrying type II cells. In light of the fact that IL-21 is already in clinical trials for the treatment of multiple malignancies, the in vivo modulation of EBV gene expression by IL-21 might have therapeutic benefits for the EBV-carrying malignancies. PMID:20080768

  11. Characterization of Three mnp Genes of Fomitiporia mediterranea and Report of Additional Class II Peroxidases in the Order Hymenochaetales ? †

    PubMed Central

    Morgenstern, Ingo; Robertson, Deborah L.; Hibbett, David S.

    2010-01-01

    We report the sequence-based characterization and expression patterns of three manganese peroxidase genes from the white rot fungus and grape vine pathogen Fomitiporia mediterranea (Agaricomycotina, Hymenochaetales), termed Fmmnp1, Fmmnp2, and Fmmnp3. The predicted open reading frames (ORFs) are 1,516-, 1,351-, and 1,345-bp long and are interrupted by seven, four, and four introns, respectively. The deduced amino acid sequences encode manganese peroxidases (EC 1.11.1.13) containing 371, 369, and 371 residues, respectively, and are similar to the manganese peroxidases of the model white rot organism Phanerochaete chrysosporium. The expression of the genes is most likely differentially regulated, as revealed by real-time PCR analysis. Phylogenetic analysis reveals that other members of the order Hymenochaetales harbor mnp genes encoding proteins that are related only distantly to those of F. mediterranea. Furthermore, multiple partial lip- and mnp-like sequences obtained for Pycnoporus cinnabarinus (Agaricomycotina, Polyporales) suggest that lignin degradation by white rot taxa relies heavily on ligninolytic peroxidases and is not efficiently achieved by laccases only. PMID:20675443

  12. Selective pressures on MHC class II genes in the guppy (Poecilia reticulata) as inferred by hierarchical analysis of population structure.

    PubMed

    Herdegen, M; Babik, W; Radwan, J

    2014-11-01

    Genes of the major histocompatibility complex, which are the most polymorphic of all vertebrate genes, are a pre-eminent system for the study of selective pressures that arise from host-pathogen interactions. Balancing selection capable of maintaining high polymorphism should lead to the homogenization of MHC allele frequencies among populations, but there is some evidence to suggest that diversifying selection also operates on the MHC. However, the pattern of population structure observed at MHC loci is likely to depend on the spatial and/or temporal scale examined. Here, we investigated selection acting on MHC genes at different geographic scales using Venezuelan guppy populations inhabiting four regions. We found a significant correlation between MHC and microsatellite allelic richness across populations, which suggests the role of genetic drift in shaping MHC diversity. However, compared to microsatellites, more MHC variation was explained by differences between populations within larger geographic regions and less by the differences between the regions. Furthermore, among proximate populations, variation in MHC allele frequencies was significantly higher compared to microsatellites, indicating that selection acting on MHC may increase population structure at small spatial scales. However, in populations that have significantly diverged at neutral markers, the population-genetic signature of diversifying selection may be eradicated in the long term by that of balancing selection, which acts to preserve rare alleles and thus maintain a common pool of MHC alleles. PMID:25244157

  13. The Arabidopsis Mediator Complex Subunits MED16, MED14, and MED2 Regulate Mediator and RNA Polymerase II Recruitment to CBF-Responsive Cold-Regulated Genes[C][W][OPEN

    PubMed Central

    Hemsley, Piers A.; Hurst, Charlotte H.; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R.; De Cothi, Elizabeth A.; Steele, John F.; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation–induced freezing tolerance. In addition, these three subunits are required for low temperature–induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. PMID:24415770

  14. Salt Stress Inhibits the Repair of Photodamaged Photosystem II by Suppressing the Transcription and Translation of psbA Genes in Synechocystis1

    PubMed Central

    Allakhverdiev, Suleyman I.; Nishiyama, Yoshitaka; Miyairi, Sachio; Yamamoto, Hiroshi; Inagaki, Noritoshi; Kanesaki, Yu; Murata, Norio

    2002-01-01

    Light stress and salt stress are major environmental factors that limit the efficiency of photosynthesis. However, we have found that the effects of light and salt stress on photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 are completely different. Strong light induced photodamage to PSII, whereas salt stress inhibited the repair of the photodamaged PSII and did not accelerate damage to PSII directly. The combination of light and salt stress appeared to inactivate PSII very rapidly as a consequence of their synergistic effects. Radioactive labeling of cells revealed that salt stress inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern- and western-blotting analyses demonstrated that salt stress inhibited the transcription and the translation of psbA genes, which encode D1 protein. DNA microarray analysis indicated that the light-induced expression of various genes was suppressed by salt stress. Thus, our results suggest that salt stress inhibits the repair of PSII via suppression of the activities of the transcriptional and translational machinery. PMID:12428009

  15. An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation

    PubMed Central

    Sabbattini, Pierangela; Sjoberg, Marcela; Nikic, Svetlana; Frangini, Alberto; Holmqvist, Per-Henrik; Kunowska, Natalia; Carroll, Tom; Brookes, Emily; Arthur, Simon J.; Pombo, Ana; Dillon, Niall

    2014-01-01

    Methylated histones H3K9 and H3K27 are canonical epigenetic silencing modifications in metazoan organisms, but the relationship between the two modifications has not been well characterized. H3K9me3 coexists with H3K27me3 in pluripotent and differentiated cells. However, we find that the functioning of H3K9me3 is altered by H3S10 phosphorylation in differentiated postmitotic osteoblasts and cycling B cells. Deposition of H3K9me3/S10ph at silent genes is partially mediated by the mitogen- and stress-activated kinases (MSK1/2) and the Aurora B kinase. Acquisition of H3K9me3/S10ph during differentiation correlates with loss of paused S5 phosphorylated RNA polymerase II, which is present on Polycomb-regulated genes in embryonic stem cells. Reduction of the levels of H3K9me3/S10ph by kinase inhibition results in increased binding of RNAPIIS5ph and the H3K27 methyltransferase Ezh1 at silent promoters. Our results provide evidence of a novel developmentally regulated methyl-phospho switch that modulates Polycomb regulation in differentiated cells and stabilizes repressed states. PMID:24430871

  16. ANG II receptor subtype 1a gene knockdown in the subfornical organ prevents increased drinking behavior in bile duct-ligated rats.

    PubMed

    Walch, Joseph D; Nedungadi, T Prashant; Cunningham, J Thomas

    2014-09-15

    Bile duct ligation (BDL) causes congestive liver failure that initiates hemodynamic changes, resulting in dilutional hyponatremia due to increased water intake and vasopressin release. This project tested the hypothesis that angiotensin signaling at the subfornical organ (SFO) augments drinking behavior in BDL rats. A genetically modified adeno-associated virus containing short hairpin RNA (shRNA) for ANG II receptor subtype 1a (AT1aR) gene was microinjected into the SFO of rats to knock down expression. Two weeks later, BDL or sham surgery was performed. Rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. The rats were euthanized 28 days after BDL surgery for analysis. A group of rats was perfused for immunohistochemistry, and a second group was used for laser-capture microdissection for analysis of SFO AT1aR gene expression. BDL rats showed increased water intake that was attenuated in rats that received SFO microinjection of AT1aR shRNA. Among BDL rats treated with scrambled (control) and AT1aR shRNA, we observed an increased number of vasopressin-positive cells in the supraoptic nucleus that colocalized with ?FosB staining, suggesting increased vasopressin release in both groups. These results indicate that angiotensin signaling through the SFO contributes to increased water intake, but not dilutional hyponatremia, during congestive liver failure. PMID:25009217

  17. Chromatin-wide Profiling of DYRK1A Reveals a Role as a Gene-Specific RNA Polymerase II CTD Kinase.

    PubMed

    Di Vona, Chiara; Bezdan, Daniela; Islam, Abul B M M K; Salichs, Eulàlia; López-Bigas, Nuria; Ossowski, Stephan; de la Luna, Susana

    2015-02-01

    DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the kinase is recruited preferentially to promoters of genes actively transcribed by RNA polymerase II (RNAPII), which are functionally associated with translation, RNA processing, and cell cycle. DYRK1A-bound promoter sequences are highly enriched in a conserved palindromic motif, which is necessary to drive DYRK1A-dependent transcriptional activation. DYRK1A phosphorylates the C-terminal domain (CTD) of RNAPII at Ser2 and Ser5. Depletion of DYRK1A results in reduced association of RNAPII at the target promoters as well as hypophosphorylation of the RNAPII CTD along the target gene bodies. These results are consistent with DYRK1A being a transcriptional regulator by acting as a CTD kinase. PMID:25620562

  18. Slc39a1 to 3 (Subfamily II) Zip Genes in Mice have Unique Cell-Specific Functions during Adaptation to Zinc Deficiency

    PubMed Central

    Kambe, Taiho; Geiser, Jim; Lahner, Brett; Salt, David E.; Andrews, Glen K.

    2008-01-01

    Subfamily II of the solute-carrier (Slc)39a family contains three highly conserved members (ZIPs 1 to 3) that share a twelve amino acid signature sequence present in the putative fourth transmembrane domain and function as zinc transporters in transfected cells. The physiological significance of this genetic redundancy is unknown. Herein, we report that the complete elimination of all three of these Zip genes, by targeted mutagenesis and cross-breeding mice, causes no overt phenotypic effect. When fed a zinc-adequate diet, several indicators of zinc status were indistinguishable between wild-type and triple-knockout mice; including embryonic morphogenesis and growth, alkaline phosphatase activity in the embryo, and ZIP4 protein in the visceral yolk sac and initial rates (30 min) of accumulation/retention of 67Zn in liver and pancreas. When fed a zinc-deficient diet, embryonic membrane-bound alkaline phosphatase activity was reduced to a much greater extent and 80% of the embryos in the triple-knock mice developed abnormally compared to 12% of the embryos in wild-type mice. During zinc deficiency, the accumulation/retention (3 hr) of 67Zn in the liver and pancreas of weanlings was significantly impaired in the triple-knockout mice compared to wild-type mice. Thus, none of these three mammalian Zip genes apparently plays a critical role in zinc homeostasis when zinc is replete, but they play important, non-compensatory roles when this metal is deficient. PMID:18353881

  19. Ca2+/calmodulin-dependent kinase II triggers cell membrane injury by inducing complement factor B gene expression in the mouse heart.

    PubMed

    Singh, Madhu V; Kapoun, Ann; Higgins, Linda; Kutschke, William; Thurman, Joshua M; Zhang, Rong; Singh, Minati; Yang, Jinying; Guan, Xiaoqun; Lowe, John S; Weiss, Robert M; Zimmermann, Kathy; Yull, Fiona E; Blackwell, Timothy S; Mohler, Peter J; Anderson, Mark E

    2009-04-01

    Myocardial Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-kappaB pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-kappaB activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb-/- mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes. PMID:19273909

  20. Heat-shock protein 70-1 and HLA class II gene polymorphisms associated with celiac disease susceptibility in Navarra (Spain).

    PubMed

    Ramos-Arroyo, M A; Feijoó, E; Sánchez-Valverde, F; Aranburu, E; Irisarri, N; Olivera, J E; Valiente, A

    2001-08-01

    Heat shock proteins (HSP) are thought to play a role in the immune response making probable their contribution to celiac disease (CD). We studied the polymorphisms in the 5' regulatory region of the HSP70-1 gene and performed genomic HLA-DQ and -DR typing in 128 CD patients and 94 healthy controls from Navarra (Spain). The frequency of the C allele of the HSP70-1, characterized by the intermediate electrophoretic mobility of DNA, was significantly increased among CD patients (64.5% vs 37.2%. p <1 x 10(-7)). When subjects were stratified by the HLA II genotype, differences were statistically significant between DR3-negative or DR3-DQB1*02-negative CD patients and matched controls. Homozygosity for the DQB1*02 allele was present in 48.4% of CD patients and 12.8% of controls (OR = 6.4; CI:3.1 to 13.8; p <1 x 10(-7)). Similar increased risk was observed for DQB1*02/*02, DRB1*03/-, or DRB1*03/07 patients. Furthermore, those individuals expressing the classical HLA alleles in CD (DQB1*02/*02, DRB1*03/*07) who also carried the HSP70-1 CC genotype were twelve times more likely to develop the disease than the matched controls. We therefore conclude that although HSP70-1 gene does not seem to be primarily associated with CD, it might be a component of the high risk haplotype, playing a role as an additional predisposing gene for the disease. PMID:11476906

  1. Association between the c.*229C>T polymorphism of the topoisomerase II? binding protein 1 (TopBP1) gene and breast cancer.

    PubMed

    Forma, Ewa; Brzezia?ska, Ewa; Krze?lak, Anna; Chwatko, Gra?yna; Jó?wiak, Pawe?; Szymczyk, Agnieszka; Smolarz, Beata; Romanowicz-Makowska, Hanna; Ró?a?ski, Waldemar; Bry?, Magdalena

    2013-05-01

    Topoisomerase II? binding protein 1 (TopBP1) is involved in cell survival, DNA replication, DNA damage repair and cell cycle checkpoint control. The biological function of TopBP1 and its close relation with BRCA1 prompted us to investigate whether alterations in the TopBP1 gene can influence the risk of breast cancer. The aim of this study was to examine the association between five polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 3'UTR region of the TopBP1 gene and breast cancer risk as well as allele-specific gene expression. Five hundred thirty-four breast cancer patients and 556 population controls were genotyped for these SNPs. Allele-specific TopBP1 mRNA and protein expressions were determined by using real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with breast cancer. Compared to homozygous common allele carriers, heterozygous and homozygous for the T variant had significantly increased risk of breast cancer (adjusted odds ratio = 3.81, 95% confidence interval: 1.63-8.34, p = 0.001). Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT or TT genotype. There was a significant association between the rs115160714 and tumor grade and stage. Most carriers of minor allele had a high grade (G3) tumors classified as T2-T4N1M0. Our study raises a possibility that a genetic variation of TopBP1 may be implicated in the etiology of breast cancer. PMID:23277395

  2. Development of a gene knockout system using mobile group II introns (Targetron) and genetic disruption of acid production pathways in Clostridium beijerinckii.

    PubMed

    Wang, Yi; Li, Xiangzhen; Milne, Caroline B; Janssen, Holger; Lin, Weiyin; Phan, Gloria; Hu, Huiying; Jin, Yong-Su; Price, Nathan D; Blaschek, Hans P

    2013-10-01

    Clostridium beijerinckii is a well-known solvent-producing microorganism with great potential for biofuel and biochemical production. To better understand and improve the biochemical pathway to solvents, the development of genetic tools for engineering C. beijerinckii is highly desired. Based on mobile group II intron technology, a targetron gene knockout system was developed for C. beijerinckii in this study. This system was successfully employed to disrupt acid production pathways in C. beijerinckii, leading to pta (encoding phosphotransacetylase)- and buk (encoding butyrate kinase)-negative mutants. In addition to experimental characterization, the mutant phenotypes were analyzed in the context of our C. beijerinckii genome-scale model. Compared to those of the parental strain (C. beijerinckii 8052), acetate production in the pta mutant was substantially reduced and butyrate production was remarkably increased, while solvent production was dependent on the growth medium. The pta mutant also produced much higher levels of lactate, suggesting that disrupting pta influenced the energy generation and electron flow pathways. In contrast, acetate and butyrate production in the buk mutant was generally similar to that of the wild type, but solvent production was consistently 20 to 30% higher and glucose consumption was more rapid and complete. Our results suggest that the acid and solvent production of C. beijerinckii can be effectively altered by disrupting the acid production pathways. As the gene disruption method developed in this study does not leave any antibiotic marker in a disrupted allele, multiple and high-throughput gene disruption is feasible for elucidating genotype and phenotype relationships in C. beijerinckii. PMID:23872562

  3. The Type II Secretion Pathway in Vibrio cholerae Is Characterized by Growth Phase-Dependent Expression of Exoprotein Genes and Is Positively Regulated by ?E

    PubMed Central

    Zielke, Ryszard A.; Simmons, Ryan S.; Park, Bo R.; Nonogaki, Mariko; Emerson, Sarah

    2014-01-01

    Vibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that under laboratory growth conditions, the T2S complex was continuously expressed throughout V. cholerae growth, whereas there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure of V. cholerae to different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S. Subsequent screening of a V. cholerae genomic library suggested that ?E stress response, phosphate metabolism, and the second messenger 3?,5?-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on ?E, we discovered that the upstream region of the T2S operon possesses both the consensus ?E and ?70 signatures, and deletion of the ?E binding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of ?E stimulated transcription of T2S in wild-type and isogenic ?rpoE strains of V. cholerae, providing additional support for the idea that the T2S complex belongs to the ?E regulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness of V. cholerae in different ecological niches. PMID:24733097

  4. Purifying Selection and Birth-and-Death Evolution in the Class II Hydrophobin Gene Families of the Ascomycete Trichoderma/Hypocrea

    SciTech Connect

    kubicek, Christian P.; Baker, Scott E.; Gamauf, Christian; Kenerley, Chuck; Druzhinina, Irina S.

    2008-01-10

    Hydrophobins are proteins containing eight conserved cysteine residues that occur uniquely in mycelial fungi, where their main function is to confer hydrophobicity to fungal surfaces in contact with air and during attachment of hyphae to hydrophobic surfaces of hosts, symbiotic partners or of themselves resulting in morphogenetic signals. Based on their hydropathy patterns and their solubility characteristics, they are classified in class I and class II hydrophobins, the latter being found only in ascomycetes. Here we have investigated the mechanisms driving the evolution of the class II hydrophobins in nine species of the mycoparasitic ascomycetous genus Trichoderma/Hypocrea, using three fully sequenced genomes (H. jecorina=T. reesei, H. atroviridis=T. atroviride; H. virens=T. virens) and a total of 14.000 ESTs of six others (T. asperellum, H. lixii=T. harzianum, T. aggressivum var. europeae, T. longibrachiatum, T. cf. viride). The former three contained six, ten and nine members, which is the highest number found in any other ascomycete so far. They all showed the conserved four beta-strands/one helix structure, which is stabilized by four disulfide bonds. In addition, a small number of these HFBs contained an extended N-terminus rich in either praline and aspartate, or glycine-asparagine. Phylogenetic analysis reveals a mosaic of terminal clades contain duplicated genes and shows only three reasonably supported clades. Calculation of the ratio of differences in synonymous vs. non-synonymous nucleotide substitutions provides evidence for strong purifying selection (KS/Ka >> 1). A genome database search for class II HFBs from other ascomycetes retrieved a much smaller number of hydrophobins (2-4) from each species, and most of them were from Pyrenomycetes. A combined phylogeny of these sequences with those of Trichoderma showed that the Trichoderma HFBs mostly formed their own clades, whereas those of other pyrenomycetes occured in shared clades. Our study shows that the genus Trichoderma/Hypocrea has a proliferated arsenal of class II hydrophobins which arose by purifying selection and birth-and-death evolution.

  5. Analysis of Gln223Agr Polymorphism of Leptin Receptor Gene in Type II Diabetic Mellitus Subjects among Malaysians

    PubMed Central

    Etemad, Ali; Ramachandran, Vasudevan; Pishva, Seyyed Reza; Heidari, Farzad; Aziz, Ahmad Fazli Abdul; Yusof, Ahmad Khairuddin Mohamed; Pei, Chong Pei; Ismail, Patimah

    2013-01-01

    Leptin is known as the adipose peptide hormone. It plays an important role in the regulation of body fat and inhibits food intake by its action. Moreover, it is believed that leptin level deductions might be the cause of obesity and may play an important role in the development of Type 2 Diabetes Mellitus (T2DM), as well as in cardiovascular diseases (CVD). The Leptin Receptor (LEPR) gene and its polymorphisms have not been extensively studied in relation to the T2DM and its complications in various populations. In this study, we have determined the association of Gln223Agr loci of LEPR gene in three ethnic groups of Malaysia, namely: Malays, Chinese and Indians. A total of 284 T2DM subjects and 281 healthy individuals were recruited based on International Diabetes Federation (IDF) criteria. Genomic DNA was extracted from the buccal specimens of the subjects. The commercial polymerase chain reaction (PCR) method was carried out by proper restriction enzyme MSP I to both amplify and digest the Gln223Agr polymorphism. The p-value among the three studied races was 0.057, 0.011 and 0.095, respectively. The values such as age, WHR, FPG, HbA1C, LDL, HDL, Chol and Family History were significantly different among the subjects with Gln223Agr polymorphism of LEPR (p < 0.05). PMID:24051404

  6. Analysis of Gln223Agr polymorphism of Leptin Receptor Gene in type II diabetic mellitus subjects among Malaysians.

    PubMed

    Etemad, Ali; Ramachandran, Vasudevan; Pishva, Seyyed Reza; Heidari, Farzad; Aziz, Ahmad Fazli Abdul; Yusof, Ahmad Khairuddin Mohamed; Pei, Chong Pei; Ismail, Patimah

    2013-01-01

    Leptin is known as the adipose peptide hormone. It plays an important role in the regulation of body fat and inhibits food intake by its action. Moreover, it is believed that leptin level deductions might be the cause of obesity and may play an important role in the development of Type 2 Diabetes Mellitus (T2DM), as well as in cardiovascular diseases (CVD). The Leptin Receptor (LEPR) gene and its polymorphisms have not been extensively studied in relation to the T2DM and its complications in various populations. In this study, we have determined the association of Gln223Agr loci of LEPR gene in three ethnic groups of Malaysia, namely: Malays, Chinese and Indians. A total of 284 T2DM subjects and 281 healthy individuals were recruited based on International Diabetes Federation (IDF) criteria. Genomic DNA was extracted from the buccal specimens of the subjects. The commercial polymerase chain reaction (PCR) method was carried out by proper restriction enzyme MSP I to both amplify and digest the Gln223Agr polymorphism. The p-value among the three studied races was 0.057, 0.011 and 0.095, respectively. The values such as age, WHR, FPG, HbA1C, LDL, HDL, Chol and Family History were significantly different among the subjects with Gln223Agr polymorphism of LEPR (p < 0.05). PMID:24051404

  7. Leaky splicing mutation in the acid maltase gene is associated with delayed onset of glycogenosis type II

    SciTech Connect

    Boerkoel, C.F.; Exelbert, R.; Nicastri, C.; Nichols, R.C.; Plotz, P.H.; Raben, N. [National Institutes of Health, Bethesda, MD (United States); Miller, F.W. [Food and Drug Administration, Rockville, MD (United States)

    1995-04-01

    An autosomal recessive deficiency of acid {alpha}-glucosidase (GAA), type II glycogenosis, is genetically and clinically heterogeneous. The discovery of an enzyme-inactivating genomic deletion of exon 18 in three unrelated genetic compound patients - two infants and and adult - provided a rare opportunity to analyze the effect of the second mutation in patients who displayed dramatically different phenotypes. A deletion of Lys-903 in one patient and a substitution of Arg for Leu-299 in another resulted in the fatal infantile form. In the adult, a T-to-G base change at position-13 of intron 1 resulted in alternatively spliced transcripts with deletion of exon 2, the location of the start codon. The low level of active enzyme (12% of normal) generated from the leakage of normally spliced mRNA sustained the patient to adult life. 61 refs., 9 figs., 3 tabs.

  8. An endemic frog harbors multiple expression loci with different patterns of variation in the MHC class II B gene.

    PubMed

    Shu, Yi-Lin; Hong, Pei; Yang, Yi-Wen; Wu, Hai-Long

    2013-12-01

    The Chinese concave-eared torrent frog (Odorrana tormota) was the first non-mammalian vertebrate demonstrated to both produce and perceive ultrasonic frequencies. Due to habitat degradation caused by human activity, currently, the species is at the edge of extinction. In this study, we demonstrated that the species harbors at least four expressed major histocompatibility complex (MHC) class II B loci. Four distinct intron 2 sequences and three intron 1 sequences were isolated from genomic DNA. Additionally, eight alleles were recovered from three cDNA samples, of which five could be assigned to three individual loci while the locus identity of the remaining three alleles could not be determined. Based on the intron sequences, locus-specific primers were designed for Odto-A, Odto-B, and Odto-C. Of these loci, Odto-B was monomorphic in Huangshan population (n?=?32), and Odto-C displayed low allelic polymorphism with only three similar sequences obtained from partial individuals. In contrast, Odto-A showed high allelic diversity with 20 unique alleles recovered from the population. No inter- or intra-allele recombination was detected. However, 10 positive selected amino acid residues and an excess of non-synonymous substitutions were detected in the putative antigen binding sites (ABS), these results combined with trans-species evolution suggested some form of balancing selection that maintained higher allelic polymorphism for the locus. Our findings demonstrate that despite the effects of a severely shrinking population, O. tormota exhibits high polymorphism compared to other amphibians, both in terms of the allele number and diversity across the MHC class II B loci. PMID:23900979

  9. Transgenic almond (Prunus dulcis Mill.) plants obtained by Agrobacterium-mediated transformation of leaf explants

    Microsoft Academic Search

    C. M. Miguel; M. M. Oliveira

    1999-01-01

    Almond (Prunus dulcis Mill.) leaves were transformed with the marker genes gusA (?-glucuronidase) and nptII (neomycin phosphotransferase II) via Agrobacterium-mediated transformation. Bacterial strains and preculture of explants affected efficiency of gene transfer evaluated by transient\\u000a expression assays. Following transformation, shoots were induced from primary explants on medium without kanamycin and exposed\\u000a to selection 20 days after cocultivation. From 1419 original

  10. An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens

    Microsoft Academic Search

    Julie Belles-Isles; Mathieu Dusabenyagasani; Francine M. Tremblay

    2001-01-01

    An efficient and reproducible procedure for the transformation of white spruce (Picea glauca (Moench) Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, con- taining the neomycin phosphotransferase II (nptII) gene providing kanamycin

  11. Agrobacterium -mediated transformation and regeneration of kiwi fruit

    Microsoft Academic Search

    Chiyomi Uematsu; Makoto Murase; Hiroaki Ichikawa; Jun Imamura

    1991-01-01

    Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the ß-glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25µg\\/ml kanamycin

  12. HLA class II gene associations in African American Type 1 diabetes reveal a protective HLA-DRB1*03 haplotype

    PubMed Central

    Howson, J M M; Roy, M S; Zeitels, L; Stevens, H; Todd, J A

    2013-01-01

    Aims Owing to strong linkage disequilibrium between markers, pinpointing disease associations within genetic regions is difficult in European ancestral populations, most notably the very strong association of the HLA-DRB1*03-DQA1*05:01-DQB1*02:01 haplotype with Type 1 diabetes risk, which is assumed to be because of a combination of HLA-DRB1 and HLA-DQB1. In contrast, populations of African ancestry have greater haplotype diversity, offering the possibility of narrowing down regions and strengthening support for a particular gene in a region being causal. We aimed to study the human leukocyte antigen (HLA) region in African American Type 1 diabetes. Methods Two hundred and twenty-seven African American patients with Type 1 diabetes and 471 African American control subjects were tested for association at the HLA class II genes, HLA-DRB1, HLA-DQA1, HLA-DQB1 and 5147 single nucleotide polymorphisms across the major histocompatibility complex region using logistic regression models. Population admixture was accounted for with principal components analysis. Results Single nucleotide polymorphism marker associations were explained by the HLA associations, with the major peak over the class II loci. The HLA association overall was extremely strong, as expected for Type 1 diabetes, even in African Americans in whom diabetes diagnosis is heterogeneous. In addition, there were unique features: the HLA-DRB1*03 haplotype was split into HLA-DRB1*03:01, which confers greatest susceptibility in these samples (odds ratio 3.17, 95% CI 1.72–5.83) and HLA-DRB1*03:02, an allele rarely observed in Europeans, which confers the greatest protection in these African American samples (odds ratio 0.22, 95% CI 0.09–0.55). Conclusions The unique diversity of the African HLA region we have uncovered supports a specific and major role for HLA-DRB1 in HLA-DRB1*03 haplotype-associated Type 1 diabetes risk. PMID:23398374

  13. Generation of BAC transgenic tadpoles enabling live imaging of motoneurons by using the urotensin II-related peptide (ust2b) gene as a driver.

    PubMed

    Bougerol, Marion; Auradé, Frédéric; Lambert, François M; Le Ray, Didier; Combes, Denis; Thoby-Brisson, Muriel; Relaix, Frédéric; Pollet, Nicolas; Tostivint, Hervé

    2015-01-01

    Xenopus is an excellent tetrapod model for studying normal and pathological motoneuron ontogeny due to its developmental morpho-physiological advantages. In mammals, the urotensin II-related peptide (UTS2B) gene is primarily expressed in motoneurons of the brainstem and the spinal cord. Here, we show that this expression pattern was conserved in Xenopus and established during the early embryonic development, starting at the early tailbud stage. In late tadpole stage, uts2b mRNA was detected both in the hindbrain and in the spinal cord. Spinal uts2b+ cells were identified as axial motoneurons. In adult, however, the uts2b expression was only detected in the hindbrain. We assessed the ability of the uts2b promoter to drive the expression of a fluorescent reporter in motoneurons by recombineering a green fluorescent protein (GFP) into a bacterial artificial chromosome (BAC) clone containing the entire X. tropicalis uts2b locus. After injection of this construction in one-cell stage embryos, a transient GFP expression was observed in the spinal cord of about a quarter of the resulting animals from the early tailbud stage and up to juveniles. The GFP expression pattern was globally consistent with that of the endogenous uts2b in the spinal cord but no fluorescence was observed in the brainstem. A combination of histological and electrophysiological approaches was employed to further characterize the GFP+ cells in the larvae. More than 98% of the GFP+ cells expressed choline acetyltransferase, while their projections were co-localized with ?-bungarotoxin labeling. When tail myotomes were injected with rhodamine dextran amine crystals, numerous double-stained GFP+ cells were observed. In addition, intracellular electrophysiological recordings of GFP+ neurons revealed locomotion-related rhythmic discharge patterns during fictive swimming. Taken together our results provide evidence that uts2b is an appropriate driver to express reporter genes in larval motoneurons of the Xenopus spinal cord. PMID:25658845

  14. Non-coding RNA derived from the region adjacent to the human HO-1 E2 enhancer selectively regulates HO-1 gene induction by modulating Pol II binding

    PubMed Central

    Maruyama, Atsushi; Mimura, Junsei; Itoh, Ken

    2014-01-01

    Recent studies have disclosed the function of enhancer RNAs (eRNAs), which are long non-coding RNAs transcribed from gene enhancer regions, in transcriptional regulation. However, it remains unclear whether eRNAs are involved in the regulation of human heme oxygenase-1 gene (HO-1) induction. Here, we report that multiple nuclear-enriched eRNAs are transcribed from the regions adjacent to two human HO-1 enhancers (i.e. the distal E2 and proximal E1 enhancers), and some of these eRNAs are induced by the oxidative stress-causing reagent diethyl maleate (DEM). We demonstrated that the expression of one forward direction (5? to 3?) eRNA transcribed from the human HO-1 E2 enhancer region (named human HO-1enhancer RNA E2-3; hereafter called eRNA E2-3) was induced by DEM in an NRF2-dependent manner in HeLa cells. Conversely, knockdown of BACH1, a repressor of HO-1 transcription, further increased DEM-inducible eRNA E2-3 transcription as well as HO-1 expression. In addition, we showed that knockdown of eRNA E2-3 selectively down-regulated DEM-induced HO-1 expression. Furthermore, eRNA E2-3 knockdown attenuated DEM-induced Pol II binding to the promoter and E2 enhancer regions of HO-1 without affecting NRF2 recruitment to the E2 enhancer. These findings indicate that eRNAE2-3 is functional and is required for HO-1 induction. PMID:25404134

  15. Growth-inhibitory activity and downregulation of the class II tumor-suppressor gene H-rev107 in tumor cell lines and experimental tumors.

    PubMed

    Sers, C; Emmenegger, U; Husmann, K; Bucher, K; Andres, A C; Schäfer, R

    1997-02-24

    The H-rev107 gene is a new class II tumor suppressor, as defined by its reversible downregulation and growth-inhibiting capacity in HRAS transformed cell lines. Overexpression of the H-rev107 cDNA in HRAS-transformed ANR4 hepatoma cells or in FE-8 fibroblasts resulted in 75% reduction of colony formation. Cell populations of H-rev107 transfectants showed an attenuated tumor formation in nude mice. Cells explanted from tumors or maintained in cell culture for an extended period of time no longer exhibited detectable levels of the H-rev107 protein, suggesting strong selection against H-rev107 expression in vitro and in vivo. Expression of the truncated form of H-rev107 lacking the COOH-terminal membrane associated domain of 25 amino acids, had a weaker inhibitory effect on proliferation in vitro and was unable to attenuate tumor growth in nude mice. The H-rev107 mRNA is expressed in most adult rat tissues, and immunohistochemical analysis showed expression of the protein in differentiated epithelial cells of stomach, of colon and small intestine, in kidney, bladder, esophagus, and in tracheal and bronchial epithelium. H-rev107 gene transcription is downregulated in rat cell lines derived from liver, kidney, and pancreatic tumors and also in experimental mammary tumors expressing a RAS transgene. In colon carcinoma cell lines only minute amounts of protein were detectable. Thus, downregulation of H-rev107 expression may occur at the level of mRNA or protein. PMID:9049257

  16. Growth-inhibitory Activity and Downregulation of the Class II Tumor-suppressor Gene H-rev107 in Tumor Cell Lines and Experimental Tumors

    PubMed Central

    Sers, Christine; Emmenegger, Urban; Husmann, Knut; Bucher, Katharina; Andres, Ann-Catherine; Schäfer, Reinhold

    1997-01-01

    The H-rev107 gene is a new class II tumor suppressor, as defined by its reversible downregulation and growth-inhibiting capacity in HRAS transformed cell lines. Overexpression of the H-rev107 cDNA in HRAS-transformed ANR4 hepatoma cells or in FE-8 fibroblasts resulted in 75% reduction of colony formation. Cell populations of H-rev107 transfectants showed an attenuated tumor formation in nude mice. Cells explanted from tumors or maintained in cell culture for an extended period of time no longer exhibited detectable levels of the H-rev107 protein, suggesting strong selection against H-rev107 expression in vitro and in vivo. Expression of the truncated form of H-rev107 lacking the COOH-terminal membrane associated domain of 25 amino acids, had a weaker inhibitory effect on proliferation in vitro and was unable to attenuate tumor growth in nude mice. The H-rev107 mRNA is expressed in most adult rat tissues, and immunohistochemical analysis showed expression of the protein in differentiated epithelial cells of stomach, of colon and small intestine, in kidney, bladder, esophagus, and in tracheal and bronchial epithelium. H-rev107 gene transcription is downregulated in rat cell lines derived from liver, kidney, and pancreatic tumors and also in experimental mammary tumors expressing a RAS transgene. In colon carcinoma cell lines only minute amounts of protein were detectable. Thus, downregulation of H-rev107 expression may occur at the level of mRNA or protein. PMID:9049257

  17. Generation of BAC Transgenic Tadpoles Enabling Live Imaging of Motoneurons by Using the Urotensin II-Related Peptide (ust2b) Gene as a Driver

    PubMed Central

    Bougerol, Marion; Auradé, Frédéric; Lambert, François M.; Le Ray, Didier; Combes, Denis; Thoby-Brisson, Muriel; Relaix, Frédéric; Pollet, Nicolas; Tostivint, Hervé

    2015-01-01

    Xenopus is an excellent tetrapod model for studying normal and pathological motoneuron ontogeny due to its developmental morpho-physiological advantages. In mammals, the urotensin II-related peptide (UTS2B) gene is primarily expressed in motoneurons of the brainstem and the spinal cord. Here, we show that this expression pattern was conserved in Xenopus and established during the early embryonic development, starting at the early tailbud stage. In late tadpole stage, uts2b mRNA was detected both in the hindbrain and in the spinal cord. Spinal uts2b+ cells were identified as axial motoneurons. In adult, however, the uts2b expression was only detected in the hindbrain. We assessed the ability of the uts2b promoter to drive the expression of a fluorescent reporter in motoneurons by recombineering a green fluorescent protein (GFP) into a bacterial artificial chromosome (BAC) clone containing the entire X. tropicalis uts2b locus. After injection of this construction in one-cell stage embryos, a transient GFP expression was observed in the spinal cord of about a quarter of the resulting animals from the early tailbud stage and up to juveniles. The GFP expression pattern was globally consistent with that of the endogenous uts2b in the spinal cord but no fluorescence was observed in the brainstem. A combination of histological and electrophysiological approaches was employed to further characterize the GFP+ cells in the larvae. More than 98% of the GFP+ cells expressed choline acetyltransferase, while their projections were co-localized with ?-bungarotoxin labeling. When tail myotomes were injected with rhodamine dextran amine crystals, numerous double-stained GFP+ cells were observed. In addition, intracellular electrophysiological recordings of GFP+ neurons revealed locomotion-related rhythmic discharge patterns during fictive swimming. Taken together our results provide evidence that uts2b is an appropriate driver to express reporter genes in larval motoneurons of the Xenopus spinal cord. PMID:25658845

  18. Association between Gene Expression Profiles and Clinical Outcome of Pemetrexed-Based Treatment in Patients with Advanced Non-Squamous Non-Small Cell Lung Cancer: Exploratory Results from a Phase II Study

    PubMed Central

    Fennell, Dean A.; Myrand, Scott P.; Nguyen, Tuan S.; Ferry, David; Kerr, Keith M.; Maxwell, Perry; Moore, Stephen D.; Visseren-Grul, Carla; Das, Mayukh; Nicolson, Marianne C.

    2014-01-01

    Introduction We report exploratory gene-expression profiling data from a single-arm Phase-II-study in patients with non-squamous (ns)NSCLC treated with pemetrexed and cisplatin. Previously disclosed results indicated a significant association of low thymidylate-synthase (TS)-expression with longer progression-free and overall survival (PFS/OS). Methods Treatment-naïve nsNSCLC patients (IIIB/IV) received 4 cycles of pemetrexed/cisplatin; non-progressing patients continued on pemetrexed-maintenance. Diagnostic tissue-samples were used to assess TS-expression by immunohistochemistry (IHC) and mRNA-expression array-profiling (1,030 lung cancer-specific genes). Cox proportional-hazard models were applied to explore the association between each gene and PFS/OS. Genes significantly correlated with PFS/OS were further correlated with TS-protein expression (Spearman-rank). Unsupervised clustering was applied to all evaluable samples (n?=?51) for all 1,030 genes and an overlapping 870-gene subset associated with adenocarcinoma (ADC, n?=?47). Results 51/70 tissue-samples (72.9%) were evaluable; 9 of 1,030 genes were significantly associated with PFS/OS (unadjusted p<0.01, genes: Chromosome 16 open reading frame 89, napsin A, surfactant protein B, aquaporin 4, TRAF2- and Nck-interacting kinase, Lysophosphatidylcholine acyltransferase 1, Interleukin 1 receptor type II, NK2 homeobox 1, ABO glycosyl-transferase); expression for all except IL1R2 correlated negatively with nuclear TS-expression (statistically significant for 5/8 genes, unadjusted p<0.01). Cluster-analysis based on 1,030 genes revealed no clear trend regarding PFS/OS; the ADC-based cluster analysis identified 3 groups (n?=?21/11/15) with median (95%CI) PFS of 8.1(6.9,NE)/2.4(1.2,NE)/4.4(1.2,NE) months and OS of 20.3(17.5,NE)/4.3(1.4,NE)/8.3(3.9,NE) months, respectively. Conclusions These exploratory gene-expression profiling results describe genes potentially linked to low TS-expression. Nine genes were significantly associated with PFS/OS but could not be differentiated as prognostic or predictive as this was a single-arm study. Although these hypotheses-generating results are interesting, they provide no evidence to change the current histology-based treatment approach with pemetrexed. PMID:25250715

  19. Aminoglycoside antibiotics: structure, functions and effects on in vitro plant culture and genetic transformation protocols

    Microsoft Academic Search

    I. M. G. Padilla; L. Burgos

    2010-01-01

    Plant transformation protocols generally involve the use of selectable marker genes for the screening of transgenic material.\\u000a The bacterial gene nptII, coding for a neomycin phosphotransferase, and the hpt gene, coding for a hygromycin phosphotransferase, are frequently used. These enzymes detoxify aminoglycoside antibiotics\\u000a by phosphorylation, thereby permitting cell growth in the presence of antibiotics. Nevertheless, the screening for transgenic\\u000a regenerated

  20. Transformation of elite white poplar (Populus alba L.) cv. '?Villafranca' and evaluation of herbicide resistance

    Microsoft Academic Search

    M. Confalonieri; B. Belenghi; A. Balestrazzi; S. Negri; G. Facciotto; G. Schenone; M. Delledonne

    2000-01-01

    Transgenic white poplar plants (Populus alba L.) expressing the nptII gene and the bar gene from Streptomyces hygroscopicus have been produced using Agrobacterium tumefaciens-mediated gene transfer. Eleven kanamycin-resistant plant lines were obtained with a transformation frequency of 7%. Successful\\u000a genetic transformation was confirmed by Southern and northern analyses. The level of resistance to the commercial preparation\\u000a of phosphinothricin (Basta; Roussel-Hoechst

  1. Matrix attachment regions (MARs) enhance transformation frequency and transgene expression in poplar

    Microsoft Academic Search

    KYUNG-HWAN HAN; CAIPING MA; STEVEN H. STRAUSS

    1997-01-01

    We tested the value of a matrix attachment region (MAR) fragment derived from a tobacco gene for increasing the frequency of Agrobacterium-mediated transformation. A binary vector that carried a GUS reporter gene containing an intron and an nptII gene was modified to contain flanking MAR elements within the T-DNA borders. Vectors containing or lacking MARs were then used to transform

  2. Developing an Agrobacterium tumefaciens -mediated genetic transformation for a selenium-hyperaccumulator Astragalus racemosus

    Microsoft Academic Search

    Diane E. Darlington; Chiu-Yueh Hung; Jiahua Xie

    2009-01-01

    Agrobacterium\\u000a tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The\\u000a optimal concentration of kanamycin that could effectively inhibit cell growth and division in

  3. Polymorphism in the tumour necrosis factor receptor II gene is associated with circulating levels of soluble tumour necrosis factor receptors in rheumatoid arthritis

    PubMed Central

    Glossop, John R; Dawes, Peter T; Nixon, Nicola B; Mattey, Derek L

    2005-01-01

    Levels of soluble tumour necrosis factor receptors (sTNFRs) are elevated in the circulation of patients with rheumatoid arthritis (RA). Although these receptors can act as natural inhibitors of tumour necrosis factor-?, levels of sTNFRs in RA appear to be insufficient to prevent tumour necrosis factor-? induced inflammation. The factors that regulate circulating levels of sTNFRs are unclear, but polymorphisms in the tumour necrosis factor receptor genes may play a role. We investigated the relationship between polymorphisms in the tumour necrosis factor receptor I (TNF-RI) and II (TNF-RII) genes and levels of sTNFRs in two groups of Caucasian RA patients: one with early (disease duration ?2 years; n = 103) and one with established disease (disease duration ?5 years; n = 151). PCR restriction fragment length polymorphism analysis was used to genotype patients for the A36G polymorphism in the TNF-RI gene and the T676G polymorphism in TNF-RII. Levels of sTNFRs were measured using ELISA. We also isolated T cells from peripheral blood of 58 patients with established RA with known TNF-R genotypes, and release of sTNFRs into the culture medium was measured in cells incubated with or without phytohaemagglutinin. Serum levels of the two sTNFRs (sTNF-RI and sTNF-RII) were positively correlated in both populations, and the level of each sTNFR was significantly higher in the patients with established disease (P < 0.0001). Multiple regression analyses corrected for age, sex and disease duration revealed a significant trend toward decreasing sTNF-RI and sTNF-RII levels across the TNF-RII genotypes (TT > TG > GG) of patients with established disease (P for trend = 0.01 and P for trend = 0.03, respectively). A similar nonsignificant trend was seen for early disease. No relationship with the TNF-RI A36G polymorphism was observed. sTNFRs released by isolated T cells exhibited a similar trend toward decreasing levels according to TNF-RII genotype, although only the association with levels of sTNF-RII was significant. Strong correlations were found between levels of circulating sTNFRs and levels released by T cells in vitro. Our data indicate that the T676G polymorphism in TNF-RII is associated with levels of sTNFRs released from peripheral blood T cells, and with circulating levels of sTNFR in patients with RA. PMID:16277675

  4. A new mutation of the PCNT gene in a Colombian patient with microcephalic osteodysplastic primordial dwarfism type II: a case report

    PubMed Central

    2014-01-01

    Introduction Microcephalic osteodysplastic primordial dwarfism is a syndrome characterized by the presence of intrauterine growth restriction, post-natal growth deficiency and microcephaly. Microcephalic osteodysplastic primordial dwarfism type II is the most distinctive syndrome in this group of entities. Individuals affected by this disease present at an adult height of less than 100cm, a post-pubertal head circumference of 40cm or less, mild mental retardation, an outgoing personality and bone dysplasia. Case presentation We report the first case of a five-year-old Colombian boy of mixed race ancestry (mestizo), with clinical features of microcephaly, prominent and narrow nose, arched palate, amelogenesis imperfecta, short stature, tall and narrow pelvis, disproportionate shortening of fore-arms and legs, and mild coxa vara. Analysis of the PCNT gene by sequencing showed the presence of a nucleotide change in exon 10, c. 1468C>T, evidencing a new mutation not reported in the literature for microcephalic osteodysplastic primordial dwarfism. Conclusion The new mutation identified in this case could be associated with the severity of the phenotypic expression of the disease, resulting in the extreme short stature of the patient. Further studies are required to reach an explanation that can justify such findings, and it is vital to emphasize the importance of detection and follow-up by the epidemiological surveillance groups in birth defects and rare diseases. PMID:24928221

  5. Mapping of the human dentin matrix acidic phosphoprotein gene (DMP1) to the dentinogenesis imperfecta type II critical region at chromosome 4q21

    SciTech Connect

    Aplin, H.M.; Hirst, K.L.; Crosby, A.H.; Dixon, M.J. [Univ. of Manchester (United Kingdom)] [Univ. of Manchester (United Kingdom)

    1995-11-20

    Dentinogenesis imperfecta type II (DGI1) is an autosomal dominant disorder of dentin formation, which has been mapped to human chromosome 4q12-q21. The region most likely to contain the DGI1 locus is a 3.2-cM region surrounding the osteopontin (SPP1) locus. Recently, a novel dentin-specific acidic phosphoprotein (dmp1) has been cloned in the rat and mapped to mouse chromosome 5q21. In the current investigation, we have isolated a cosmid containing the human DMP1 gene. The isolation of a short tandem repeat polymorphism at this locus has allowed us to map the DMP1 locus to human chromosome 4q21 and demonstrate that it is tightly linked to DGI1 in two families (Z{sub max} = 11.01, {theta} = 0.001). The creation of a yeast artificial chromosome contig around SPP1 has further allowed us to demonstrate that DMP1 is located within 150 kb of the bone sialoprotein and 490 kb of the SPP1 loci, respectively. DMP1 is therefore a strong candidate for the DGI1 locus. 12 refs., 2 figs., 1 tab.

  6. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung [Inha University College of Medicine, Incheon 400-121 (Korea, Republic of); Lee, Youn Sook [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Han, In-Oc [Inha University College of Medicine, Incheon 400-121 (Korea, Republic of); Park, Seok Hee [Inha University College of Medicine, Incheon 400-121 (Korea, Republic of) and Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)]. E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

  7. Identification of a Novel Cyclic AMP-response Element (CRE-II) and the Role of CREB-1 in the cAMP-induced Expression of the Survival Motor Neuron (SMN) Gene*

    PubMed Central

    Majumder, Sarmila; Varadharaj, Saradhadevi; Ghoshal, Kalpana; Monani, Umrao; Burghes, Arthur H. M.; Jacob, Samson T.

    2006-01-01

    Spinal muscular atrophy, an autosomal recessive disorder, is caused by loss of the SMN1 (survival motor neuron) gene while retaining the SMN2 gene. SMN1 produces a majority of full-length SMN transcript, whereas SMN2 generates mostly an isoform lacking exon 7. Here, we demonstrate a novel cAMP-response element, CRE-II, in the SMN promoter that interacts with the cAMP-response element-binding (CREB) family of proteins. In vitro DNase I protection analysis and in vivo genomic footprinting of the SMN promoter using the brain and liver nuclei from SMN2 transgenic mice revealed footprinting at the CRE-II site. Site-directed mutation of the CRE-II element caused a marked reduction in the SMN promoter activity revealed by transient transfection assay. Activation of the cAMP pathway by dibutyryl cAMP (0.5 mM) alone or in combination with forskolin (20 ?M) caused a 2–5-fold increase in the SMN promoter activity but had no effect on the CRE-II mutated promoter. Electrophoretic mobility shift assay and a UV-induced DNA-protein cross-linking experiment confirmed that CREB1 binds specifically to the CRE-II site. Transient overexpression of CREB1 protein resulted in a 4-fold increase of the SMN promoter activity. Intraperitoneal injection of epinephrine in mice expressing two copies of the human SMN2 gene resulted in a 2-fold increase in full-length SMN transcript in the liver. Combined treatment with dibutyryl cAMP and forskolin significantly increased the level of both the full-length and exon 7-deleted SMN (exon?7SMN) transcript in primary hepatocytes from mice expressing two copies of human SMN2 gene. Similar treatments of type I spinal muscular atrophy mouse and human fibroblasts as well as HeLa cells resulted in an augmented level of SMN transcript. These findings suggest that the CRE-II site in SMN promoter positively regulates the expression of the SMN gene, and treatment with cAMP-elevating agents increases expression of both the full-length and exon?7SMN transcript. PMID:14742439

  8. Missense mutations in the pancreatic islet beta cell inwardly rectifying K+ channel gene (KIR6.2\\/BIR ): a meta-analysis suggests a role in the polygenic basis of Type II diabetes mellitus in Caucasians

    Microsoft Academic Search

    E. H. Hani; P. Boutin; E. Durand; H. Inoue; M. A. Permutt; G. Velho; P. Froguel

    1998-01-01

    Summary   The K+ inwardly rectifier channel (KIR) is one of the two sub-units of the pancreatic islet ATP-sensitive potassium channel complex\\u000a (IKATP), which has a key role in glucose-stimulated insulin secretion and thus is a potential candidate for a genetic defect in\\u000a Type II (non-insulin-dependent) diabetes mellitus. We did a molecular screening of the KIR6.2 gene by single strand conformational

  9. Characterization of pECL18 and pKPN2: a proposed pathway for the evolution of two plasmids that carry identical genes for a Type II restriction-modification system

    Microsoft Academic Search

    M. V. Zakharova; I. V. Beletskaya; M. M. Denjmukhametov; T. V. Yurkova; L. M. Semenova; M. G. Shlyapnikov; A. S. Solonin

    2002-01-01

    The primary structures of the plasmids pECL18 (5571 bp) and pKPN2 (4196 bp) from Escherichia coli and Klebsiella pneumoniae, respectively, which carry genes for a Type II restriction-modification system (RMS2) with the specificity 5'-CCNGG-3', were determined in order to elucidate the structural relationship between them. The data suggest a possible role for recombination events at bom (basis of mobility) regions

  10. Contribution of two missense mutations (G71R and Y486D) of the bilirubin UDP glycosyltransferase ( UGT1A1) gene to phenotypes of Gilbert's syndrome and Crigler–Najjar syndrome type II

    Microsoft Academic Search

    Kazuo Yamamoto; Hiroshi Sato; Yoshihide Fujiyama; Yukio Doida; Tadao Bamba

    1998-01-01

    In our mutation analyses of bilirubin UDP glycosyltransferase (UGT1A1) gene, we encountered six patients with Crigler–Najjar syndrome type II who were double homozygotes for G71R and Y486D, a patient with Gilbert's syndrome who was a single homozygote for G71R and six patients with Gilbert's syndrome who were single heterozygote for G71R. To clarify the role of each mutation in the

  11. The Pro12Ala polymorphism in the peroxisome proliferator-activated receptor ?2 gene is not associated with postprandial responses to glucose or fat tolerance tests in young healthy subjects: the European Atherosclerosis Research Study II

    Microsoft Academic Search

    O. Poirier; V. Nicaud; F. Cambien; L. Tiret

    2000-01-01

    This study investigated whether the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor Ŵ (PPARŴ) gene is associated with glucose and lipid metabolism in young healthy subjects participating in the European Atherosclerosis Research Study II. Men aged 18-28 years (n=675) were recruited from 14 university student populations in 11 European countries. At their first visit subjects had an oral glucose tolerance

  12. Genetic transformation of selected mature cork oak (Quercus suber L.) trees.

    PubMed

    Alvarez, R; Alonso, P; Cortizo, M; Celestino, C; Hernández, I; Toribio, M; Ordás, R J

    2004-10-01

    A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil. PMID:15185122

  13. Molecular relationships and classification of several tufted capuchin lineages (Cebus apella, Cebus xanthosternos and Cebus nigritus, Cebidae), by means of mitochondrial cytochrome oxidase II gene sequences.

    PubMed

    Ruiz-García, Manuel; Castillo, Maria Ignacia; Lichilín-Ortiz, Nicolás; Pinedo-Castro, Myreya

    2012-01-01

    The morphological systematics of the tufted capuchins is confusing. In an attempt to clarify the complex systematics and phylogeography of this taxon, we provide a first molecular analysis. We obtained mitochondrial cytochrome oxidase II (mtCOII) gene sequences from 49 tufted capuchins that had exact geographic origins from diverse lineages in Colombia, Peru, Bolivia, French Guyana, Brazil, Argentina and Paraguay and that belonged to clearly recognized morphological taxa. This project had 4 main findings: (1) we determined 2 established and related taxa in the northern Amazon River area, which we named C. a. apella and C. a. fatuellus. C. a. apella is distributed from French Guyana until, at least, the Negro River in the northern Brazilian Amazon, whereas C. a. fatuellus is distributed throughout the Colombian Eastern Llanos and the northern Colombian Amazon. We also determined 2 other southern C. apella taxa, which we named C. a. macrodon and C. a. cay. C. a. macrodon has a western and southern Amazon distribution, while C. a. cay has a more southern distribution outside the Amazon basin. (2) In the upper Amazon basin, there is a unique lineage (C. a. macrocephalus) with 1 widely distributed haplotype. The 4 morphological subspecies (C. a. maranonis, C. a. macrocephalus, C. a. peruanus, C. a. pallidus), and maybe a fifth unknown subspecies, described in this area were molecularly undifferentiated at least for the mitochondrial gene analyzed. (3) Our molecular analysis determined that 1 individual of C. robustus fell into the lineage of C. a. macrocephalus. Therefore, this form does not receive any specific name. (4) The animals classified a priori as C. nigritus and C. xanthosternos (because of their morphological phenotypes and by their geographical origins) were clearly differentiated from the other specimens analyzed with the molecular marker employed. Therefore, we consider that these 2 lineages could be assigned the status of full species following the biological species definition. (5) In 2001, Groves described 4 tufted capuchin species (C. apella, C. libidinosus, C. nigritus and C. xanthosternos), while Silva Jr. determined 7 species (C. apella, C. macrocephalus, C. libidinosus, C. cay, C. nigritus, C. robustus and C. xanthosternos). The tests of Swofford-Olsen-Waddell-Hillis, of Shimodaira and Hasegawa and of Templeton did not fit with either of these two classificatory schemes, although Groves' scheme was better with regard to our data than that of Silva Jr. (6) All the temporal splits among the tufted capuchin taxa studied were estimated to have occurred during the last phase of the Pleistocene by using the ? statistic applied to the median joining haplotype network. PMID:23128150

  14. Genetic control of T cell responsiveness to the Friend murine leukemia virus envelope antigen. Identification of class II loci of the H-2 as immune response genes

    PubMed Central

    1988-01-01

    T cells primed specifically for the envelope glycoprotein of Friend murine leukemia helper virus (F-MuLV) were prepared by immunizing mice with a recombinant vaccinia virus that expressed the entire env gene of F-MuLV. Significant proliferative responses of F-MuLV envelope- specific, H-2a/b T cells were observed when the T cells were stimulated with antigen-pulsed peritoneal exudate cells (PEC) having the b allele at the K, A beta, A alpha, and E beta loci of the H-2. On the other hand, PEC having only the kappa allele at these loci did not induce the envelope-specific T cell proliferation, even when the PEC had the b allele at the E alpha, S, or D loci. F-MuLV envelope-specific proliferation of H-2a/b T cells under the stimulation of antigen- pulsed, H-2a/b PEC was specifically blocked with anti-I-Ab and anti-I- Ek mAbs but not with anti-Kb, anti-Kk, or anti-I-Ak mAbs. Moreover, (B10.MBR x A/WySn)F1 mice that have the b allele only at the K locus but not in I-A subregion were nonresponders to the envelope glycoprotein, and the bm12 mutation at the A beta locus completely abolished the T cell responsiveness to this antigen. These results indicate that proliferative T cells recognize a limited number of epitopes on F-MuLV envelope protein in the context of I-Ab, hybrid I- Ak/b, and/or hybrid I-Ek/b class II MHC molecules but fail to recognize the same envelope protein in the context of I-Ak or I-Ek molecules. This influence of the H-2I region on T cell recognition of the envelope glycoprotein appeared to control in vivo induction of protective immunity against Friend virus complex after immunization with the vaccinia-F-MuLV env vaccine. Thus, these results provide, for the first time, direct evidence for Ir gene-controlled responder/nonresponder phenotypes influencing the immune response to a pathogenic virus of mice. PMID:3141552

  15. The pcz1 Gene, which Encodes a Zn(II)2Cys6 Protein, Is Involved in the Control of Growth, Conidiation, and Conidial Germination in the Filamentous Fungus Penicillium roqueforti

    PubMed Central

    Medina, Exequiel; Vaca, Inmaculada; García-Rico, Ramón O.; Villagrán, Sebastián; Levicán, Gloria; Chávez, Renato

    2015-01-01

    Proteins containing Zn(II)2Cys6 domains are exclusively found in fungi and yeasts. Genes encoding this class of proteins are broadly distributed in fungi, but few of them have been functionally characterized. In this work, we have characterized a gene from the filamentous fungus Penicillium roqueforti that encodes a Zn(II)2Cys6 protein, whose function to date remains unknown. We have named this gene pcz1. We showed that the expression of pcz1 is negatively regulated in a P. roqueforti strain containing a dominant active G?i protein, suggesting that pcz1 encodes a downstream effector that is negatively controlled by G?i. More interestingly, the silencing of pcz1 in P. roqueforti using RNAi-silencing technology resulted in decreased apical growth, the promotion of conidial germination (even in the absence of a carbon source), and the strong repression of conidiation, concomitant with the downregulation of the genes of the central conidiation pathway brlA, abaA and wetA. A model for the participation of pcz1 in these physiological processes in P. roqueforti is proposed. PMID:25811807

  16. Rhizobia with 16S rRNA and nifH Similar to Mesorhizobium huakuii but Novel recA, glnII, nodA and nodC Genes Are Symbionts of New Zealand Carmichaelinae

    PubMed Central

    Tan, Heng Wee; Weir, Bevan S.; Carter, Noel; Heenan, Peter B.; Ridgway, Hayley J.; James, Euan K.; Sprent, Janet I.; Young, J. Peter W.; Andrews, Mitchell

    2012-01-01

    New Zealand became geographically isolated about 80 million years ago and this separation gave rise to a unique native flora including four genera of legume, Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, tribe Sophoreae, sub-family Papilionoideae. Ten bacterial strains isolated from NZ Carmichaelinae growing in natural ecosystems grouped close to the Mesorhizobium huakuii type strain in relation to their 16S rRNA and nifH gene sequences. However, the ten strains separated into four groups on the basis of their recA and glnII sequences: all groups were clearly distinct from all Mesorhizobium type strains. The ten strains separated into two groups on the basis of their nodA sequences but grouped closely together in relation to nodC sequences; all nodA and nodC sequences were novel. Seven strains selected and the M. huakuii type strain (isolated from Astragalus sinicus) produced functional nodules on Carmichaelia spp., Clianthus puniceus and A. sinicus but did not nodulate two Sophora species. We conclude that rhizobia closely related to M. huakuii on the basis of 16S rRNA and nifH gene sequences, but with variable recA and glnII genes and novel nodA and nodC genes, are common symbionts of NZ Carmichaelinae. PMID:23118889

  17. The pcz1 Gene, which Encodes a Zn(II)2Cys6 Protein, Is Involved in the Control of Growth, Conidiation, and Conidial Germination in the Filamentous Fungus Penicillium roqueforti.

    PubMed

    Gil-Durán, Carlos; Rojas-Aedo, Juan F; Medina, Exequiel; Vaca, Inmaculada; García-Rico, Ramón O; Villagrán, Sebastián; Levicán, Gloria; Chávez, Renato

    2015-01-01

    Proteins containing Zn(II)2Cys6 domains are exclusively found in fungi and yeasts. Genes encoding this class of proteins are broadly distributed in fungi, but few of them have been functionally characterized. In this work, we have characterized a gene from the filamentous fungus Penicillium roqueforti that encodes a Zn(II)2Cys6 protein, whose function to date remains unknown. We have named this gene pcz1. We showed that the expression of pcz1 is negatively regulated in a P. roqueforti strain containing a dominant active G?i protein, suggesting that pcz1 encodes a downstream effector that is negatively controlled by G?i. More interestingly, the silencing of pcz1 in P. roqueforti using RNAi-silencing technology resulted in decreased apical growth, the promotion of conidial germination (even in the absence of a carbon source), and the strong repression of conidiation, concomitant with the downregulation of the genes of the central conidiation pathway brlA, abaA and wetA. A model for the participation of pcz1 in these physiological processes in P. roqueforti is proposed. PMID:25811807

  18. The Chloroplast Gene ycf9 Encodes a Photosystem II (PSII) Core Subunit, PsbZ, That Participates in PSII Supramolecular Architecture

    PubMed Central

    Swiatek, Magdalena; Kuras, Richard; Sokolenko, Anna; Higgs, David; Olive, Jacqueline; Cinque, Gianfelice; Müller, Bernd; Eichacker, Lutz A.; Stern, David B.; Bassi, Roberto; Herrmann, Reinhold G.; Wollman, Francis-André

    2001-01-01

    We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII. PMID:11402165

  19. Do Biochemical Markers and Apa I Polymorphism in IGF-II Gene Play a Role in the Association of Birth Weight and Later BMI?

    PubMed Central

    WU, Junqing; REN, Jingchao; LI, Yuyan; WU, Yinjie; GAO, Ersheng

    2013-01-01

    Background The aim of the study was to explore the mechanisms underlying the association of birth weight with later body mass index (BMI) from the biochemical markers related to metabolism and the Apa I polymorphism in IGF-II gene. Methods: A total of 300 children were selected randomly from the Macrosomia Birth Cohort in Wuxi, China. The height and weight were measured and blood samples were collected. Plasma concentrations of 8 biochemical markers were detected. Apa I polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: Biochemical markers were detected for 296 subjects and 271 subjects were genotyped for the Apa I polymorphism. No association was found between birth weight and 8 biochemical markers. In boys, the BMIs of AA, AG and GG genotypes were 16.10 ± 2.24 kg/m2, 17.40 ± 3.20 kg/m2, 17.65 ± 2.66 kg/m2. And there was statistical difference among the three genotypes. But in girls, there was no statistical difference. The birth weights of AA, AG and GG genotypes were 3751.13 ± 492.43 g, 3734.00 ± 456.88 g, 3782.00 ± 461.78 g. And there was no statistical difference among the three genotypes. Conclusion: Biochemical markers are not associated with birth weight. Apa I polymorphism may be related to childhood BMI, but it may be not associated with birth weight. Therefore, biochemical markers and Apa I polymorphism might not play a role in the association of birth weight and BMI. PMID:23802105

  20. The properties of the single chicken MHC classical class II a chain (BLA) gene indicate an ancient origin for the DR\\/E-like isotype of class II molecules

    Microsoft Academic Search

    Jan Salomonsen; Denise Marston; David Avila; Nat Bumstead; Brit Johansson; Helle Juul-Madsen; Gerda D. Olesen; Patricia Riegert; Karsten Skjødt; Olli Vainio; Michael V. Wiles; Jim Kaufman

    2003-01-01

    In mammals, there are MHC class II molecules with distinctive sequence features, such as the classical isotypes DR, DQ and DP. These particular isotypes have not been reported in non-mammalian vertebrates. We have isolated the class II (B-L) a chain from outbred chickens as the basis for the cloning and sequencing of the cDNA. We found only one class II

  1. A stable transformation system for the ornamental plant, Datura meteloides D. C

    Microsoft Academic Search

    I. S. Curtis; J. B. Power; P. Hedden; D. A. Ward; A. Phillips; K. C. Lowe; M. R. Davey

    1999-01-01

    A transformation system is described for Datura meteloides using the supervirulent Agrobacterium tumefaciens strain 1065, carrying both the ?-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) genes between the T-DNA border sequences of the binary vector. The importance of conditions such as the preculture period\\u000a of the plant tissues, wounding, bacterial dilution and incubation time were evaluated in terms of transgenic

  2. Genetic transformation of selected mature cork oak ( Quercus suber L.) trees

    Microsoft Academic Search

    R. Álvarez; P. Alonso; M. Cortizo; C. Celestino; I. Hernández; M. Toribio; R. J. Ordás

    2004-01-01

    A transformation system for selected mature cork oak ( Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II ( nptII) and ?-glucuronidase ( uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly

  3. Factors affecting transformation efficiency of embryogenic callus of Upland cotton ( Gossypium hirsutum ) with Agrobacterium tumefaciens

    Microsoft Academic Search

    Shuangxia Jin; Xianlong Zhang; Shaoguang liang; Yichun Nie; Xiaoping Guo; Chao Huang

    2005-01-01

    A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the p?gusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone

  4. Agrobacterium -mediated transformation and regeneration of cotton plants

    Microsoft Academic Search

    S. Unlu Yuceer; N. K. Koc

    2006-01-01

    Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and ?-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as\\u000a explants. Explants were cultured in a Murashige

  5. Comparative genomic analysis reveals independent expansion of a lineage-specific gene family in vertebrates: The class II cytokine receptors and their ligands in mammals and fish

    E-print Network

    Lutfalla, Georges

    Background: The high degree of sequence conservation between coding regions in fish and mammals can be exploited to identify genes in mammalian genomes by comparison with the sequence of similar genes in fish. Conversely, ...

  6. Transformation of Montmorency sour cherry ( Prunus cerasus L.) and Gisela 6 ( P. cerasus × P. canescens ) cherry rootstock mediated by Agrobacterium tumefaciens

    Microsoft Academic Search

    Guo-Qing Song; K. C. Sink

    2006-01-01

    Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus × P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ß-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars

  7. Agrobacterium tumefaciens -mediated genetic transformation of sesame ( Sesamum indicum L.)

    Microsoft Academic Search

    Manju Yadav; Darshna Chaudhary; Manish Sainger; Pawan K. Jaiwal

    2010-01-01

    Sesame (Sesamum indicum) is an important oil seed crop that has not yet been transformed genetically. We report herein for the first time successful\\u000a recovery of fertile transgenic plants of sesame from cotyledon explants inoculated with Agrobacterium tumefaciens carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a ?-glucuronidase (GUS) gene (uidA) interrupted with an intron.

  8. Genetic transformation of cork oak ( Quercus suber L.) for herbicide resistance

    Microsoft Academic Search

    Rubén Álvarez; José M. Álvarez; Jaime M. Humara; Ángeles Revilla; Ricardo J. Ordás

    2009-01-01

    The bar gene was introduced into the cork oak genome. Cork oak embryogenic masses were transformed using the Agrobacterium strain AGL1 which carried the plasmid pBINUbiBar. This vector harbours the genes, nptII and bar, the latter under control of the maize ubiquitin promoter. The transgenic embryogenic lines were cryopreserved. Varying activities\\u000a of phosphinothricin acetyl transferase were detected among the lines,

  9. Regeneration of transgenic Picea glauca, P. Mariana , and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens

    Microsoft Academic Search

    Krystyna Klimaszewska; Denis Lachance; Gervais Pelletier; Marie-Anne Lelu; Armand Séguin

    2001-01-01

    Summary  Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58\\/pMP90\\/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (?-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation\\u000a procedure. Of the three

  10. Agrobacterium rhizogenes and A. tumefaciens co-transformation to obtain grapevine hairy roots producing the coat protein of grapevine chrome mosaic nepovirus

    Microsoft Academic Search

    L. Torregrosa; A. Bouquet

    1997-01-01

    Hairy root cultures of grapevine were obtained from plantlets co-inoculated by virulent Agrobacterium rhizogenes strains and\\u000a disarmed A. tumefaciens strains harbouring the binary vectors pKHG4 and pKVHG 2+. These plasmids contain the nptII, hpt and\\u000a gus genes and differ for the presence of the gene encoding for the grapevine chrome mosaic virus coat protein. For the cultivar\\u000a ‘Gravesac’, 72% of

  11. Agrobacterium tumefaciens -mediated transgenic plant production via direct shoot bud organogenesis from pre-plasmolyzed leaf explants of Catharanthus roseus

    Microsoft Academic Search

    Priyanka Verma; Ajay Kumar Mathur

    2011-01-01

    Production of Agrobacterium tumefaciens-mediated transgenic plants, via direct shoot bud organogenesis from leaves of Catharanthus roseus, is reported. A. tumefaciens harbouring the plasmid pBI121 with GUS gene uidA and kanamycin resistance gene nptII was used. Highest transformation efficiency of 1.4 transgenic shoots\\/responded explant was obtained when pre-plasmolysed\\u000a leaves, pre-incubated on shoot bud induction medium for 10 days, were subjected to sonication

  12. Unexpected effects of chitinases on the peach-potato aphid ( Myzus persicae Sulzer) when delivered via transgenic potato plants ( Solanum tuberosum Linné) and in vitro

    Microsoft Academic Search

    Julien Saguez; Romaric Hainez; Anas Cherqui; Olivier Van Wuytswinkel; Haude Jeanpierre; Gaël Lebon; Nathalie Noiraud; Antony Beaujean; Lise Jouanin; Jean-Claude Laberche; Charles Vincent; Philippe Giordanengo

    2005-01-01

    With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting

  13. hH-Rev107, a class II tumor suppressor gene, is expressed by post-meiotic testicular germ cells and CIS cells but not by human testicular germ cell tumors.

    PubMed Central

    Siegrist, Sylvie; Féral, Chloé; Chami, Mounia; Solhonne, Brigitte; Mattéi, Marie-Geneviève; Rajpert-De Meyts, Ewa; Guellaën, Georges; Bulle, Frédérique

    2001-01-01

    By systematic analysis of a human testis library, we have isolated the hH-Rev107-3 cDNA, identical to hH-Rev107-1 cDNA, which was previously described as a class II tumor suppressor gene (Husmann et al., 1998). In this study, two transcripts (1 and 0,8 kb) were detected by Northern blot in all human tissues, excepted in thymus. The strongest expression was found in testis, skeletal muscle and heart. These two mRNA are probably transcribed from only one gene that we mapped to the q12–q13 region of the chromosome 11. In human testis, hH-Rev107 gene expression was localized, by in situ hybridization, within the round spermatids. To investigate a possible role for hH-Rev107 protein in testicular malignant growth, we examined the expression of this gene in germ cell tumors. A strong hH-Rev107 gene expression was observed in normal testis as well as in samples with preinvasive carcinoma in situ but was completely absent in overt tumors, both seminomas and non-seminomas. By in situ hybridization, CIS was found hH-Rev107 positive and tumor negative. A semi-quantitative assessment of hH-Rev107 mRNA level in testicular germ cell tumors, by RT-PCR, exhibited a 9-fold decrease in the gene expression. No gross structural aberrations of hH-Rev107 gene were detected in these human primary tumors. The results suggest that down-regulation of hH-Rev107 may be associated with invasive progression of testicular germ cell tumors. PMID:11526504

  14. HLA class II genes in primary sclerosing cholangitis and chronic inflammatory bowel disease: no HLA-DRw52a association in Swedish patients with sclerosing cholangitis

    Microsoft Academic Search

    H Zetterquist; U Broomé; K Einarsson; O Olerup

    1992-01-01

    The familial predisposition to chronic inflammatory bowel disease and the increased concordance rate in monozygotic twins with Crohn's disease, suggest that genetic factors influence disease susceptibility. A 100% association with the supertypic HLA class II specificity DRw52a was recently described in white North American patients with primary sclerosing cholangitis, with or without concurrent ulcerative colitis. HLA class II alleles of

  15. A potato alpha-glucosidase gene encodes a glycoprotein-processing alpha-glucosidase II-like activity. Demonstration of enzyme activity and effects of down-regulation in transgenic plants.

    PubMed

    Taylor, M A; Ross, H A; McRae, D; Stewart, D; Roberts, I; Duncan, G; Wright, F; Millam, S; Davies, H V

    2000-11-01

    In order to elucidate more fully the function of a potato gene (MAL1) encoding alpha-glucosidase activity, transgenic plants in which MAL1 expression was down-regulated were generated using antisense technology. In transgenic lines severely down-regulated in the expression of MAL1, total alpha-glucosidase activity was not decreased in leaves and tubers, and the contents of starch, glucose, fructose and sucrose remained unchanged in tubers. Phylogenetic analysis indicated that the MAL1 gene product was more similar to the glycoprotein-processing alpha-glucosidase II of mammalian and yeast origin than to other plant alpha-glucosidases. Using [14C-Glc]-labelled Glc2Man9GlcNAc2 as a substrate, it was demonstrated that glucosidase II activity was markedly down-regulated in microsomes isolated from tubers of four independent antisense lines studied in detail, strongly suggesting that MAL1 encodes glucosidase II activity. In field trials (but not in the glasshouse), MAL1 down-regulation produced an extremely stunted phenotype - the leaves were curled and tuber yield was decreased by 90% compared to control values. Microscopic analysis of leaves revealed significant differences between the antisense and control samples. Plants with down-regulated glucosidase II activity showed a greater degree of plasmolysis, and an increase in the size of mesophyll intracellular spaces. Analysis of cell walls also indicated changes in structure as a result of MAL1 down-regulation. In leaves from four antisense lines, the steady-state transcript level corresponding to the endoplasmic reticulum chaperone, BiP, was enhanced. This is diagnostic of stress in the endoplasmic reticulum. PMID:11069704

  16. Development of transgenic CryIA(c) + GNA cotton plants via pollen tube pathway method confers resistance to Helicoverpa armigera and Aphis gossypii Glover.

    PubMed

    Liu, Zhi; Zhu, Zhen; Zhang, Tianzhen

    2013-01-01

    Elite cotton cultivar Sumian16 was transformed with p7RPSBK-mGNA-NPTII containing Bt (CryIA(c)), Galanthus nivalis agglutinin (GNA) resistance genes and selectable marker NptII gene via the pollen tube pathway method and two fertile transgenic Bt + GNA plants were obtained in the present study. The integration and expression of the Bt and GNA genes were confirmed by molecular biology techniques and insect bioassays. Insect bioassays showed that the transformed plants were highly toxic to bollworm larvae as well as obviously retarding development of aphid populations. PCR analyses and identification of resistance to Kanamycin and bollworm showed that the resistance to bollworm for the two transgenic plants was dominantly inherited in a Mendelian manner and the two resistance genes and selectable marker co-segregated from primary transformed parents to the first self-fertilized progeny plants. PMID:23143495

  17. Association of Angiotensin-Converting Enzyme Intron 16 Insertion/Deletion and Angiotensin II Type 1 Receptor A1166C Gene Polymorphisms with Preeclampsia in South East of Iran

    PubMed Central

    Salimi, Saeedeh; Mokhtari, Mojgan; Yaghmaei, Minoo; Jamshidi, Mohammad; Naghavi, Anoosh

    2011-01-01

    Some evidence suggests that a variety of genetic factors contributed in pathogenesis of the preeclampsia. The aim of this study was to assess the association between the angiotensin-converting enzyme (ACE) I/D and angiotensin II type1 receptor A1166C polymorphisms with preeclampsia. This study was performed in 125 preeclamptic pregnant women and 132 controls. The I/D Polymorphism of the ACE gene was assessed by polymerase chain reaction and the A1166C Polymorphism of the AT1R gene was determined by restriction fragment length polymorphism. The genotype and allele frequencies of I/D polymorphism differed between two groups. The risk of preeclampsia was 3.2-fold in pregnant women with D allele (OR, 3.2 [95% CI, 1.1 to 3.8]; P = 0.01). The distribution of the AT1R gene A1166C polymorphism was similar in affected and control groups. Our results supported that presence of the I/D polymorphism of ACE gene is a marker for the increased risk of preeclampsia. PMID:21808598

  18. Genes and Gene Therapy

    MedlinePLUS

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  19. Gene clusters encoding the cytotoxic necrotizing factor type 1, Prs-fimbriae and ?-hemolysin form the pathogenicity island II of the uropathogenic Escherichia coli strain J96

    Microsoft Academic Search

    Gabriele Blum; Vincenzo Falbo; Alfredo Caprioli; Jörg Hacker

    1995-01-01

    The uropathogenic Escherichia coli strain J96 (04:K6) is able to produce four adherence factors [P-fimbriae (pap and prs), F1C-fimbriae (foc) and Type 1-fimbriae (fim)], two ?-hemolysins (hfyI and II) and the cytotoxic necrotizing factor type 1 (cnf1). Using phenotypic test systems and genotypic analysis, it has been shown that the mutant strain J96-M1 has lost the hlyII, prs and cnf1

  20. Cloning and Sequencing of cDNA Encoding Human DNA Topoisomerase II and Localization of the Gene to Chromosome Region 17q21-22

    Microsoft Academic Search

    Monika Tsai-Pflugfelder; Leroy F. Liu; Angela A. Liu; Kathleen M. Tewey; Jacqueline Whang-Peng; Turid Knutsen; Kay Huebner; Carlo M. Croce; James C. Wang

    1988-01-01

    Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11

  1. Single-nucleotide polymorphisms in base excision repair, nucleotide excision repair, and double strand break genes as markers for response to radiotherapy in patients with Stage I to II head-and-neck cancer

    SciTech Connect

    Carles, Joan [Department of Medical Oncology, Hospital del Mar, University Autonoma of Barcelona, Barcelona (Spain)]. E-mail: jcarles@imas.imim.es; Monzo, Mariano [Department of Human Anatomy, Faculty of Medicine, University of Barcelona, Barcelona (Spain); Amat, Marta [Department of Otolaryngology, Hospital del Mar, University Autonoma of Barcelona, Barcelona (Spain); Jansa, Sonia [Department of Human Anatomy, Faculty of Medicine, University of Barcelona, Barcelona (Spain); Artells, Rosa [Department of Human Anatomy, Faculty of Medicine, University of Barcelona, Barcelona (Spain); Navarro, Alfons [Department of Human Anatomy, Faculty of Medicine, University of Barcelona, Barcelona (Spain); Foro, Palmira [Department of Radiation Oncology, Hospital del Mar, University Autonoma of Barcelona, Barcelona (Spain); Alameda, Francesc [Department of Pathology, Hospital del Mar, University Autonoma of Barcelona, Barcelona (Spain); Gayete, Angel [Department of Radiology, Hospital del Mar, University Autonoma of Barcelona, Barcelona (Spain); Gel, Bernat [Department of Human Anatomy, Faculty of Medicine, University of Barcelona, Barcelona (Spain); Miguel, Maribel [Department of Human Anatomy, Faculty of Medicine, University of Barcelona, Barcelona (Spain); Albanell, Joan [Department of Medical Oncology, Hospital del Mar, University Autonoma of Barcelona, Barcelona (Spain); Fabregat, Xavier [Department of Medical Oncology, Hospital del Mar, University Autonoma of Barcelona, Barcelona (Spain)

    2006-11-15

    Purpose: Polymorphisms in DNA repair genes can influence response to radiotherapy. We analyzed single-nucleotide polymorphisms (SNP) in nine DNA repair genes in 108 patients with head-and-neck cancer (HNSCC) who had received radiotherapy only. Methods and Materials: From May 1993 to December 2004, patients with Stage I and II histopathologically confirmed HNSCC underwent radiotherapy. DNA was obtained from paraffin-embedded tissue, and SNP analysis was performed using a real-time polymerase chain reaction allelic discrimination TaqMan assay with minor modifications. Results: Patients were 101 men (93.5%) and 7 (6.5%) women, with a median age of 64 years (range, 40 to 89 years). Of the patients, 76 (70.4%) patients were Stage I and 32 (29.6%) were Stage II. The XPF/ERCC1 SNP at codon 259 and XPG/ERCC5 at codon 46 emerged as significant predictors of progression (p 0.00005 and 0.049, respectively) and survival (p = 0.0089 and 0.0066, respectively). Similarly, when variant alleles of XPF/ERCC1, XPG/ERCC5 and XPA were examined in combination, a greater number of variant alleles was associated with shorter time to progression (p = 0.0003) and survival (p 0.0002). Conclusions: Genetic polymorphisms in XPF/ERCC1, XPG/ERCC5, and XPA may significantly influence response to radiotherapy; large studies are warranted to confirm their role in HNSCC.

  2. Structure and stability of Co(II)-complexes formed by wild-type and metal-ligand substitution mutants of T4 gene 32 protein

    E-print Network

    Guo, Juqian

    1996-01-01

    Phage T4 gene 32 protein (gp32) is a zinc metalloprotein that binds cooperatively and preferentially to single-stranded nucleic acids and functions as a replication and recombination accessory protein. We have previously shown that the ZN...

  3. Insulin-like growth factor-II (IGF-II), type 2 IGF receptor, and IGF-binding protein-2 gene expression in rat lung alveolar epithelial cells: relation to proliferation.

    PubMed

    Mouhieddine, O B; Cazals, V; Maitre, B; Le Bouc, Y; Chadelat, K; Clement, A

    1994-07-01

    Pulmonary alveolar type 2 cells act as a reservoir of stem cells which can be induced to proliferate during periods of lung growth and repair after lung injury. Despite the importance of this process, the mechanisms that regulate type 2 cell proliferation have not been well characterized. We show in this study that insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) accumulates to high levels in culture medium of growth-arrested type 2 cells. This is associated with an increased expression of IGFBP-2 messenger RNA (mRNA). Study of the other components of the IGF system also reveals induction of IGF-II and type 2 IGF receptor mRNA during the process of type 2 cell block of proliferation. When growth-arrested cells are allowed to resume proliferation by the addition of serum, the level of expression of IGFBP-2, type 2 IGF receptor, and IGF-II rapidly decreased. Despite the similarities in the timing of induction, it is likely that these components are not necessarily linked to mediate effects through a single pathway. Indeed, we show that the addition of conditioned medium from growth-arrested cells on proliferative cells results in down-regulation of IGFBP-2 and increased expression of IGF-II and type 2 IGF receptor mRNA. Treatment of the cells with various concentrations of IGF-II affects only the level of expression of type 2 IGF receptor, whereas IGF-I and insulin appear to influence only the expression of IGFBP-2. From the results presented in this study, it can be suggested that IGFBP-2, IGF-II, and type 2 IGF receptor play an important role in the transition of lung alveolar epithelial cells in and out of the cell cycle. PMID:7516870

  4. Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III

    PubMed Central

    Carpenter, Margaret A.; Joyce, Nigel I.; Genet, Russell A.; Cooper, Rebecca D.; Murray, Sarah R.; Noble, Alasdair D.; Butler, Ruth C.; Timmerman-Vaughan, Gail M.

    2015-01-01

    Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch phosphorylation is a complex trait which has been previously shown by antisense gene repression to be influenced by a number of genes including those involved in starch synthesis and degradation. We have used an association mapping approach to discover genetic markers associated with the degree of starch phosphorylation. A diverse collection of 193 potato lines was grown in replicated field trials, and the levels of starch phosphorylation at the C6 and C3 positions of the glucosyl residues were determined by mass spectrometry of hydrolyzed starch from tubers. In addition, the potato lines were genotyped by amplicon sequencing and microsatellite analysis, focusing on candidate genes known to be involved in starch synthesis. As potato is an autotetraploid, genotyping included determination of allele dosage. Significant associations (p < 0.001) were found with SNPs in the glucan water dikinase (GWD), starch branching enzyme I (SBEI) and the starch synthase III (SSIII) genes, and with a SSR allele in the SBEII gene. SNPs in the GWD gene were associated with C6 phosphorylation, whereas polymorphisms in the SBEI and SBEII genes were associated with both C6 and C3 phosphorylation and the SNP in the SSIII gene was associated with C3 phosphorylation. These allelic variants have potential as genetic markers for starch phosphorylation in potato. PMID:25806042

  5. Interferon-? lipofection II. Mechanisms involved in cell death and bystander effect induced by cationic lipid-mediated interferon-? gene transfer to human tumor cells.

    PubMed

    Villaverde, M S; Gil-Cardeza, M L; Glikin, G C; Finocchiaro, L M E

    2012-06-01

    We evaluated the cytotoxic effects (apoptosis, necrosis and early senescence) of human interferon-? (hIFN?) gene lipofection. The cytotoxicity of hIFN? gene lipofection resulted equivalent to that of the corresponding addition of the recombinant protein (rhIFN?) on human tumor cell lines derived from Ewing's sarcoma (EW7 and COH) and colon (HT-29) carcinomas. However, it was stronger than rhIFN? on melanoma (M8) and breast adenocarcinoma (MCF7). To reveal the mechanisms involved in these differences, we compared the effects of hIFN? gene and rhIFN? protein on EW7 and M8 (sensitive and resistant to rhIFN? protein, respectively). Lipofection with hIFN? gene caused a mitochondrial potential decrease simultaneous with an increase of oxidative stress in both cell lines. However, rhIFN? protein displayed the same pattern of response only in EW7-sensitive cell line. The great bystander effect of the hIFN? gene lipofection, involving the production of reactive oxygen species, would be among the main causes of its success. In EW7, this effect killed >60% of EW7 cell population, even though only 1% of cells were expressing the transgene. As hIFN? gene was effective even in the rhIFN? protein-resistant M8 cell line and in a way not limited by low lipofection efficiency, these results strongly support the clinical potential of this approach. PMID:22555508

  6. Mapping of the HSD17B2 gene encoding type II 17{beta}-hydroxysteroid dehydrogenase close to D16S422 on chromosome 16q24.1-q24.2

    SciTech Connect

    Durocher, F.; Labrie, Y.; Labrie, F. [CHUL Research Center and Laval Univ., Quebec (Canada)] [and others] [CHUL Research Center and Laval Univ., Quebec (Canada); and others

    1995-02-10

    The enzymes of the 17{Beta}-hydroxysteroid dehydrogenase (17{Beta}-HSD) gene family are responsible for a key step in the formation and degradation of androgens and estrogens: catalyzing the interconversion of 17-ketosteroids and their active 17{Beta}-hydroxysteroid counterparts. The structure of human type II 17{Beta}-HSD cDNA was recently reported. This enzyme catalyzes the interconversion of {Delta}{sup 4}-androstenedione and testosterone, and rostanedione and dihydrotestosterone, and estrone and 17p-estradiol, whereas type 17{Beta}-HSD catalyzes exclusively the interconversion of estrogens. To locate the HSD17B2 gene, the novel dinucleotide CA repeat sequence found 571 hp downstream from the end of exon 1 was genotyped into eight CEPH reference families by PCR. Two-point linkage analysis was performed between the latter polymorphism and the 2066 microsatellite markers of Genethon. The maximal pairwise lod score (Z{sub max} = 33.3) with a maximal recombination fraction ({theta}{sub max}) of 0.008 was obtained with the marker D16S422 located on 16q24.1-q24.2. To define further the localization of the HSD17B2 gene, we constructed a high-resolution genetic map of the region flanking the polymorphic HSD17B2 gene including eight Genethon markers. The order of the HSD17132 gene and markers is qter- D16S516 - D16S504 - D16S507 - D16S505 - D16S511 - HSD17B2-Dl6S4221-Dl6S520-Dl6S413-tel. 15 refs., 2 figs., 1 tab.

  7. Sox9/Sox6 and Sp1 are involved in the insulin-like growth factor-I-mediated upregulation of human type II collagen gene expression in articular chondrocytes.

    PubMed

    Renard, Emmanuelle; Porée, Benoît; Chadjichristos, Christos; Kypriotou, Magdalini; Maneix, Laure; Bigot, Nicolas; Legendre, Florence; Ollitrault, David; De Crombrugghe, Benoît; Malléin-Gérin, Frédéric; Moslemi, Safa; Demoor, Magali; Boumediene, Karim; Galéra, Philippe

    2012-06-01

    Type II collagen is a marker of articular cartilage encoded by the COL2A1 gene. The nature of the trans factors involved in the upregulation of this gene by insulin-like growth factor-I (IGF-I) remains unclear. We found that IGF-I increased type II collagen synthesis by a transcriptional control mechanism involving a 715-bp region within the COL2A1 first-intron specific enhancer. The overproduction of L-Sox5/Sox6/Sox9 and Sp1 and decoy experiments targeting these factors demonstrated their action in concert in IGF-I trans-activation. These results were supported by the data obtained in knockdown experiments in which siRNA against Sox9/Sox6 and Sp1 prevented the IGF-I-induced increase in collagen II production. Indeed, each of these trans-activators increased the expression of others. IGF-I increased the binding of Sox9 and Sp1/Sp3 to their cis elements in the enhancer, and we provide the first evidence of Sox9 interaction with the promoter by chromatin immunoprecipitation. Interactions with COL2A1 were also observed for Sp1, p300/CBP, and Tip60. Finally, a physical interaction between Sox9, p300, Sp3, and Sp1 was detected. These data demonstrate the role of Sox9, Sp1/Sp3, and euchromatin-associated factors (p300, Tip60) in the IGF-I-induced upregulation of COL2A1, indicating possible use of this growth factor in articular cartilage engineering applications to promote repair in patients with degenerative diseases, such as osteoarthritis. PMID:22215151

  8. Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences

    PubMed Central

    Li, Qianhong; Guo, Yiru; Ou, Qinghui; Wu, Wen-Jian; Chen, Ning; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Dawn, Buddhadeb; Luo, Li; Hunt, Gregory N.

    2013-01-01

    Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 ± 2.1%, n = 12, vs. 44.7 ± 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 ± 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., “immunization against infarction”). PMID:21785893

  9. Regeneration of transgenic plants by Agrobacterium -mediated transformation of somatic embryos of juvenile and mature Quercus robur

    Microsoft Academic Search

    N. Vidal; R. Mallón; S. Valladares; A. M. Meijomín; A. M. Vieitez

    2010-01-01

    A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with\\u000a five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable

  10. Genotype-independent leaf disc transformation of potato ( Solanum tuberosum ) using Agrobacterium tumefaciens

    Microsoft Academic Search

    M. Block

    1988-01-01

    Leaves of the in vitro grown potato cultivars ‘Bintje’, ‘Berolina’, ‘Desiree’, and ‘Russet Burbank’ were wounded and co-cultivated with Agrobacterium strains having chimeric bar and nptII genes on a disarmed T-DNA. Each leaf from these cultivars formed numerous calli on kanamycin-containing medium, and almost all calli regenerated shoots. For ‘Russet Burbank’, it was necessary to include AgNO3 in the medium

  11. Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols

    Microsoft Academic Search

    Helena Mathews; Valerie Dewey; Wendy Wagoner; Richard K. Bestwick

    1998-01-01

    Transgenic plants of strawberry cultivar Totem were developed by Agrobacterium-mediated transformation using a plasmid vector containing gus and nptII genes. Parallel experiments were carried out with and without repeated subculturing (iterative cultures) for generation of transgenic shoots on selection medium. The selection levels in the non-iterative pathway were kept constant, while in the iterative protocol, stepwise increase of selection pressure

  12. Rapid transformation and regeneration of alfalfa (Medicago falcata L.) via direct somatic embryogenesis

    Microsoft Academic Search

    C. Y. Shao; E. Russinova; A. Iantcheva; A. Atanassov; A. McCormac; D. F. Chen; M. C. Elliott; A. Slater

    2000-01-01

    Two simple, rapid and efficient protocols for theregeneration of transformed tetraploid lines ofalfalfa (Medicago falcata L.) have beendeveloped and compared. Leaf explants fromembryogenic lines 47\\/1-150 and 47\\/1-5 were inoculatedwith Agrobacterium tumefaciens containingconstructs carrying the nptII selectable markergene and promoter:gusA gene fusions under thecontrol of the CaMV 35S or Arabidopsis cdc2a,CycB1 and CycA2 promoters. In the firstregeneration system (the MSH system),

  13. Agrobacterium -mediated transformation of Citrus stem segments and regeneration of transgenic plants

    Microsoft Academic Search

    G. A. Moore; C. C. Jacono; J. L. Neidigh; S. D. Lawrence; K. Cline

    1992-01-01

    A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker ß-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage (= 5%) of the shoots regenerated in the presence of 100 µg\\/ml kanamycin were GUS+.

  14. A Microarray-based Detection System for Genetically Modified (GM) Food Ingredients

    Microsoft Academic Search

    Serge Leimanis; Marta Hernández; Sophie Fernández; Francine Boyer; Malcolm Burns; Shirin Bruderer; Thomas Glouden; Neil Harris; Othmar Kaeppeli; Patrick Philipp; Maria Pla; Pere Puigdomènech; Marc Vaitilingom; Yves Bertheau; José Remacle

    2006-01-01

    A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs),\\u000a five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was\\u000a performed directly with PCR amplified products.

  15. An improved transformation system for Lombardy poplar ( Populus nigra var. italica )

    Microsoft Academic Search

    Mitsuru Nishiguchi; Kazumasa Yoshida; Takeshi Mohri; Tomohiro Igasaki; Kenji Shinohara

    2006-01-01

    We report an improved transformation system for Lombardy poplar (Populus nigra var. italica). A new binary vector, pBF2, with 11 unique restriction enzyme sites and the normal neomycin phosphotransferase II (NPTII) gene was constructed for the transformation of Lombardy poplar. Genetically transformed adventitious shoots were directly\\u000a regenerated after cocultivation of stem segments with Agrobacterium tumefaciens EHA105 that harbored a binary

  16. Transcriptional activation of the intercellular adhesion molecule 1 (CD54) gene by human T lymphotropic virus types I and II Tax is mediated through a palindromic response element.

    PubMed

    Owen, S M; Rudolph, D L; Dezzutti, C S; Shibata, N; Naik, S; Caughman, S W; Lal, R B

    1997-11-01

    In vitro infection of T cells with human T lymphotropic virus types I and II (HTLV-I and HTLV-II) resulted in constitutive expression of ICAM-1. Higher levels of ICAM-1 mRNA were expressed in HTLV-transformed cell lines (MT-2, MoT, C8166) when compared with uninfected T cell lines (A301). We demonstrate that this activation is conferred through a site on the ICAM-1 promoter that is activated in trans by the Tax protein of HTLV-I and HTLV-II. Enhanced promoter activity was detected when the ICAM-1 construct (-1162/+1) was transfected into HTLV-I-infected (MT-2), HTLV-II-infected (MoT, AI 1050), or an HTLV-I Tax-only-expressing (C8166) cell line as compared to the uninfected T cell line (A3.01). Cotransfection of the uninfected T cell line A3.01 with the ICAM construct along with Tax-I and Tax-II expression plasmid also resulted in increased promoter activity. Furthermore, experiments with deletion constructs of the ICAM-1 promoter region indicated that a region between -88 and -53 bp relative to the transcription start site is sufficient for Tax-inducible CAT expression. This segment includes an 11-bp palindromic segment (TTTCCGGGAAA) that has homology with the IFN-gamma and IL-6 response element. An 11-bp segment containing this regulatory region proved to be sufficient to confer Tax-I and Tax-II inducibility on a heterologous promoter (TK-CAT). Taken together these findings indicate that constitutive expression of ICAM-1 by HTLV-infected cells is influenced by the viral trans-activator protein Tax. This increased expression of ICAM-1 in response to the Tax protein may play an important role in the lymphoproliferation associated with HTLV infection. PMID:9359663

  17. Safety and Efficacy of Autologous CD34+ Hematopoietic Progenitor Cells Transduced with an Anti-Tat Ribozyme in a Multi-Center, Randomized, Placebo-Controlled, Phase II Gene Therapy Trial for the Human Immunodeficiency Virus

    PubMed Central

    Mitsuyasu, Ronald T; Merigan, Thomas C; Carr, Andrew; Zack, Jerome A; Winters, Mark A; Workman, Cassy; Bloch, Mark; Lalezari, Jacob; Becker, Stephen; Thornton, Lorna; Akil, Bisher; Khanlou, Homayoon; Finlayson, Robert; McFarlane, Robert; Smith, Don E; Garsia, Roger; Ma, David; Law, Matthew; Murray, John M.; von Kalle, Christof; Ely, Julie A; Patino, Sharon M; Knop, Alison E; Wong, Philip; Todd, Alison V; Haughton, Margaret; Fuery, Caroline; Macpherson, Janet L; Symonds, Geoff P; Evans, Louise A; Pond, Susan M; Cooper, David A

    2009-01-01

    SUMMARY Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system, and avoids lifetime highly active antiretroviral therapy. This study, the first randomized, double-blind, placebo-controlled, phase II cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1 infected adults who received a tat/vpr specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistical difference in viral load between the OZ1 and placebo group at the primary end-point (average at weeks 47 and 48) but time weighted areas under the curve from weeks 40-48 and 40-100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study provides the first indication that cell-delivered gene transfer is safe and biologically active in HIV patients and can be developed as a conventional therapeutic product. PMID:19219022

  18. Deciphering the modulation of gene expression by type I and II interferons combining 4sU-tagging, translational arrest and in silico promoter analysis

    PubMed Central

    Trilling, Mirko; Bellora, Nicolás; Rutkowski, Andrzej J.; de Graaf, Miranda; Dickinson, Paul; Robertson, Kevin; Prazeres da Costa, Olivia; Ghazal, Peter; Friedel, Caroline C.; Albà, M. Mar; Dölken, Lars

    2013-01-01

    Interferons (IFN) play a pivotal role in innate immunity, orchestrating a cell-intrinsic anti-pathogenic state and stimulating adaptive immune responses. The complex interplay between the primary response to IFNs and its modulation by positive and negative feedback loops is incompletely understood. Here, we implement the combination of high-resolution gene-expression profiling of nascent RNA with translational inhibition of secondary feedback by cycloheximide. Unexpectedly, this approach revealed a prominent role of negative feedback mechanisms during the immediate (?60 min) IFN? response. In contrast, a more complex picture involving both negative and positive feedback loops was observed on IFN? treatment. IFN?-induced repression of genes associated with regulation of gene expression, cellular development, apoptosis and cell growth resulted from cycloheximide-resistant primary IFN? signalling. In silico promoter analysis revealed significant overrepresentation of SP1/SP3-binding sites and/or GC-rich stretches. Although signal transducer and activator of transcription 1 (STAT1)-binding sites were not overrepresented, repression was lost in absence of STAT1. Interestingly, basal expression of the majority of these IFN?-repressed genes was dependent on STAT1 in IFN-naïve fibroblasts. Finally, IFN?-mediated repression was also found to be evident in primary murine macrophages. IFN-repressed genes include negative regulators of innate and stress response, and their decrease may thus aid the establishment of a signalling perceptive milieu. PMID:23832230

  19. The human antibody repertoire: Heavy and light chain variable region gene usage in six alloantibodies specific for human HLA class I and class II alloantigens

    Microsoft Academic Search

    Jennifer S. Andris; Sheena R. Abraham; Virginia Pascual; Maria P. Pistillo; Stefano Mantero; Giovanni B. Ferrara; J. Donald Capra

    1995-01-01

    Peripheral blood B lymphocytes have been isolated from healthy individuals who were immunized with lymphocytes from HLA-incompatible donors and transformed with Epstein-Barr virus to produce human monoclonal cell lines specific for human HLA molecules. The cell lines have been previously characterized and are known to bind to various class I and class II alloantigens. In this report we describe the

  20. High Ambient Glucose Augments Angiotensin II-Induced Proinflammatory Gene mRNA Expression in Human Mesangial Cells: Effects of Valsartan and Simvastatin

    Microsoft Academic Search

    Masayo Naito; Ananth Shenoy; Isao Aoyama; Joseph S. Koopmeiners; Radko Komers; H. William Schnaper; Karol Bomsztyk

    2009-01-01

    Background: Hyperglycemia may potentiate the adverse renal effects of angiotensin II (AII). In the kidney, the major target of AII action is the glomerular mesangial cell, where its hemodynamic and proinflammatory action contributes to renal injury. AII action is mediated by several types of cell receptors. Among those, the AT1 receptor has been best studied using specific AII receptor blockers

  1. Gene therapy in the cornea

    Microsoft Academic Search

    Rajiv R. Mohan; Ajay Sharma; Marcelo V. Netto; Sunilima Sinha; Steven E. Wilson

    2005-01-01

    Technological advances in the field of gene therapy has prompted more than three hundred phase I and phase II gene-based clinical trials for the treatment of cancer, AIDS, macular degeneration, cardiovascular, and other monogenic diseases. Besides treating diseases, gene transfer technology has been utilized for the development of preventive and therapeutic vaccines for malaria, tuberculosis, hepatitis A, B and C

  2. An RNA-splicing mutation (G+5IVS20) in the type II collagen gene (COL2A1) in a family with spondyloepiphyseal dysplasia congenita.

    PubMed Central

    Tiller, G E; Weis, M A; Polumbo, P A; Gruber, H E; Rimoin, D L; Cohn, D H; Eyre, D R

    1995-01-01

    Defects in type II collagen have been demonstrated in a phenotypic continuum of chondrodysplasias that includes achondrogenesis II, hypochondrogenesis, spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia, and Stickler syndrome. We have determined that cartilage from a terminated fetus with an inherited form of SEDC contained both normal alpha 1(II) collagen chains and chains that lacked amino acids 256-273 of the triple-helical domain. PCR amplification of this region of COL2A1, from genomic DNA, yielded products of normal size, while amplification of cDNA yielded a normal sized species and a shorter fragment missing exon 20. Sequence analysis of genomic DNA from the fetus revealed a G-->T transversion at position +5 of intron 20; the affected father was also heterozygous for the mutation. Allele-specific PCR and heteroduplex analysis of a VNTR in COL2A1 independently confirmed the unaffected status of a fetus in a subsequent pregnancy. Thermodynamic calculations suggest that the mutation prevents normal splicing of exon 20 by interfering with binding of U1 small-nuclear RNA to pre-mRNA, thus leading to skipping of exon 20 in transcripts from the mutant allele. Electron micrographs of diseased cartilage showed intracellular inclusion bodies, which were stained by an antibody to alpha 1(II) procollagen. Our findings support the hypothesis that alpha-chain length alterations that preserve the Gly-X-Y repeat motif of the triple helix result in partial intracellular retention of alpha 1(II) procollagen and produce mild to moderate chondrodysplasia phenotypes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7847372

  3. Characterization of the thrombospondin (TSP)-II gene in Penaeus monodon and a novel role of TSP-like proteins in an induction of shrimp sperm acrosome reaction.

    PubMed

    Magerd, Sirilug; Asuvapongpatana, Somluk; Vanichviriyakit, Rapeepun; Chotwiwatthanakun, Charoonroj; Weerachatyanukul, Wattana

    2013-05-01

    We have recently shown that water-soluble materials from the egg extracellular cortical rods (wsCRs) exert the ability to induce the sperm acrosome reaction in Penaeus monodon. In this study, we further demonstrated that the thrombospondin protein family (TSP) existed in wsCRs, and that their mRNA transcripts were detected in developing oocytes as early as stage I. Full sequence analysis revealed that our pmTSP sequence was considerably different from the recently reported pmTSP in the 5' nonconserved region and in many TSP signature domains, hence, the name pmTSP-II was given to our variant. The transcripts of pmTSP-II were detected only in early developing oocytes (stage-I and -II) while TSP-like proteins were detected in all developing oocytes, particularly at the outer rim of cortical rods situated in the extracellular crypts of the mature, stage-IV oocytes. In addition, wsCRs contained anti-TSP-reactive proteins, suggesting that TSP-like proteins are dissolved in and are part of the egg water during spawning. The functional importance of TSP-like proteins was evident by the interference of a wsCR-induced acrosome reaction response with anti-TSP in a concentration-dependent manner. In summary, we found that pmTSP-II transcripts were present in the developing oocytes and pmTSP-II protein accumulated in cortical rods, which are partly secreted and thus solubilized to produce dissolved TSP-like proteins that participate in induction of the sperm acrosome reaction-a novel reproductive role for TSP protein family. PMID:23559158

  4. Establishment of a genetic transformation system for the marine pennate diatom Fistulifera sp. strain JPCC DA0580--a high triglyceride producer.

    PubMed

    Muto, Masaki; Fukuda, Yorikane; Nemoto, Michiko; Yoshino, Tomoko; Matsunaga, Tadashi; Tanaka, Tsuyoshi

    2013-02-01

    A genetic transformation system for the marine pennate diatom, Fistulifera sp. JPCC DA0580, was established using microparticle bombardment methods. Strain JPCC DA0580 has been recently identified as the highest triglyceride (60 % w/w) producer from a culture collection of 1,393 strains of marine microalgae, and it is expected to be a feasible source of biodiesel fuel. The transformation conditions for strain JPCC DA0580 were optimised using the green fluorescent protein gene (gfp) and the gene encoding neomycin phosphotransferase II (nptII). The most efficient rate of transformation was attained when tungsten particles (0.6 ?m in diameter) were used for microparticle bombardment. The effect of endogenous and exogenous promoters on the expression of nptII was examined. Endogenous promoters were more efficient for obtaining transformants compared with exogenous promoters. Southern hybridisation analysis suggested that nptII integrated into the nuclear genome. This genetic manipulation technique should allow us to understand the mechanisms of high triglyceride accumulation in this strain, thereby contributing to improving BDF production. PMID:22555859

  5. DNA SEQUENCE OF THE TABD GENE WITHIN THE TABTOXIN BIOSYNTHETIC GENE CLUSTER OF PSEUDOMONAS SYRINGAE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA sequence of the tabD gene within the tabtoxin biosynthetic gene cluster of Pseudomonas syringae (GenBank accession number AY091643). The predicted gene product of the P. syringae tabD gene is a probable aminotransferase, class I and II,pfam00155 (1e-12); most similar to aat-like gene products fr...