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Sample records for gene clone library

  1. [Construction of Frankia genomic libraries and isolation of clones homologous to nodulation genes from Rhizobium leguminosarum].

    Science.gov (United States)

    Cui, Y H; Qin, M; Wang, Y L; Ding, J; Ma, Q S

    1990-01-01

    High molecular genomic DNAs were isolated by using the lysozyme plus achromopeptidase system from Frankia strains At4, Ccol and Hr16, the root nodule endophytes of Alnus, Casuarina and Hippophae respectively, and used to construct genomic libraries in pLAFR1, a broad host range cosmid vector within many gram-negative hosts. The genomic libraries were screened by in situ colony hybridization to identify clones homologous to common nodulation genes of Rhizobium leguminosarum, based on the sequence homology of EcoRI-digested Frankia total DNA to nodABC from Rhizobium meliloti. Several clones showing relatively strong hybridization were found, the recombinant plasmid was isolated, and their homology with Rhizobium nodulation genes was confirmed by spot hybridization. Further work on these positive clones is now underway.

  2. Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning.

    Science.gov (United States)

    Liu, Yao-Guang; Liu, Hongmei; Chen, Letian; Qiu, Weihua; Zhang, Qunyu; Wu, Hao; Yang, Chunyi; Su, Jing; Wang, Zhonghua; Tian, Dongsheng; Mei, Mantong

    2002-01-09

    The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.

  3. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    Science.gov (United States)

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  4. Cloning of a third nitrate reductase gene from the cyanobacterium Anacystis nidulans R2 using a shuttle cosmid library

    NARCIS (Netherlands)

    Kuhlemeier, C.J.; Teeuwsen, V.J.P.; Janssen, M.J.T.; Arkel, G.A. van

    1984-01-01

    A strategy for gene cloning in the cyanobacterium Anacystis nidulans R2 was developed which made use of a gene library constructed in a shuttle cosmid vector. The method involved phenotypic complementation of mutants with pooled cosmid DNA. The development of the procedure and its application to the

  5. Construction and characterization of two Citrus BAC libraries and identification of clones containing the phytoene synthase gene.

    Science.gov (United States)

    Baig, M N R; Yu, An; Guo, Wenwu; Deng, Xiuxin

    2009-05-01

    Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

  6. Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

    DEFF Research Database (Denmark)

    Nielsen, Jeppe Lund; Schramm, Andreas; Bernhard, Anne E.

    2004-01-01

      FLOW CYTOMETRY-ASSISTED CLONING OF SPECIFIC SEQUENCE MOTIFS FROM COMPLEX 16S RRNA GENE LIBRARIES Jeppe L. Nielsen,1 Andreas Schramm,1,2 Anne E. Bernhard,1 Gerrit J. van den Engh,3 and David A. Stahl1* Department of Civil and Environmental Engineering, University of Washington,1 and Institute...... for Systems Biology,3 Seattle, Washington, and Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany2 A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting......-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.  ...

  7. Gene Cloning of Penicillin V Acylase from Bacillus sp BAC4 by Genomic Library

    Directory of Open Access Journals (Sweden)

    ELFI SUSANTI VH

    2004-01-01

    Full Text Available This research was aimed to clone and identify penicillin V acylase (PVA gene of Bacillus sp. BAC4 by genomic library. Chromosome DNA of Bacillus sp. BAC4 was isolated by Wang method. pHB201 of E. coli was isolated by alkali lyses method. Recombinant DNA of Bacillus sp. BAC4 chromosome fragment and pHB201 was made by ligase process using T4 DNA ligase. Transformation of E. coli using this recombinant plasmid was carried out according to Mandel-Higa method. The results indicated that chromosome DNA fragment of Bacillus sp. BAC4 was bigger 23 kb with purity 1,3. Plasmid DNA fragment of E coli was 6,5 kb. Transformants laboring pHB201 recombinant plasmid was screen as blue-white colonies in a medium containing IPTG/X-gal and chloramphenicol.

  8. Flow Cytometry-assisted Cloning of Specific Sequence Motifs fromComplex 16S ribosomal RNA Gene Libraries.

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, J.L.; Schramm, A.; Bernhard, A.E.; van den Engh, G.J.; Stahl, D.A.

    2004-07-21

    A flow cytometry method was developed for rapid screeningand recovery of cloned DNA containing common sequence motifs. Thisapproach, termed fluorescence-activated cell sorting-assisted cloning,was used to recover sequences affiliated with a unique lineage within theBacteroidetes not abundant in a clone library of environmental 16S rRNAgenes. Retrieval and sequence analysis of phylogenetically informativegenes has become a standard cultivation-independent technique toinvestigate microbial diversity in nature (7, 18). Genes encoding the 16SrRNA, because of the relative ease of their selective amplification, havebeen most frequently employed for general diversity surveys (16).Environmental studies have also focused on specific subpopulationsaffiliated with a phylogenetic group or identified by genes encodingspecific metabolic functions (e.g., ammonia oxidation, sulfaterespiration, and nitrate reduction) (8,15,20). However, specificpopulations may be of low abundance (1,23), or the genes encodingspecific metabolic functions may be insufficiently conserved to providepriming sites for general PCR amplification. Three general approacheshave been used to obtain 16S rRNA sequence information from low-abundancepopulations: screening hundreds to thousands of clones in a general 16SrRNA gene library (21), flow cytometric sorting of a subpopulation ofenvironmentally derived cells labeled by fluorescent in situhybridization (FISH) (27), or selective PCR amplification using primersspecific for the subpopulation (2,23). While the first approach is simplytime-consuming and tedious, the second has been restricted to fairlylarge and strongly fluorescent cells from aquatic samples (5, 27). Thethird approach often generates fragments of only a few hundred bases dueto the limited number of specific priming sites. Partial sequenceinformation often degrades analysis, obscuring or distorting thephylogenetic placement of the new sequences (11, 20). A more robustcharacterization of environ

  9. Cloning of Bt cry Genes by Rapid Screening of DNA Libraries with PCR-RFLP

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhong-yi; WU Xian; ZHANG Jie; SONG Fu-ping; GUAN Yu; HUANG Da-fang

    2003-01-01

    Bacillus thuringiensis (Bt) strain C002 contains crylAa, cry2Ab, cry1Ca insecticidal crystal genes and an unkown gene cryX, among which crylCa is located in a 6 -9 kb EcoR Ⅰ fragment of the chromosomal DNA. The total DNA and the plasmids DNA libraries of C002 were constructed in Bt-E. coli shuttle plasmid pHT315 by inserting 6 - 9 kb chromosomal and plasmid DNA fragments prepared respectively with EcoR Ⅰ complete and Sau3A Ⅰ partial digestion. On the basis of every 50 transformants pooled together from 5 - 10 tubes, the pools containing about 2 000 transformants from the plasmids DNA library and 400 transformants from the total DNA library were rapidly screened by PCR-RFLP. Clones containing crylAa, cryX, crylCa, and cry2Ab were isolated and named as pHT-1Aa, pHT-X, pHT-1Ca and pHT-2Ab respectively. Restriction analysis indicated that pHT-1Aa, pHT-1Ca and pHT-2Ab had the typical physical map of the homologous cry genes. Furthermore, each plasmid was transferred into Bt acrystalliferous strain cryB- by eletroporation. SDS-PAGE result showed that transformant of pHT-1Ca expressed 130 kDa protein and bioassay result proved its high toxicity against Spodotera exigua 1st instar larvae with 100% corrected motality.

  10. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras.

    Science.gov (United States)

    Ashelford, Kevin E; Chuzhanova, Nadia A; Fry, John C; Jones, Antonia J; Weightman, Andrew J

    2006-09-01

    A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

  11. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    Science.gov (United States)

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  12. Cloning chromosome specific genes by reciprocal probing of arrayed cDNA and cosmid libraries

    Energy Technology Data Exchange (ETDEWEB)

    Yazdani, A.; Lee, C.C.; Wehnert, M. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    A human gene map will greatly facilitate the association of genes to single locus diseases and provide candidates for genes involved in complex genetic traits. Given the estimated 100,000 human genes an integrated strategy with a high throughput approach for isolation and mapping of expressed sequences is needed to create such a gene map. We have developed an approach that allows high throughput gene isolation and mapping using arrayed genomic and cDNA lambda libraries. Reciprocal probing of the arrayed genomic and cDNA cosmic libraries can rapidly establish cDNA-cosmid associations. Fluorescence in situ hybridization (FISH) chromosomal mapping and expressed sequence tag/sequence tag site (EST/STS) primers generated from DNA sequence of PCR-based mapping using somatic hybrid cell line mapping panels were utilized to characterize further the hybridization-based cDNA cosmid association. We have applied this approach to chromosome 17 using a placental cDNA library and have identified a total of 30 genes out of which 11 are novel. Furthermore seven cDNAs were mapped to 17q21 in this study, providing novel candidate genes for BRCA-1 gene for early onset breast cancer. The results of our study clearly show that an integration of an expression map into physical and genetic maps can provide candidate genes for human diseases that have been mapped to specific regions. This approach combined with the current physical mapping efforts could efficiently provide a detailed human gene map.

  13. Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei.

    Science.gov (United States)

    So, Kum-Kang; Kim, Jung-Mi; Nguyen, Ngoc-Luong; Park, Jin-Ah; Kim, Beom-Tae; Park, Seung-Moon; Hwang, Ki-Jun; Kim, Dae-Hyuk

    2012-12-01

    In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.

  14. Construction of SMART cDNA Library of Sheep Ovary and Identification of Candidate Gene by Homologous Cloning

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5' end of RNA transcript (SMART) approach. This library had a plaque titer of 1 × 109pfu mL-1 and a 96% recombinant ratio of which the fragment length of inserted average eDNA sequences was 1.0 kb. Based on bioinformatics analysis of the sequences, we obtained 338 expressed sequence tags (ESTs) from 380 cDNA clones which indicated 191 contigs. These contigs consist of 89 unmatched ESTs, 9 homologous known genes in sheep, and 93 homologous sequences in species of mouse, bovine, and human beings, including 19 sequences expressed in the ovary or follicle and 14 unknown sequences.Several candidate genes associated with sheep reproduction trait such as epidermal growth factor (EGF), estrogen receptor (ESR), Inhibin, follicle stimulating hormone receptor (FSHR), prostaglandin (PG), and transforming growth factor-β (TGF-β) were identified and the homologous were cloned from this library, which will contribute to compile expression profies and find the major genes of prolificacy of Small Tail Han sheep.

  15. Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost.

    Science.gov (United States)

    Yasir, Muhammad; Khan, Haji; Azam, Syed Sikander; Telke, Amar; Kim, Seon Won; Chung, Young Ryun

    2013-06-01

    In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25-55°C) and pH (5.5-8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.

  16. Expression cloning of different bacterial phosphatase-encoding genes by histochemical screening of genomic libraries onto an indicator medium containing phenolphthalein diphosphate and methyl green.

    Science.gov (United States)

    Riccio, M L; Rossolini, G M; Lombardi, G; Chiesurin, A; Satta, G

    1997-02-01

    A system for expression cloning of bacterial phosphatase-encoding genes has been developed, and its potential has been investigated. The system is based on histochemical screening of bacterial genomic libraries, constructed in an Escherichia coli multicopy plasmid vector, for phosphatase-producing clones using an indicator medium (named TPMG) made of Tryptose-Phosphate agar supplemented with the phosphatase substrate phenolphthalein diphosphate and the stain methyl green. To test the performance of this system, three genomic libraries were constructed from bacterial strains of different species which showed different patterns of phosphatase activity, and were screened using the TPMG medium. Following a partial screening, three different phosphatase-encoding genes (respectively encoding a class A non-specific acid phosphatase, an acid-hexose phosphatase and a non-specific alkaline phosphatase) were shotgun-cloned from the above libraries, indicating that the TPMG-based expression cloning system can be useful for rapid isolation of different bacterial phosphatase-encoding genes.

  17. A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes.

    Science.gov (United States)

    Osiewacz, H D

    1994-07-01

    A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15-20 micrograms of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pAN-sCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.

  18. Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

    Science.gov (United States)

    Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

    2015-01-01

    Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries.

  19. Analysis of microbiota associated with peri-implantitis using 16S rRNA gene clone library

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    Tatsuro Koyanagi

    2010-05-01

    Full Text Available Background: Peri-implantitis (PI is an inflammatory disease which leads to the destruction of soft and hard tissues around osseointegrated implants. The subgingival microbiota appears to be responsible for peri-implant lesions and although the complexity of the microbiota has been reported in PI, the microbiota responsible for PI has not been identified. Objective: The purpose of this study was to identify the microbiota in subjects who have PI, clinically healthy implants, and periodontitis-affected teeth using 16S rRNA gene clone library analysis to clarify the microbial differences. Design: Three subjects participated in this study. The conditions around the teeth and implants were evaluated based on clinical and radiographic examinations and diseased implants, clinically healthy implants, and periodontally diseased teeth were selected. Subgingival plaque samples were taken from the deepest pockets using sterile paper points. Prevalence and identity of bacteria was analyzed using a 16S rRNA gene clone library technique. Results: A total of 112 different species were identified from 335 clones sequenced. Among the 112 species, 51 (46% were uncultivated phylotypes, of which 22 were novel phylotypes. The numbers of bacterial species identified at the sites of PI, periodontitis, and periodontally healthy implants were 77, 57, and 12, respectively. Microbiota in PI mainly included Gram-negative species and the composition was more diverse when compared to that of the healthy implant and periodontitis. The phyla Chloroflexi, Tenericutes, and Synergistetes were only detected at PI sites, as were Parvimonas micra, Peptostreptococcus stomatis, Pseudoramibacter alactolyticus, and Solobacterium moorei. Low levels of periodontopathic bacteria, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were seen in peri-implant lesions. Conclusions: The biofilm in PI showed a more complex microbiota when compared to periodontitis and

  20. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    Science.gov (United States)

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  1. Isolation of BAC Clones Containing Conserved Genes from Libraries of Three Distantly Related Moths: A Useful Resource for Comparative Genomics of Lepidoptera

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    Yuji Yasukochi

    2011-01-01

    Full Text Available Lepidoptera, butterflies and moths, is the second largest animal order and includes numerous agricultural pests. To facilitate comparative genomics in Lepidoptera, we isolated BAC clones containing conserved and putative single-copy genes from libraries of three pests, Heliothis virescens, Ostrinia nubilalis, and Plutella xylostella, harboring the haploid chromosome number, =31, which are not closely related with each other or with the silkworm, Bombyx mori, (=28, the sequenced model lepidopteran. A total of 108–184 clones representing 101–182 conserved genes were isolated for each species. For 79 genes, clones were isolated from more than two species, which will be useful as common markers for analysis using fluorescence in situ hybridization (FISH, as well as for comparison of genome sequence among multiple species. The PCR-based clone isolation method presented here is applicable to species which lack a sequenced genome but have a significant collection of cDNA or EST sequences.

  2. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

    Directory of Open Access Journals (Sweden)

    Gong Xia

    2012-10-01

    Full Text Available Abstract Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

  3. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

    Full Text Available Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new

  4. Comparative analysis of eukaryotic marine microbial assemblages from 18S rRNA gene and gene transcript clone libraries by using different methods of extraction.

    Science.gov (United States)

    Koid, Amy; Nelson, William C; Mraz, Amy; Heidelberg, Karla B

    2012-06-01

    Eukaryotic marine microbes play pivotal roles in biogeochemical nutrient cycling and ecosystem function, but studies that focus on the protistan biogeography and genetic diversity lag-behind studies of other microbes. 18S rRNA PCR amplification and clone library sequencing are commonly used to assess diversity that is culture independent. However, molecular methods are not without potential biases and artifacts. In this study, we compare the community composition of clone libraries generated from the same water sample collected at the San Pedro Ocean Time Series (SPOTs) station in the northwest Pacific Ocean. Community composition was assessed using different cell lysis methods (chemical and mechanical) and the extraction of different nucleic acids (DNA and RNA reverse transcribed to cDNA) to build Sanger ABI clone libraries. We describe specific biases for ecologically important phylogenetic groups resulting from differences in nucleic acid extraction methods that will inform future designs of eukaryotic diversity studies, regardless of the target sequencing platform planned.

  5. Molecular diversity of Bacteroides spp. in human fecal microbiota as determined by group-specific 16S rRNA gene clone library analysis.

    Science.gov (United States)

    Li, Min; Zhou, Haokui; Hua, Weiying; Wang, Baohong; Wang, Shengyue; Zhao, Guoping; Li, Lanjuan; Zhao, Liping; Pang, Xiaoyan

    2009-05-01

    Bacteroides spp. represent a prominent bacterial group in human intestinal microbiota with roles in symbiosis and pathogenicity; however, the detailed composition of this group in human feces has yet to be comprehensively characterized. In this study, the molecular diversity of Bacteroides spp. in human fecal microbiota was analyzed from a seven-member, four-generation Chinese family using Bacteroides spp. group-specific 16S rRNA gene clone library analysis. A total of 549 partial 16S rRNA sequences amplified by Bacteroides spp.-specific primers were classified into 52 operational taxonomic units (OTUs) with a 99% sequence identity cut-off. Twenty-three OTUs, representing 83% of all clones, were related to 11 validly described Bacteroides species, dominated by Bacteroides coprocola, B. uniformis, and B. vulgatus. Most of the OTUs did not correspond to known species and represented hitherto uncharacterized bacteria. Relative to 16S rRNA gene universal libraries, the diversity of Bacteroides spp. detected by the group-specific libraries was much higher than previously described. Remarkable inter-individual differences were also observed in the composition of Bacteroides spp. in this family cohort. The comprehensive observation of molecular diversity of Bacteroides spp. provides new insights into potential contributions of various species in this group to human health and disease.

  6. Analysis of bacterial community structure in Saba-Narezushi (Narezushi of Mackerel) by 16S rRNA gene clone library.

    Science.gov (United States)

    Matsui, Hiroki; Tsuchiya, Rie; Isobe, Yuka; Narita, Miyo

    2013-08-01

    Narezushi, a derivation of sushi, is a traditional Japanese food made by fermenting salted fish meat and cooked rice together. In this study, the microbial diversity of saba-narezushi (narezushi of mackerel, Scomber japonicus) was analyzed by the 16S ribosomal RNA gene clone library method. Chemical composition was also analyzed to compare with different kinds of narezushi. The chemical composition of the narezushi was similar to those obtained from samma-narezushi. Ninety-four clones were randomly selected and DNA sequences of cloned fragments (approx. 890 bp) were analyzed. The DNA sequences obtained were phylogenetically analyzed. The expected operational taxonomy units (OTUs) by Chao1 estimates and Shannon-Wiener index (H') at 97% identity threshold were 48 and 1.822, respectively. The sequence similarity of the cloned fragment was equal to or higher than 98% of the sequence of cultivated bacterial species in the public database. Most of the clones (85%) belonged to lactic acid bacteria (LAB). Lactobacillus curvatus was the most abundant species followed by Lactococcus piscium and Leuconostoc gasicomitatum, suggesting that these bacteria play important roles in the fermentation of saba-narezushi.

  7. Molecular cloning and characterization of multidomain xylanase from manure library

    Science.gov (United States)

    The gene (manf-x10) encoding xylanase from an environmental genomic DNA library was cloned and expressed in Escherichia coli. The encoded enzyme was predicted to be 467 amino acids with a molecular mass of 50.3 kD. The recombinant ManF-X10 was purified by HisTrap affinity column and showed activit...

  8. RAPD-based screening of genomic libraries for positional cloning.

    Science.gov (United States)

    Dioh, W; Tharreau, D; Lebrun, M H

    1997-12-15

    RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.

  9. A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

    Science.gov (United States)

    2011-01-01

    Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

  10. A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocyte-derived immunoglobulin genes in a bacteriophage lambda library.

    Science.gov (United States)

    Portolano, S; Seto, P; Chazenbalk, G D; Nagayama, Y; McLachlan, S M; Rapoport, B

    1991-08-30

    A human Fab fragment (SP2) which binds specifically to human thyroid peroxidase has been generated by expressing random combinations of heavy and light chain immunoglobulin genes (derived from Graves' thyroid cDNA) in a bacteriophage lambda library. In common with many serum TPO autoantibodies, the cloned Fab fragment is IgG1 kappa and has a high affinity for TPO (approximately 10(-9) M). On the basis of their nucleotide sequences, the heavy and light chain genes coding for SP2 belong to families VHI, (D), JH3 and VKI, JK2, respectively. These data provide the first characterization at a molecular level of a human thyroid peroxidase antibody associated with autoimmune thyroid disease.

  11. Mapping genomic library clones using oligonucleotide arrays

    Energy Technology Data Exchange (ETDEWEB)

    Sapolsky, R.J.; Lipshutz, R.J. [Affymetrix, Santa Clara, CA (United States)

    1996-05-01

    We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adaptors. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density olligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection. 28 refs., 9 figs., 2 tabs.

  12. YAC-base transfer of fibroin gene from Anthemea yamamal to domestic silkworm Bombyx mod (I)——Identification of fibroin clones from a YAC library of Antheraeu yamamai constructed from its posterior silk gland

    Institute of Scientific and Technical Information of China (English)

    唐恒立; 柴建华; 李振刚

    1995-01-01

    Antheraea yamamai (Japanese oak silkworm) is a kind of silkworm of great economic value. and the process of the expression of its silkprotem genes is a perfect model for the study of molecular regulation during the development and differentiation. So studying its fibroin and allied genes is of both theoretic and practical magnitude A YAC library with an average size of 570kb is constructed from the posterior silk gland, using pYAC4 as a vector. The library was screened by means of polymerase chain reaction, and clones representing fibroin gene were isolated and characterized.

  13. Cloning and sequencing genes related to preeclampsia

    Institute of Scientific and Technical Information of China (English)

    SHI Juan-zi; LIU Yan-fang; YAO Yuan-qing; YAN Wei; ZHU Feng; ZHAO Zhong-liang

    2001-01-01

    To clone genes specifically expressed in the placenta of patients with preeclampsia, and to explain the mechanism in the etiopathology ofpreeclampsia. Methods: The placentae ofpreeclamptic and normotensive subjects with pregnancy were used as models, and the cDNA Library was constructed and 20 differentially expressed fragments were cloned after a new version of PCR-based subtractive hybridization. The false positive clones were identified by reverse dot blot analysis. With one of the obtained gene taken as the probe, the placentas of 10 normal pregnant women and 10 preeclamptic patients were studied by using dot hybridization methods. Results: Six false positive clones were identified by reverse dot blot, and the rest 14 clones were identified as preeclampsia-related genes. These clones were sequenced, and analyzed with BLAST analysis system. Eleven of 14 clones were genes already known, among which one belongs to necdin family; the rest 3 were identified as novel genes. These 3 genes were acknowledged by GenBank, with the accession numbers AF232216, AF232217, AF233648. The results of dot hybridization using necdin gene as probe were as follows: (1) There was this mRNA in the placental tissues of normal pregnancy as well as in that ofpreeclampsia.(2) The intensity of transcription of this mRNA in the placental tissues of preeclampsia increased significantly compared with that of the normal pregnancy (P<0.05). Conclusions: This study for the first time reported this group of genes, especially necdin-expressing gene, which are related to the etiopathology of preeclampsia. In addition, the overtranscription ofnecdin gene has been found in preeclampsia. It is helpful in further studies of the etiology ofpreeclampsia.

  14. Sequencing and identification of expressed Schistosoma mansoni genes by random selection of cDNA clones from a directional library

    Directory of Open Access Journals (Sweden)

    Glória R. Franco

    1995-04-01

    Full Text Available We have initiated a gene discovery program in Schistosoma mansoni based on the technique of Expressed Sequence Tags (ESTs, i.e. partial sequences of cDNAs obtained from single passes in automatic DNA sequencers. ESTs can be used to identify genese onf the basis of their homology whith sequences from other species deposited in DNA or protein databases. Trasncripts with sequences without matches in teh databases may represent novel parasite-specific genes. This approach has shown to be very efficient and in less than two years a broad range of novel genes has already been ascertained, more than doubling the number of known S. mansoni genes.

  15. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    OpenAIRE

    Alamar Santiago; Arribas Raquel; Forment Javier; Alonso-Cantabrana Hugo; Marques M Carmen; Conejero Vicente; Perez-Amador Miguel A

    2009-01-01

    Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information an...

  16. Positional cloning of deafness genes

    NARCIS (Netherlands)

    Kremer, H.; Cremers, F.P.M.

    2009-01-01

    The identification of the majority of the known causative genes involved in nonsyndromic sensorineural hearing loss (NSHL) started with linkage analysis as part of a positional cloning procedure. The human and mouse genome projects in combination with technical developments on genotyping, transcript

  17. Rice's Salt Tolerance Gene Cloned

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    @@ In cooperation with US colleagues, CAS researchers have made significant progress in their studies into functional genes for key agronomic traits by cloning SKC1, a salt-tolerant functional gene of rice and making clear its biological functions and mechanisms. This pioneering work,which was reported in the Oct. issue of Nature Genetics (37:1141-1146), is believed to hold promise to increase the output of the crop plant in this country.

  18. Structural analysis and molecular modeling of two antitrichosanthin IgE clones from phage antibody library

    Institute of Scientific and Technical Information of China (English)

    LIZONGDONG; YURENYUAN; 等

    1997-01-01

    Recently we constructed a murine IgE phage surface display library and screened out two IgE (Fab) clones with specific binding activity to Trichosanthin (TCS).In this work,the Vε and Vκ genes of the two clones were sequenced and their putative germline gene usages were studied.On the basis of the known 3D structure of Trichosanthin and antibody,molecular modeling was carried out to study the antigen-antibody interaction.The possible antigenic determinant sites on the surface of TCS recognized by both the clones were analyzed,and the reaction forces between TCS and two Fab fragments were also analyzed respectively.

  19. Cloning and developmental expression of the murine neurofilament gene family.

    NARCIS (Netherlands)

    J-P. Julien (Jean-Pierre); D.N. Meijer (Dies); D. Flavell (David); J. Hurst; F.G. Grosveld (Frank)

    1986-01-01

    textabstractDNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a lambda gt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the e

  20. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    Science.gov (United States)

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.

  1. Database for exchangeable gene trap clones: pathway and gene ontology analysis of exchangeable gene trap clone mouse lines.

    Science.gov (United States)

    Araki, Masatake; Nakahara, Mai; Muta, Mayumi; Itou, Miharu; Yanai, Chika; Yamazoe, Fumika; Miyake, Mikiko; Morita, Ayaka; Araki, Miyuki; Okamoto, Yoshiyuki; Nakagata, Naomi; Yoshinobu, Kumiko; Yamamura, Ken-ichi; Araki, Kimi

    2014-02-01

    Gene trapping in embryonic stem (ES) cells is a proven method for large-scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox-mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [http://egtc.jp]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene-trap mouse lines. Because we used a promoter-trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

  2. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in

  3. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in

  4. Comparison of soil bacterial communities of Pinus patula of Nilgiris, western ghats with other biogeographically distant pine forest clone libraries.

    Science.gov (United States)

    Rohini-Kumar, M; Osborne, Jabez W; Saravanan, V S

    2013-07-01

    The bacterial community structure of the rhizosphere and non-rhizosphere soil of Pinus patula, found in the Nilgiris region of Western Ghats, was studied by constructing 16S rRNA gene clone libraries. In the rhizosphere and non-rhizosphere soil clone libraries constructed, 13 and 15 bacterial phyla were identified, respectively. The clone libraries showed the predominance of members of culturally underrepresented phyla like Acidobacteria and Verrucomicrobia. The Alphaproteobacteria and Acidobacteria clones were predominant in rhizosphere and non-rhizosphere soil samples, respectively. In rhizosphere, amongst Alphaproteobacteria members, Bradyrhizobium formed the significant proportion, whereas in non-rhizosphere, members of subdivision-6 of phylum Acidobacteria were abundant. The diversity analysis of P. patula soil libraries showed that the phylotypes (16S rRNA gene similarity cutoff, ≥97 %) of Acidobacteria and Bacteroidetes were relatively predominant and diverse followed by Alphaproteobacteria and Verrucomicrobia. The diversity indices estimated higher richness and abundance of bacteria in P. patula soil clone libraries than the pine forest clone libraries retrieved from previous studies. The tools like principal co-ordinate analysis and Jackknife cluster analysis, which were under UniFrac analysis indicated that variations in soil bacterial communities were attributed to their respective geographical locations due to the phylogenetic divergence amongst the clone libraries. Overall, the P. patula rhizosphere and non-rhizosphere clone libraries were found significantly unique in composition, evenly distributed and highly rich in phylotypes, amongst the biogeographically distant clone libraries. It was finally hypothesised that the phylogenetic divergence amongst the bacterial phylotypes and natural selection plays a pivotal role in the variations of bacterial communities across the geographical distance.

  5. Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics

    Directory of Open Access Journals (Sweden)

    Hui eYuan

    2015-09-01

    Full Text Available Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of Pyrococcus furiosus proteins at whole genome level, we constructed expression plasmids of each Pyrococcus furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3pLysS. In summary, this recombinant expression library of Pyrococcus furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms.

  6. Analysis of bacterial community diversity in anaerobic fluidized bed bioreactors treating 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) using 16S rRNA gene clone libraries.

    Science.gov (United States)

    Arnett, Clint M; Rodriguez, Giselle; Maloney, Stephen W

    2009-01-01

    Clone libraries were used to evaluate the effects of 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) on bacterial populations within three anaerobic bioreactors. Prior to the addition of DNAN and MNA greater than 69% of the clones in each reactor were identified as a single Desulfuromonales species. However, after 60 days of treatment the Desulfuromonales distribution decreased to less than 13% of the distribution and a clone identified as a Levilinea sp. became the dominant organism at greater than 27% of the clone distribution in each reactor suggesting the species may play an important roll in the reduction of DNAN and MNA.

  7. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones.

    Science.gov (United States)

    Ginolhac, Aurélien; Jarrin, Cyrille; Gillet, Benjamin; Robe, Patrick; Pujic, Petar; Tuphile, Karine; Bertrand, Hélène; Vogel, Timothy M; Perrière, Guy; Simonet, Pascal; Nalin, Renaud

    2004-09-01

    The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.

  8. A diverse bacterial community in an anoxic quinoline-degrading bioreactor determined by using pyrosequencing and clone library analysis.

    Science.gov (United States)

    Zhang, Xiaojun; Yue, Siqing; Zhong, Huihui; Hua, Weiying; Chen, Ruijia; Cao, Youfang; Zhao, Liping

    2011-07-01

    There is a concern of whether the structure and diversity of a microbial community can be effectively revealed by short-length pyrosequencing reads. In this study, we performed a microbial community analysis on a sample from a high-efficiency denitrifying quinoline-degrading bioreactor and compared the results generated by pyrosequencing with those generated by clone library technology. By both technologies, 16S rRNA gene analysis indicated that the bacteria in the sample were closely related to, for example, Proteobacteria, Actinobacteria, and Bacteroidetes. The sequences belonging to Rhodococcus were the most predominant, and Pseudomonas, Sphingomonas, Acidovorax, and Zoogloea were also abundant. Both methods revealed a similar overall bacterial community structure. However, the 622 pyrosequencing reads of the hypervariable V3 region of the 16S rRNA gene revealed much higher bacterial diversity than the 130 sequences from the full-length 16S rRNA gene clone library. The 92 operational taxonomic unit (OTUs) detected using pyrosequencing belonged to 45 families, whereas the 37 OTUs found in the clone library belonged to 25 families. Most sequences obtained from the clone library had equivalents in the pyrosequencing reads. However, 64 OTUs detected by pyrosequencing were not represented in the clone library. Our results demonstrate that pyrosequencing of the V3 region of the 16S rRNA gene is not only a powerful tool for discovering low-abundance bacterial populations but is also reliable for dissecting the bacterial community structure in a wastewater environment.

  9. Characterization of the airborne bacteria community at different distances from the rotating brushes in a wastewater treatment plant by 16S rRNA gene clone libraries

    Institute of Scientific and Technical Information of China (English)

    Yunping Han; Lin Li; Junxin Liu

    2013-01-01

    Biological risks of bioaerosols emitted from wastewater treatment processes have attracted wide attention in the recent years.However,the culture-based analysis method has been mostly adopted for detecting the bacterial community in bioaerosols,which may result in the underestimation of total microorganism concentration as not all microorganisms are cultivable.In this study,oligonucleotide fingerprinting of 16S rRNA genes was applied to reveal the composition and structure of the bacterial community in bioaerosols from an Orbal oxidation ditch in a Beijing wastewater treatment plant (WWTP).Bioaerosols were collected at different distances from the aerosol source,rotating brushes,and the sampling height was 1.5 m which is the common respiratory height of a human being.The bacterial communities of bioaerosols were diverse,and the lowest bacterial diversity was found at the sampling site just after the rotating brush rotating brush.A large proportion of bacteria in bioaerosols were affiliated with Proteobacteria and Bacteroidetes.Numerous bacteria present in the bioaerosols also emerged in water,indicating that the bacterial community in the bioaerosols was related to that of the aerosols' sources.The forced aeration of rotating brushes brought about observably distinct bacterial communities between sampling sites situated before and after the rotating brush.Isolation sources of closest relatives in bioaerosols done libraries were associated with the aqueous environment in the WWTP.Common potential pathogens in bioaerosols as well as those not reported in previous research were also analyzed in this study.Measures should be adopted to reduce the emission of bioaerosols and prevent their exposure to workers.

  10. A five-fold pig bacterial artificial chromosome library:a resource for positional cloning and physical mapping

    Institute of Scientific and Technical Information of China (English)

    LIU Wei; LI Ning; ZHANG Ying; LIU Zhaoliang; GUO Li; WANG Xiaobo; FEI Jing; FENG Jidong; ZHAO Rui; HU Xiaoxiang

    2006-01-01

    A pig BAC library was constructed with genomic DNA from a male Erhualian pig. After partial digestion with Hind Ⅲ or BamH I the fragments obtained were cloned into the pBeloBAC11 vector. The library consists of 184320 clones which stored in 480pieces 384-well plates (20 plates per superpool). A two-step 4-dimension PCR screening system was established to screen the positive clones. An average insert size of 128 kb was estimated from 105 randomly isolated clones, which indicates that the library is more than five times of genomic coverage. For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 10single-copy genes were screened out and the positive clones were yielded between 1 and 8 with an average of 3.6. Positive superpools were obtained for 32 microsatellite markers selected from different regions of pig genome. The number of positive superpools for each marker varies from 1 to 9 with an average of 4.78. This BAC library provides an additional resource for pig physical mapping and gene identification.

  11. Cloning and expression of Kluyveromyces fragilis LAC4 gene

    Institute of Scientific and Technical Information of China (English)

    霍克克; 李育阳

    1995-01-01

    The genomic library of Kluyveromyces fragilis was constructed in E.coli TG1,and 5β-galactosidase gene(LAC4)clones have been obtained from the library by complementation of theKluyveromyces lactis lac4-8 mutation.The studies on the structure and the function of the LAC4 gene revealedthat(i)the gene can also complement E.coli lacZ mutation;(ii)the physical map of the K.fragilis LAC4gene was very similar to that of K.lactis;(iii)the β-galactosidase levels expressed by the clone strains weremuch higher than that expressed by the original strain;(iv)the variation of the β-galactosidase level of differ-ent clone strains induced by lactose or galactose was related to the retained degree of the 5’ flanking region ofLAC4 gene,suggesting that there migth he a lactose specific transcription activating element in the region.

  12. Consensus maps of cloned plant cuticle genes

    Institute of Scientific and Technical Information of China (English)

    Eviatar; Nevo

    2010-01-01

    Plant cuticle,which covers the plant surface,consists of waxes and cutins,and is associated with plant drought,cold,and salt resistance.Hitherto,at least 47 genes participating in the formation of plant cuticle have been cloned from Arabidopsis thaliana,Oryza sativa,Zea mays,Ricinus communis,Brassica napus,and Medicago truncatula;and about 85% of them encode proteins sharing above 50% identities with their rice homologous sequences.These cloned cuticle genes were mapped in silico on different chromosomes of rice and Arabidopsis,respectively.The mapping results revealed that plant cuticle genes were not evenly distributed in both genomes.About 40% of the mapped cuticle genes were located on chromosome 1 in Arabidopsis,while 20% of the mapped cuticle genes were located on chromosome 2 but none on chromosome 12 in rice.Some cloned plant cuticle genes have several rice homologous sequences,which might be produced by chromosomal segment duplication.The consensus map of cloned plant cuticle genes will provide important clues for the selection of candidate genes in a positional cloning of an unknown cuticle gene in plants.

  13. Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries

    Energy Technology Data Exchange (ETDEWEB)

    Gingrich, J.C.; Boehrer, D.M.; Garnes, J.A. [Lawrence Livermore National Lab., CA (United States)] [and others

    1996-02-15

    This article discusses the construction and characterization of three human chromosome 2-specific clone libraries. A chromosome 2-specific PAC library was also constructed from a hybrid cell line. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. 47 refs., 3 figs., 1 tab.

  14. A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance.

    Science.gov (United States)

    Tomkins, J P; Mahalingam, R; Smith, H; Goicoechea, J L; Knap, H T; Wing, R A

    1999-09-01

    We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73 728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.

  15. Cloning arbuscule-related genes from mycorrhizas

    DEFF Research Database (Denmark)

    Burleigh, Stephen

    2000-01-01

    Until recently little was known about the identity of the genes expressed in the arbuscules of mycorrhizas, due in part to problems associated with cloning genes from the tissues of an obligate symbiont. However, the combination of advanced molecular techniques, innovative use of the materials...... available and fortuitous cloning has resulted in the recent identification of a number of arbuscule-related genes. This article provides a brief summary of the genes involved in arbuscule development, function and regulation, and the techniques used to study them. Molecular techniques include differential...

  16. MOLECULAR CLONING OF HUMAN NEUROTROPHIN-4 GENE

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Cloning and sequencing of the human neurotrophin-4(hNT-4) gene.Methods With the chromosomal DNA of human blood lymphocytes as template,hNT-4 coding genes were amplified by polymerase chain reaction(PCR) and recombinated into phage vector pGEM-T Easy,which were sequenced by using Sanger's single stranded DNA terminal termination method.Results The sequence of the cloned gene is completely the same as that reported in the literature(GenBank data base,M86528).Conclusion This study successfully cloning and sequenced the gene of mhNT-4,and it would be convenient for us to study the expression of mhNT-4 in eukaryote,and to continue the research on the gene therapy of Alzheimer's disease intensively.This study indicate that the hNT-4 is conservative in different races and individuals.

  17. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    Directory of Open Access Journals (Sweden)

    CLAUDIA TEREZIA SOCOL

    2013-12-01

    Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

  18. Identification and cloning of a new category of DNA fragments which are poorly represented in human genomic libraries.

    Science.gov (United States)

    Wong, P; Myal, Y; Shui, R; Tenniswood, M

    1993-01-29

    We have developed an alternative strategy for the preparation of genomic libraries that ensures better representation of genomic sequences commonly underrepresented in genomic libraries constructed using standard protocols. To overcome the apparent bias against genomic sequences containing clusters of restriction sites we have used nonoptimized restriction digestions to generate a mixture of DNA fragments which have been cloned into the EMBL3 vector. To validate this protocol we have screened the EMBL3 library to identify a full length genomic clone of the prolactin-inducible gene (PIP). Screening 4 other, commercially available, genomic libraries prepared using standard protocols for restriction digestion of the genomic DNA failed to identify any full length clones. We show that this increase in the representation of the full length PIP gene in the EMBL3 genomic library is attributable to the method of insert preparation used and suggests that an additional subset of sequences that may be poorly represented in, or absent from, established libraries may be cloned using this modified protocol.

  19. Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

    Institute of Scientific and Technical Information of China (English)

    Wan-Song Ni; Zhi-Yong Lei; Xi Chen; David J. Oliver; Cheng-Bin Xiang

    2007-01-01

    Salt cress (Thellungiella halophila), a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt-tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombinationassisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination-assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.

  20. [Structural organization of the human p53 gene. I. Molecular cloning of the human p53 gene].

    Science.gov (United States)

    Bukhman, V L; Ninkina, N N; Chumakov, P M; Khilenkova, M A; Samarina, O P

    1987-09-01

    Human p53 gene was cloned from the normal human placenta DNA and DNA from the strain of human kidney carcinoma transplanted into nude mice. Representative gene library from tumor strain of human kidney carcinoma and library of 15 kb EcoRI fragments of DNA from normal human placenta were constructed. Maniatis gene library was also used. Five clones were isolated from kidney carcinoma library; they covered 27 kb and included full-length p53 gene of 19.5 kb and flanking sequences. From normal placenta libraries three overlapped clones were obtained. Restriction map of cloned sequences was constructed and polarity of the p53 gene determined. The first intron of the gene is large (10.4 kb); polymorphic BglII site was observed in this intron, which allows to discriminate between allelic genes. One of these (BglII-) is ten times more abundant that the other (BglII+). Both allelic genes are able to synthesize the 2.8 kb p53 gene.

  1. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  2. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning

  3. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  4. Isolation and characterization of sequences homologous to the tobacco clone axi 1 (auxin independent) from a Vicia sativa nodule cDNA library

    NARCIS (Netherlands)

    Yalçin-Mendi, Y.; Çetiner, S.; Bisseling, T.

    2001-01-01

    In this research, partial nucleotide sequences of the axi 1 gene, which is related to auxin perception and transduction, isolated from Vicia sativa using cDNA library screening were investigated. Four V. sativa cDNA clones representing homologous of the tobacco axi 1 (auxin independent) cDNA clone w

  5. Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

    Science.gov (United States)

    Jiang, Likun; You, Weiwei; Zhang, Xiaojun; Xu, Jian; Jiang, Yanliang; Wang, Kai; Zhao, Zixia; Chen, Baohua; Zhao, Yunfeng; Mahboob, Shahid; Al-Ghanim, Khalid A; Ke, Caihuan; Xu, Peng

    2016-02-01

    The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

  6. RecA-assisted rapid enrichment of specific clones from model DNA libraries.

    Science.gov (United States)

    Teintze, M; Arzimanoglou, I I; Lovelace, C I; Xu, Z J; Rigas, B

    1995-06-26

    An approach to library screening is being developed, in which the desired clone is "fished" out of a mixture of all the recombinants in a library with a RecA-coated probe. In the current embodiment of this method, we used as a probe the (+) strand of an M13 phage containing a fragment of the human albumin gene and a (dA)49 stretch. We screened a library of two plasmids, one containing the same albumin fragment as the probe, and one heterologous to the probe in 50-100 fold molar excess. The plasmids were linearized. Probe and library were reacted in the presence of RecA, the mixture was loaded onto an oligo(dT) column, which retained the probe-target complex by base-pairing to the dAs of the probe, the uncaptured plasmids were washed, and the probe-target complex was released from the column, religated and propagated into E. coli. Recovery of the homologous target was 15-28%, and enrichment for the homologous plasmid was 200 to 400-fold. This approach may provide a general method for expedited DNA library screening.

  7. THE CLONING OF HUMAN NEUROTROPHIN-3 GENE

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    In the present study, we have cloned the gene of human neurotrophin-3 (hNT-3) from the genomic DNA of white blood cells (WBC) by polymerase chain reaction (PCR). The amplification products were cloned into pUC19 and sequenced. Genomic sequence comparison of the cloned fragment and the reported hNT-3 (GenBank M61180) reveals 7 base differences: 1 in the signal peptide, 3 in the prepro peptide, and 3 in the mature hNT-3. Except the 2 varied bases (16th, T to G; 285th, A to C) in the signal peptide and pro-sequence resulted in the change of their encoded amino-acids (Tyr→Asp; Gln→His), the other varied bases have no influence on their respective encoded amino-acids, and all the changes have no influence on the open reading frame (ORF) of the hNT-3.

  8. BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.

    Directory of Open Access Journals (Sweden)

    Wu Cheng-Cang

    2011-05-01

    Full Text Available Background Although second generation sequencing (2GS technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. Result Five new BAC libraries were constructed for barley (Hordeum vulgare L. cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast, empty wells and off-scale clones (clones with 250 fragments. Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. Conclusion BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.

  9. A polymerase chain reaction-based method for isolating clones from a complimentary DNA library in sheep.

    Science.gov (United States)

    Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon; Hutmacher, Dietmar W

    2014-10-01

    The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes.

  10. Cloning of Leishmania Major P4 Gene

    Directory of Open Access Journals (Sweden)

    Minoo Shaddel

    2008-01-01

    Full Text Available Objective: Leishmania major P4 gene is normally expressed during amastigote form ofthe parasite and can be good candidate for producing an effective vaccine. In this study wecloned this gene in suitable vector (pQE-30 for further vaccine preparation studies.Materials and Methods: Leishmania promastigotes were grown in N.N.N.medium and culturein RPMI 1640 cell culture medium. Total genomic DNA was extracted by centrifugationof promastigotes. The pellet was suspended in lysis buffer and followed by boiling method.PCR was carried out using P4 gene specific primers. PCR product was detected by agarosgel electrophoresis and cloned into Bluescript plasmid via T/A cloning method. Reactionwas transformed into XL1- Blue competent cell and recombinant plasmid screened usingagar plate contained X-gal and IPTG. The product was extracted, digested by restrictionenzyme and electrophoresed on agarose gel.Results: Plasmid was extracted and cloned gene was released by restriction enzyme andsubcloned into pQE-30 expression vector.Conclusion: This construct is ready for protein expression in in-vitro.

  11. 16S rRNA gene clone library analysis of bacterial communities of the tick with infection of 4 species of pathogens%4种病原菌特异基因片段阳性蜱的16S rRNA基因克隆文库分析

    Institute of Scientific and Technical Information of China (English)

    张守印; 俞东征; 孙继民; 贺金荣; 付秀萍; 张景山; 张建华; 蔡虹; 马凤琴; 海荣

    2009-01-01

    Objective To develop the method of 16S rRNA gene clone library for tick bacterial flora analysis, and to analyze the detection effective of pathogens in tick and capacity of bacterial flora diversity. Methods Primers were designed according to the specific gene of Borrelia burgdorferi, Bartonella henselae, Anaplasma phagocytophilum, Ehrlichia chaffeensis and templates were choosen by positive PCR result to amplify the DNA extracted from the ticks. One set of primers targeting 16S rRNA gene conserved region were chosen to amplify certain fragments, DNA extraction, PCR reaction, cloning and sequencing. Nucleotide sequences were compared with GenBank database. Calculated Coverage values of clone library and Shannon-Wiener diversity index. Results Sixteen defined genus-or species-bacteria were detected in 103 valid sequences. Eight species were edge type (Clone No. > 5). Three kinds of pathogens were identified (Borrelia burgdorferi, Bartonella henselae and Rickettsia sp). Three kinds of pathogens were not edge type(Clone No. 5个);检测到伯氏疏螺旋体、汉赛巴通体和立克次体3种病原菌,但这3种病原菌均不是优势类型(克隆子数均<5个).Coverage值为96.11%,Shannon-Wiener多样性指数为2.40.克隆序列分析结果表明,蜱寄生细菌主要为α、γ变形菌纲,占56.25%(9/16).结论 16S rRNA基因序列分析可以对蜱标本进行菌群相对定量研究,可以同时检出多种病原菌,是一种较好的细菌菌群多样性分析和病原菌筛检方法.

  12. Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain

    Science.gov (United States)

    Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.

    1985-12-01

    A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

  13. Rapid Characterization of S. mansoni Expression Library Clones of Potential Interest

    Directory of Open Access Journals (Sweden)

    KANAMURA Herminia Yohko

    1997-01-01

    Full Text Available A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing

  14. Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Mikhail Nefedov

    2011-01-01

    Full Text Available We have developed a new approach to screen bacterial artificial chromosome (BAC libraries by recombination selection. To test this method, we constructed an orangutan BAC library using an E. coli strain (DY380 with temperature inducible homologous recombination (HR capability. We amplified one library segment, induced HR at 42∘C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies.

  15. Construction of A Deep Coverage Rice BAC Library and Identification of Clones Associated with Disease-resistant Genes%高覆盖率水稻BAC库的构建及抗病基因相关克隆的筛选

    Institute of Scientific and Technical Information of China (English)

    王文明; 江光怀; 王世全; 朱立煌; 翟文学

    2001-01-01

    A BAC library for IRBB56, an accession pyramiding Xa4, xa5 and xa13 three bacterial blight resistance genes, was constructed. The library contains 55 296 clones with an average insert size of 132kb. Based on a haploid genome size of 450Mb, the coverage of the library was about 14 genome equivalents that make it oneof the most comprehensive BAC libraries available in rice and provide 99.99% possibility to isolate any interested rice genes or sequences in the library. To determine the representation of organelle DNA homologues in the library, the library was screened with three different choloroplast genes and four mitochondrial genes, respectively. Results from this screening showed that less than 1% of clones in the library contain organelle genomic DNA homologues. Then, DNA markers on three different chromosomes linked to Xa4, xa5, and xa13, and a PCR fragment of rice UROD gene, were used to screen the library resulting in a range of 11~106 hits that will promote the isolation of these genes. The deep coverage and the large insert size of the library will facilitate physical mapping, isolation, and cloning of rice genes.%利用含Xa4、xa5和xa13 3个水稻白叶枯病抗性基因的累加系IRBB56构建了一个水稻细菌人工染色体文库。该文库包含55 296个克隆,平均插入片段为132kb。按水稻基因组为450Mb 计,该文库覆盖14倍基因组,筛选出任一水稻基因或序列的概率为99.99%。用均匀分布的3个叶绿体基因和4个线粒体基因克隆作探针筛选文库,结果显示该文库中含细胞器基因组DNA同源序列的克隆数小于1%。用分布于水稻3条不同染色体、分别与Xa4、xa5和xa13连锁的DNA标记筛选文库,分别检测出11~106个阳性克隆,为克隆这些基因打下了基础。 该文库对水稻基因组的高度覆盖率和较大的插入片段,非常适合于物理作图和基因的分离和克隆。

  16. Construction of gene targeting vectors from lambda KOS genomic libraries.

    Science.gov (United States)

    Wattler, S; Kelly, M; Nehls, M

    1999-06-01

    We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones. The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked by negative-selection cassettes. The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert. We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.

  17. 水稻白叶枯病菌GX1329基因组文库的构建及含编码TAL效应物基因的克隆的分离%Construction of a Genomic Library of Xanthomonas oryzae pv.oryzae Strain GX1329 and Isolation of Clones Containing the Genes Encoding TAL Effectors

    Institute of Scientific and Technical Information of China (English)

    张子宇; 赵帅; 莫伟兰; 罗雪梅; 玉延华; 段承杰; 冯家勋

    2011-01-01

    Bacterial leaf blight (BLB), caused by Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo),is a serious threat to yield losses in the main regions of rice growth including Asia, North America and Africa.BLB could lead to a crop loss of up to 50%. It is known that the virulence of Xoo mainly relies on type Ⅲ secretion system (T3SS) and its secreted effectors. To know the numbers of genes encoding avrBs3/pthA family members in Guangxi Xoo strain GX1329, a genomic DNA library containing 736 clones was successfully constructed by partially digesting the genomic DNA with A lu Ⅰ . Restriction enzyme BamH Ⅰ digestion analysis of plasmids from 15 randomly chosen library clones showed that the cloned DNA in the genomic library was highly random.The size of the smallest cloned DNA in one clone was 27.7kb, the size of the biggest cloned DNA in one clone was 58.5 kb, and the average size of cloned DNA in one clone was 39.9 kb. The cloning capacity of the library is about 2.8x103 Mb with high randomness, and the probability of any one gene contained in the library was about 99.4%.Thirty-seven positive clones were screened out from the GX1329 genomic library by colony in situ hybridization using the 252th to 486th bp sequence of avrXa10 from Xoo strain PXO86 as probe. Southern hybridization analysis of the 17 clones showed that they contain at least 13 different avrBs3/pthA genes. The results also showed that the avrB.s3/pthA family genes occurred in individual or clusters in the genome of strain GX1329. This work defined the number of avrBs3/pthA family genes in the genome of GX1329, which may provide a solid basis for further studying the function of the genes.%由革兰氏阴性细菌水稻白叶枯病菌引起的水稻白叶枯病是亚洲、北美以及非洲部分地区最严重的水稻病害之一,水稻白叶枯病可使水稻减产高达50%以上.研究表明水稻白叶枯病菌的毒力主要依靠三型分泌系统所分泌的效应物.为了解

  18. Requirements in screening cDNA libraries for new genes and solutions offered by SBH technology

    Energy Technology Data Exchange (ETDEWEB)

    Drmanac, R.; Drmanac, S.; Labat, I.; Stavropoulos, N.

    1993-12-31

    Under different assumptions about the total number of genes, the number of housekeeping and tissue-specific genes, and the difference in the number of mRNAs per cell for functional and nonfunctional genes, significantly different results can be expected from screening random cDNA clones. We have developed gene expression models as a guide for interpretation of experimental results. For statistical, biological, and technical reasons, the search for 100,000 plus genes and discrimination between nonfunctional, housekeeping, and tissue-specific genes requires the analysis of up to 10 million clones from 20 to 50 tissues. Oligonucleotide hybridization of dense clone blots is an inexpensive and fast way to screen such large clone sets. Our preliminary results on control clones and thousands of cDNA clones from an infant brain library demonstrate the feasibility of the method. We present several models of gene expression and analyze the main factors which can influence the hunt for new genes via the screening of random cDNA libraries. The basic steps in the preparation and use of dense DNA dot arrays are described, and some results that demonstrate the feasibility and efficiency of gene inventorying by oligonucleotide hybridization are presented. Furthermore, partial SBH and single-pass gel sequencing are compared and a gene analysis scheme that combines the two approaches is discussed.

  19. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH)

    NARCIS (Netherlands)

    Leveau, J.H.J.; Gerards, S.; De Boer, W.; Van Veen, J.A.

    2004-01-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia

  20. Recovery of a soybean urease genomic clone by sequential library screening with two synthetic oligodeoxynucleotides.

    Science.gov (United States)

    Krueger, R W; Holland, M A; Chisholm, D; Polacco, J C

    1987-01-01

    We report the first isolation of a low-copy-number gene from a complex higher plant (soybean) genome by direct screening with synthetic oligodeoxynucleotide (oligo) probes. A synthetic, mixed, 21-nucleotide (nt) oligo (21-1) based on a seven amino acid (aa) sequence from soybean seed urease, was used to screen genomic libraries of soybean (Glycine max [L.] Merr.) in the lambda Charon 4 vector. Twenty homologous clones were recovered from a screen of 500,000 plaques. These were counterscreened with embryo-specific cDNA (15-2 cDNA) made by priming with a second, mixed 15-nt oligo (15-2), based on a Jack bean (Canavalia ensiformis) urease peptide [Takishima et al., J. Natl. Def. Med. Coll. 5 (1980) 19-23]. Five out of 20 clones were homologous to 15-2 cDNA and proved to be identical. Nucleotide sequence analysis of representative clone E15 confirmed that it contained urease sequences. Subclones of E15 homologous to the oligo probes contain a deduced amino acid sequence which matches 108 of 130 aa residues of an amino acid run in a recently published [Mamiya et al., Proc. Jap. Acad. 61B (1985) 359-398] complete protein sequence for Jack-bean seed urease. Using clone E15 as a probe of soybean embryonic mRNA revealed a homologous 3.8-kb species that is the size of the urease messenger. This species is absent from mRNA of embryos of a soybean seed urease-null mutant. However, both urease-positive and urease-null genomes contain the 11-kb DNA fragment bearing urease sequences.

  1. A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species.

    Science.gov (United States)

    Aakvik, Trine; Degnes, Kristin Fløgstad; Dahlsrud, Rannveig; Schmidt, Frank; Dam, Ragnar; Yu, Lihua; Völker, Uwe; Ellingsen, Trond Erling; Valla, Svein

    2009-06-01

    The majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. To enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (pRS44) for fosmid and bacterial artificial chromosome (BAC) cloning. pRS44 can be efficiently transferred to numerous hosts by conjugation. It replicates in such hosts via the plasmid RK2 origin of replication, while in Escherichia coli it replicates via the plasmid F origin. The vector was found to be remarkably stable due to the insertion of an additional stability element (parDE). The copy number of pRS44 is adjustable, allowing for easy modifications of gene expression levels. A fosmid metagenomic library consisting of 20 000 clones and BAC clones with insert sizes up to 200 kb were constructed. The 16S rRNA gene analysis of the fosmid library DNA confirmed that it represents a variety of microbial species. The entire fosmid library and the selected BAC clones were transferred to Pseudomonas fluorescens and Xanthomonas campestris (fosmids only), and heterologous proteins from the fosmid library were confirmed to be expressed in P. fluorescens. To our knowledge no other reported vector system has a comparable potential for functional screening across species barriers.

  2. Molecular cloning of genes that specify virulence in Pseudomonas solanacearum.

    Science.gov (United States)

    Xu, P L; Leong, S; Sequeira, L

    1988-02-01

    The suicide plasmid pSUP2021 was used to introduce Tn5 into the Pseudomonas solanacearum wild-type strain K60. We isolated eight avirulent mutants after screening 6,000 kanamycin-resistant transconjugants by inoculating eggplant (Solanum melongena L. cv. Black Beauty) and tobacco (Nicotiana tabacum L. cv. Bottom Special) seedlings. The Tn5-containing EcoRI fragments from the eight mutants were unique, suggesting that numerous genes specify virulence in this species. These EcoRI fragments were cloned into pBR322 or pUC12, and one of the clones, pKD810, was transformed into K60. All of the kanamycin-resistant, ampicillin-sensitive transformants were avirulent. Three randomly selected avirulent transformants were shown to carry the Tn5-containing fragment in place of the wild-type fragment and to exhibit the same hybridization pattern as the original KD810 mutant did. With pKD810 as a probe, we identified cosmids carrying the wild-type virulence genes by using a genomic library of K60 prepared in pLAFR3. Two of the homologous cosmids, pL810A and pL810C, when introduced into KD810 by transformation, restored virulence and normal growth of this mutant in tobacco. Altogether, these data indicate that the gene(s) interrupted by Tn5 insertion in KD810 is essential for the virulence of P. solanacearum. Further characterization of this gene is now being completed by subcloning, transposon mutagenesis, and complementation analysis.

  3. 水稻白叶枯病菌GX1329基因组文库的构建及含编码TAL效应物基因的克隆的分离%Construction of a Genomic Library of Xanthomonas oryzae pv. oryzae Strain GX1329 and Isolation of Clones Containing the Genes Encoding TAL Ef- fectors

    Institute of Scientific and Technical Information of China (English)

    张子宇; 赵帅; 莫伟兰; 罗雪梅; 玉延华; 段承杰; 冯家勋

    2011-01-01

    由革兰氏阴性细菌水稻白叶枯病菌引起的水稻白叶枯病是亚洲、北美以及非洲部分地区最严重的水稻病害之一,水稻白叶枯病可使水稻减产高达50%以上。研究表明水稻白叶枯病菌的毒力主要依靠三型分泌系统所分泌的效应物。为了解水稻白叶枯病菌广西菌株GX1329中含有avrBs3/pthA家族基因的情况,本研究应用Alu I部分酶切其基因组DNA,构建了含有736个克隆的菌株GX1329的基因组文库。BamHI酶切分析随机挑取的15个文库克隆表明,克隆的外源DNA随机性良好,克隆的最小片段为27.7kb,最大为58.5kb,平均大小为39.9kb,文库克隆容量约为2.8×10^3Mb,该文库中包含基因组中任一个基因的概率为99.4%。利用来自水稻白叶枯病菌菲律宾菌株PX086的无毒基因avrXa10的第252位~第486位核苷酸序列作为探针,通过菌落原位杂交从GX1329基因组文库中筛选到37个含avrBs3/pthA家族基因的克隆。再通过Southern杂交分析,得到了17个独立克隆。这17个克隆中至少含有13个不同的avrBs3/pthA家族基因。这些基因在GX1329基因组中有的单独存在,有的两个或两个以上串联存在。本工作基本上明确了菌株GX1329基因组中avrBs3/pthA家族基因的数量,为进一步研究菌株GX1329中avrBs3/pthA家族基因的功能奠定了基础。%Bacterial leaf blight (BLB), caused by Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is a serious threat to yield losses in the main regions of rice growth including Asia, North America and Africa. BLB could lead to a crop loss of up to 50%. It is known that the virulence of Xoo mainly relies on type BI secre- tion system (T3SS) and its secreted effectors. To know the numbers of genes encoding avrBs3/pthA family mem- bers in Guangxi Xoo strain GX1329, a genomic DNA library containing 736 clones was successfully constructed by partially

  4. Fosmid library construction and screening for the maize mutant gene Vestigial glume 1

    Institute of Scientific and Technical Information of China (English)

    Chaoxian Liu; Xiaoli Liu; Lei Lei; Haiying Guan; Yilin Cai

    2016-01-01

    The maize mutant gene Vestigial glume 1 (Vg1) has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and screening. The fosmid library of Vg1 consisted of 574,000 clones with an average insert size of 36.4 kb, representing 7.9-fold coverage of the maize genome. Fosmid stability assays indicated that clones were stable during propagation in the fosmid system. Using Vg1 candidate gene-specific primers, a positive clone was successfully identified. This discovery will pave the way for identifying the function of Vg1 in maize development.

  5. Fosmid library construction and screening for the maize mutant gene Vestigial glume 1

    Institute of Scientific and Technical Information of China (English)

    Chaoxian Liu; Xiaoli Liu; Lei Lei; Haiying Guan; Yilin Cai

    2016-01-01

    The maize mutant gene Vestigial glume 1(Vg1) has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and screening. The fosmid library of Vg1 consisted of574,000 clones with an average insert size of 36.4 kb, representing 7.9-fold coverage of the maize genome. Fosmid stability assays indicated that clones were stable during propagation in the fosmid system. Using Vg1 candidate gene-specific primers, a positive clone was successfully identified. This discovery will pave the way for identifying the function of Vg1 in maize development.

  6. Fosmid library construction and screening for the maize mutant gene Vestigial glume 1

    Directory of Open Access Journals (Sweden)

    Chaoxian Liu

    2016-02-01

    Full Text Available The maize mutant gene Vestigial glume 1 (Vg1 has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and screening. The fosmid library of Vg1 consisted of 574,000 clones with an average insert size of 36.4 kb, representing 7.9-fold coverage of the maize genome. Fosmid stability assays indicated that clones were stable during propagation in the fosmid system. Using Vg1 candidate gene-specific primers, a positive clone was successfully identified. This discovery will pave the way for identifying the function of Vg1 in maize development.

  7. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    Directory of Open Access Journals (Sweden)

    Kathy N Lam

    Full Text Available High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  8. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    Science.gov (United States)

    Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  9. Cloning and characterization of the gsk gene encoding guanosine kinase of Escherichia coli

    DEFF Research Database (Denmark)

    Harlow, Kenneth W.; Nygaard, Per; Hove-Jensen, Bjarne

    1995-01-01

    The Escherichia coli gsk gene encoding guanosine kinase was cloned from the Kohara gene library by complementation of the E. coli gsk-1 mutant allele. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 433 amino acids with a molecular mass of 48,113 Da. Minicell...

  10. 败血症鲢肝与肾cDNA文库构建及MHC class Ⅰ的克隆与分析%Construction of a cDNA library and cloning of the MHC class Ⅰ gene in silver carp (Hypophthalmichthys molitrix) infected with bacterial septicemia

    Institute of Scientific and Technical Information of China (English)

    汪登强; 罗晓松; 陈大庆

    2011-01-01

    Silver carp (Hypophthalmichthys molitrix) is one of the most commonly cultured freshwater species in China. However, the development of silver carp aquaculture is threatened by bacterial septicemia. To isolate and study genes relevant to the disease, we constructed a cDNA library from silver carp liver and kidney tissue using a CloneMiner? cDNA kit. The primary cDNA library titer was 1.34× 107 cfu/mL yielding 2.68×l07cfu recombi-nants with 97.5% positive clones. The exogenous inserts of the recombinants ranged in size from 0.8 to 3.5 kb. We attempted to sequence 80 positive clones from both terminals to test the completeness of the coding sequence. We successfully sequenced 74 clones, of which 49 contained the complete coding sequence. Among the clones successfully sequenced, 57 sequences were identified to 49 known genes in GenBank. The cDNA library was subsequently screened by PCR yielding a single clone containing the complete coding sequence for MHC class I. The H. Molitrix MHC class I was 1 026 bp long and encoded a 341 amino acid (aa) protein that included a leader peptide, a\\, a2, a3, and transmembrane and cytosolic domains of 16, 88, 90, 87, and 60 aa, respectively. In addition, we identified sites that were highly conserved among vertebrate MHC class I. Phylogenetic comparison of the complete coding sequences and the a3 domain ofhH, molitrix MHC class I with other vertebrate species revealed different topology, suggesting a different evolutionary history for different domains of MHC class I and the occurrence of gene recombination among cyprinidae.%采用CloneMinerTM cDNA文库构建试剂盒构建了患败血症的鲢(Hypophthalmichthys molitrix)肝和肾的cDNA文库.经检验,文库的滴度为1.34×107 cfu/mL,总库容为2.68×107 cfu,阳性克隆率为97.5%,平均插入片段大于1.2 kb.对80个随机挑选的克隆进行两端测序,结果显示有74个测序成功.经在GenBank上BLAST比对,其中57个为已知功能基因,属于45

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

    Science.gov (United States)

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

    2006-05-01

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

  12. A method for generating subtractive cDNA libraries retaining clones containing repetitive elements.

    OpenAIRE

    1997-01-01

    Here we describe a two-stepped photobiotin-based procedure to enrich a target (canine retinal) cDNA library for tissue specific clones without removing those containing repetitive ( SINE ) elements, despite the presence of these elements in the driver population. In a first hybridization excess SINE elements were hybridized to a driver (canine cerebellar) cDNA. In a second hybridization target cDNA was added to this reaction. The resulting cDNA library was enriched for retinal specific clones...

  13. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    Energy Technology Data Exchange (ETDEWEB)

    Ding, J.H.; Yang, B.Z.; Liu, H.M. [Duke Univ. Medical Center, Durham, NC (United States)] [and others

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  14. Molecular Cloning and Preliminary Analysis of a Fragile Site Associated Gene

    Institute of Scientific and Technical Information of China (English)

    YI-WEN CAO; CHUAN-LU JIANG; TAO JIANG

    2006-01-01

    Objective To analyze the molecular colning of a fragile site-associated gene. Methods Genomic Chinese hamster ovary (CHO) DNA library was constructed using high molecular weight CHO DNA partially digested with MboI restriction enzyme from cultured CHO cells. Screening of genomic DNA library followed the established procedures. Genomic CHO in the positive clones was sequenced. Appropriate primers were designed for the reverse transcriptase-polymerase chain reactions (RT-PCR). The RT-PCR products were cloned into a pCRⅡ TOPO vector and confirmed by DNA sequencing. Antibodies were prepared using synthetic peptides as antigens by immunizing the rabbits. Immunohistochemical analyses were performed to evaluate the expression of the novel gene in different tissues. Results To investigate the molecular mechanism underlying the initial events of mdrla amplification, we cloned 1q31 fragile site DNA. Strikingly, we found that this fragile site contained a novel gene which was designated as a fragile site-associated (FSA) gene. FSA encoded an unusually large mRNA of ~16 kb. Full-length human FSA cDNA was cloned. FSA mRNA was expressed in many cultured cells and tissue types. Immunohistochemical analyses also revealed an expression pattern of the encoded proteins in postmitotic, well-differentiated epithelial compartments of many organs, including colon, mammary glands, ovary, prostate, and bladder. Conclusion FSA plays an important role in regulating mammalian epithelial cell growth and differentiation.

  15. PrecisePrimer: an easy-to-use web server for designing PCR primers for DNA library cloning and DNA shuffling.

    Science.gov (United States)

    Pauthenier, Cyrille; Faulon, Jean-Loup

    2014-07-01

    PrecisePrimer is a web-based primer design software made to assist experimentalists in any repetitive primer design task such as preparing, cloning and shuffling DNA libraries. Unlike other popular primer design tools, it is conceived to generate primer libraries with popular PCR polymerase buffers proposed as pre-set options. PrecisePrimer is also meant to design primers in batches, such as for DNA libraries creation of DNA shuffling experiments and to have the simplest interface possible. It integrates the most up-to-date melting temperature algorithms validated with experimental data, and cross validated with other computational tools. We generated a library of primers for the extraction and cloning of 61 genes from yeast DNA genomic extract using default parameters. All primer pairs efficiently amplified their target without any optimization of the PCR conditions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. A gene cloning system for 'Streptomyces toyocaensis'.

    Science.gov (United States)

    Matsushima, P; Baltz, R H

    1996-02-01

    We explored different methods of introducing DNA into 'Streptomyces toyocaensis' and Streptomyces virginiae to construct stable recombinant strains. Plasmid pIJ702 isolated from Streptomyces lividans transformed protoplasts of 'S. toyocaensis' at a frequency of 7 x 10(3) transformants (mu g DNA)-1. pIJ702 prepared from 'S. toyocaensis' transformed 'S. toyocaensis' protoplasts at a frequency of 1 center dot 5 x 10(5) (mu g DNA)-1, suggesting that 'S. toyocaensis' expresses restriction and modification. Plasmid pRHB126 was transduced by bacteriophage FP43 into 'S. toyocaensis' at a frequency of 1.2 x 10(-6) (p.f.u)-1. Plasmids pOJ436 and pRHB304 were introduced into 'S. toyocaensis' by conjugation from Escherichia coli S17-1 at frequencies of about 2 x 10(-4) and 1 x 10(-4) per recipient, respectively. Analysis of several exconjugants indicated that pOJ436 and pRHB304 inserted into a unique phiC31 attB site and that some of the insertions had minimal deleterious effects on glycopeptide A47934 production. The results indicate that 'S. toyocaensis' is a suitable host for gene cloning, whereas S. virginiae does not appear to be.

  17. THE CLONING OF HRNT-1 USING A COMBINATION OF cDNA LIBRARY SCREENING WITH BIOTIN-LABELED PROBE AND RAPID AMPLIFICATION OF cDNA ENDS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To clone the human counterpart of rat ZA73, EST cloned from rat tracheal epithelial (RTE) neoplastic transformed cell model induced by (a-particles radiation by using mRNA differential display. Methods: According to the sequence of rat ZA73, a probe was biotin-labeled to screen human cDNA library, and then the gene sequence was extended by RACE (rapid amplification of cDNA ends). Result: Human gene HRNT-1 (GenBank Accession Number: AF223393) is 4.256 kb in length, with an ORF located in the region between 254 and 3013 bp. 5' UTS (untranslated sequences) is 253 bp, 3' UTS is 1243 bp. Conclusion: The combination of cDNA library screening with biotin-labeled probes and RACE is an effective method to clone full-length cDNA, especially for sequences longer than 2 kb.

  18. Functional Identification and Characterization of Genes Cloned from Halophyte Seashore Paspalum Conferring Salinity and Cadmium Tolerance

    Directory of Open Access Journals (Sweden)

    Yu eChen

    2016-02-01

    Full Text Available Salinity-affected and heavy metal-contaminated soils limit the growth of glycophytic plants. Identifying genes responsible for superior tolerance to salinity and heavy metals in halophytes has great potential for use in developing salinity- and Cd-tolerant glycophytes. The objective of this study was to identify salinity- and Cd-tolerance related genes in seashore paspalum (Paspalum vaginatum, a halophytic perennial grass species, using the yeast cDNA expression library screening method. Based on the Gateway-compatible vector system, a high quality entry library was constructed, which contained 9.9×106 clones with an average inserted fragments length of 1.48 kb representing a 100% full-length rate. The yeast expression libraries were screened in a salinity-sensitive and a Cd-sensitive yeast mutant. The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes and 20 Cd-tolerant clones, including 5 Cd-tolerance genes. qPCR analysis confirmed that most of the 18 salinity-tolerance and 5 Cd-tolerance genes were up-regulated at the transcript level in response to salinity or Cd stress in seashore paspalum. Functional analysis indicated that salinity-tolerance genes from seashore paspalum could be mainly involved in photosynthetic metabolism, antioxidant systems, protein modification, iron transport, vesicle traffic, and phospholipid biosynthesis. Cd-tolerance genes from seashore paspalum could be associated with regulating pathways involved in phytochelatin synthesis, HSFA4-relsted stress protection, CYP450 complex and sugar metabolism. The 18 salinity-tolerance genes and 5 Cd-tolerance genes could be potentially used as candidate genes for genetic modification of glycophytic grass species to improve salinity and Cd tolerance and for further analysis of molecular mechanisms regulating salinity and Cd tolerance.

  19. Functional Identification and Characterization of Genes Cloned from Halophyte Seashore Paspalum Conferring Salinity and Cadmium Tolerance

    Science.gov (United States)

    Chen, Yu; Chen, Chuanming; Tan, Zhiqun; Liu, Jun; Zhuang, Lili; Yang, Zhimin; Huang, Bingru

    2016-01-01

    Salinity-affected and heavy metal-contaminated soils limit the growth of glycophytic plants. Identifying genes responsible for superior tolerance to salinity and heavy metals in halophytes has great potential for use in developing salinity- and Cd-tolerant glycophytes. The objective of this study was to identify salinity- and Cd-tolerance related genes in seashore paspalum (Paspalum vaginatum), a halophytic perennial grass species, using yeast cDNA expression library screening method. Based on the Gateway-compatible vector system, a high-quality entry library was constructed, which contained 9.9 × 106 clones with an average inserted fragment length of 1.48 kb representing a 100% full-length rate. The yeast expression libraries were screened in a salinity-sensitive and a Cd-sensitive yeast mutant. The screening yielded 32 salinity-tolerant clones harboring 18 salinity-tolerance genes and 20 Cd-tolerant clones, including five Cd-tolerance genes. qPCR analysis confirmed that most of the 18 salinity-tolerance and five Cd-tolerance genes were up-regulated at the transcript level in response to salinity or Cd stress in seashore paspalum. Functional analysis indicated that salinity-tolerance genes from seashore paspalum could be involved mainly in photosynthetic metabolism, antioxidant systems, protein modification, iron transport, vesicle traffic, and phospholipid biosynthesis. Cd-tolerance genes could be associated with regulating pathways that are involved in phytochelatin synthesis, HSFA4-related stress protection, CYP450 complex, and sugar metabolism. The 18 salinity-tolerance genes and five Cd-tolerance genes could be potentially used as candidate genes for genetic modification of glycophytic grass species to improve salinity and Cd tolerance and for further analysis of molecular mechanisms regulating salinity and Cd tolerance. PMID:26904068

  20. Corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures.

    Science.gov (United States)

    Thomas, William D; Golomb, Miriam; Smith, George P

    2010-12-15

    Phage display is used to discover peptides or proteins with a desired target property-most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder's infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption.

  1. Corruption of phage-display libraries by target-unrelated clones: Diagnosis and countermeasures

    Science.gov (United States)

    Thomas, William D.; Golomb, Miriam; Smith, George P.

    2010-01-01

    Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior, and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phage (TUPs), that lack the target behavior. Many TUPs are propagation-related; they have mutations conferring a growth advantage, and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. PMID:20692225

  2. PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries.

    Science.gov (United States)

    Potvin, Marianne; Lovejoy, Connie

    2009-01-01

    Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray-Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.

  3. Isolation of β-globin related genes from a human cosmid library.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); H.H.M. Dahl; R.A. Flavell (Richard); E. de Boer (Ernie)

    1981-01-01

    textabstractA human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda pha

  4. A high-throughput strategy for screening of bacterial artificial chromosome libraries and anchoring of clones on a genetic map constructed with single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Deal Karin R

    2009-01-01

    Full Text Available Abstract Background Current techniques of screening bacterial artificial chromosome (BAC libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGate™ assay. Results To test the efficacy of the Golden Gate assay in BAC library screening, multidimensional pools involving 302976 Aegilops tauschii BAC clones were genotyped for the presence/absence of specific gene sequences with multiplexed Illumina GoldenGate oligonucleotide assays previously used to place single nucleotide polymorphisms on an Ae. tauschii genetic map. Of 1384 allele-informative oligonucleotide assays, 87.6% successfully clustered BAC pools into those positive for a BAC clone harboring a specific gene locus and those negative for it. The location of the positive BAC clones within contigs assembled from 199190 fingerprinted Ae. tauschii BAC clones was used to evaluate the precision of anchoring of BAC clones and contigs on the Ae. tauschii genetic map. For 41 (95% assays, positive BAC clones were neighbors in single contigs. Those contigs could be unequivocally assigned to loci on the genetic map. For two (5% assays, positive clones were in two different contigs and the relationships of these contigs to loci on the Ae. tauschii genetic map were equivocal. Screening of BAC libraries with a simple five-dimensional BAC pooling strategy was evaluated and shown to allow direct detection of positive BAC clones without the need for manual deconvolution of BAC clone pools. Conclusion The highly parallel Illumina oligonucleotide assay is shown here to be an efficient tool for screening BAC libraries and a strategy for high

  5. Tailoring enzyme-rich environmental DNA clones: a source of enzymes for generating libraries of unnatural natural products.

    Science.gov (United States)

    Banik, Jacob J; Craig, Jeffrey W; Calle, Paula Y; Brady, Sean F

    2010-11-10

    A detailed bioinformatics analysis of six glycopeptide biosynthetic gene clusters isolated from soil environmental DNA (eDNA) megalibraries indicates that a subset of these gene clusters contains collections of tailoring enzymes that are predicted to result in the production of new glycopeptide congeners. In particular, sulfotransferases appear in eDNA-derived gene clusters at a much higher frequency than would be predicted from the characterization of glycopeptides from cultured Actinomycetes . Enzymes found on tailoring-enzyme-rich eDNA clones associated with these six gene clusters were used to produce a series of new sulfated glycopeptide derivatives in both in vitro and in vivo derivatization studies. The derivatization of known natural products with eDNA-derived tailoring enzymes is likely to be a broadly applicable strategy for generating libraries of new natural product variants.

  6. Cloning and characterization of a novel barley gene,HvORG4,induced by Fusarium graminearum infection

    Institute of Scientific and Technical Information of China (English)

    Theo; Van-Der; Lee

    2007-01-01

    Barley Fusarium head blight(FHB),caused by species of the Fusarium fungus,is a devastating disease that is reemerging worldwide in recent years.In this study,a novel gene,HvORG4,was cloned from barley by using cDNA library and suppression subtractive hybridization(SSH) library strategies.The SSH library and cDNA library were constructed from the Chinese barley cultivar Jing02-461(resistance to FHB) infected by Fusarium graminearum isolate Huanggang-1.For the SSH analysis,more than 120 differentially express...

  7. Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

    Science.gov (United States)

    Ruberti, I; Fragapane, P; Pierandrei-Amaldi, P; Beccari, E; Amaldi, F; Bozzoni, I

    1982-12-11

    Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved.

  8. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    Energy Technology Data Exchange (ETDEWEB)

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  9. Functional genomics of maize submergence tolerance and cloning of the related gene Sicyp51

    Institute of Scientific and Technical Information of China (English)

    TANG Wanhu; ZHANG Zuxin; ZOU Xiling; ZHENG Yonglian

    2005-01-01

    In this study, SSH (Suppression Subtractive Hybridization) and cDNA microarray were used to identify genes associated with waterlogging response of maize roots. Mo17 and Hz32 are two maize inbred lines with differential tolerance to hypoxia. Seedlings of the inbred lines with two leaves were submerged in hypoxia buffer. SSH libraries were constructed with cDNA samples from roots. Both forward and reverse subtractions were performed for each inbred line, and 105 positive clones induced by hypoxia were selected by differential screening. The treated and control message RNA were hybridized with the cDNA microarray of Mo17, sequentially, 57 of 3-fold differentially expressed clones were obtained. A total of 162 positive clones were all sequenced. Bioinformatics analysis showed these positive clones represent 85 TUGs, including genes involved in several biochemistry pathways, such as glycolysis, protection, signal transduction, cell construction and energy metabolism and 41 EST with unknown function. Comparison between Mo17 and Hz32 indicates that genes related to hypoxia tolerance have different expression patterns in submerged roots. Several positive clones' expression patterns were revealed by Northern or RT-PCR, and a new gene (Sicyp51), which may contribute to hypoxia tolerance, was identified.

  10. Isolation and characterization of the human collagen α1(I)-like gene from a cosmid library.

    NARCIS (Netherlands)

    E.H. Weiss (Elisabeth); K.S.E. Cheah (Kathryn); F.G. Grosveld (Frank); H.H.M. Dahl; E. Solomon; R.A. Flavell (Richard)

    1994-01-01

    textabstractWe have isolated a human collagen alpha 1(I)-like gene from a cosmid library. The clone which contains 37kb of human DNA has been shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations. The collagen gene sequence extends

  11. Isolation and characterization of the human collagen α1(I)-like gene from a cosmid library.

    NARCIS (Netherlands)

    E.H. Weiss (Elisabeth); K.S.E. Cheah (Kathryn); F.G. Grosveld (Frank); H.H.M. Dahl; E. Solomon; R.A. Flavell (Richard)

    1994-01-01

    textabstractWe have isolated a human collagen alpha 1(I)-like gene from a cosmid library. The clone which contains 37kb of human DNA has been shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations. The collagen gene sequence extends t

  12. Cloning and sequencing of human lambda immunoglobulin genes by the polymerase chain reaction.

    Science.gov (United States)

    Songsivilai, S; Bye, J M; Marks, J D; Hughes-Jones, N C

    1990-12-01

    Universal oligonucleotide primers, designed for amplifying and sequencing genes encoding the rearranged human lambda immunoglobulin variable region, were validated by amplification of the lambda light chain genes from four human heterohybridoma cell lines and in the generation of a cDNA library of human V lambda sequences from Epstein-Barr virus-transformed human peripheral blood lymphocytes. This technique allows rapid cloning and sequencing of human immunoglobulin genes, and has potential applications in the rescue of unstable human antibody-producing cell lines and in the production of human monoclonal antibodies.

  13. Substrate-induced gene expression screening: a method for high-throughput screening of metagenome libraries.

    Science.gov (United States)

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-01-01

    The SIGEX (substrate-induced gene expression) method is a novel approach for the screening of gene (genome) libraries. In addition to the commonly used function- and sequence-driven approaches to screening, SIGEX provides a third option; in SIGEX, positives are identified using a reporter gene, and the library is constructed using an "operon-trap" vector. This vector contains the reporter gene immediately downstream of the cloning site for the genomic insert so that the expression of the inserted gene(s) is coupled with that of the reporter gene. This system is especially suitable for screening catabolic genes that are induced in response to metabolically relevant compounds, such as substrates. If expression of the inserted gene(s) is activated in response to the addition of these compounds, then positive clones can be identified based on the reporter signal. The most effective selection is obtained by the use of a FACS (fluorescence-activated cell sorter) in conjunction with a FACS-compatible fluorescent reporter protein, such as GFP (green fluorescent protein). Activity-based screening of metagenomic libraries often suffers from low sensitivity and low throughput. In contrast, the high throughput, high sensitivity, and versatility of SIGEX make it a particularly suitable method for screening metagenomic libraries.

  14. Characterization of Porcine Endogenous Retrovirus Clones from the NIH Miniature Pig BAC Library

    Directory of Open Access Journals (Sweden)

    Seong-Lan Yu

    2012-01-01

    Full Text Available Pigs have been considered as donors for xenotransplantation in the replacement of human organs and tissues. However, porcine endogenous retroviruses (PERVs might transmit new infectious disease to humans during xenotransplantation. To investigate PERV integration sites, 45 PERV-positive BAC clones, including 12 PERV-A, 16 PERV-B, and 17 PERV-C clones, were identified from the NIH miniature pig BAC library. The analysis of 12 selected full-length sequences of PERVs, including the long terminal repeat (LTR region, identified the expected of open reading frame length, an indicative of active PERV, in all five PERV-C clones and one of the four PERV-B clones. Premature stop codons were observed in only three PERV-A clones. Also, eleven PERV integration sites were mapped using a 5000-rad IMpRH panel. The map locations of PERV-C clones have not been reported before, thus they are novel PERV clones identified in this study. The results could provide basic information for the elimination of site-specific PERVs in selection of pigs for xenotransplantation.

  15. Characterization of new bacterial catabolic genes and mobile genetic elements by high throughput genetic screening of a soil metagenomic library.

    Science.gov (United States)

    Jacquiod, Samuel; Demanèche, Sandrine; Franqueville, Laure; Ausec, Luka; Xu, Zhuofei; Delmont, Tom O; Dunon, Vincent; Cagnon, Christine; Mandic-Mulec, Ines; Vogel, Timothy M; Simonet, Pascal

    2014-11-20

    A mix of oligonucleotide probes was used to hybridize soil metagenomic DNA from a fosmid clone library spotted on high density membranes. The pooled radio-labeled probes were designed to target genes encoding glycoside hydrolases GH18, dehalogenases, bacterial laccases and mobile genetic elements (integrases from integrons and insertion sequences). Positive hybridizing spots were affiliated to the corresponding clones in the library and the metagenomic inserts were sequenced. After assembly and annotation, new coding DNA sequences related to genes of interest were identified with low protein similarity against the closest hits in databases. This work highlights the sensitivity of DNA/DNA hybridization techniques as an effective and complementary way to recover novel genes from large metagenomic clone libraries. This study also supports that some of the identified catabolic genes might be associated with horizontal transfer events.

  16. Human nicotinamide N-methyltransferase gene: Molecular cloning, structural characterization and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Aksoy, S.; Weinshilboum, R.M. [Mayo Medical School/Mayo Clinic/Mayo Foundation, Rochester, MN (United States); Brandriff, B.F. [Lawrence Livermore National Lab., CA (United States); Ward, A.; Little, P.F.R. [Imperial College of Science, Technology and Medicine, London (United Kingdom)

    1995-10-10

    Genomic DNA clones for nicotinamide N-methyltransferase (NNMT), an enzyme that catalyzes drug and xenobiotic metabolism, were isolated from a human chromosome 11-specific DNA library. Study of one of those clones, when combined with PCR-based experiments performed with human genomic DNA, made it possible to determine the structure of the human NNMT gene. The gene was approximately 16.5 kb in length and consisted of 3 exons and 2 introns. Transcription initiation for the NNMT gene occurred 105-109 nucleotides 5{prime}-upstream from the cDNA translation initiation codon on the basis of the results of both primer extension and 5{prime}-rapid amplification of cDNA ends. NNMT mapped to chromosome band 11q23.1 by fluorescence in situ hybridization.

  17. Constructing gene-enriched plant genomic libraries using methylation filtration technology.

    Science.gov (United States)

    Rabinowicz, Pablo D

    2003-01-01

    Full genome sequencing in higher plants is a very difficult task, because their genomes are often very large and repetitive. For this reason, gene targeted partial genomic sequencing becomes a realistic option. The method reported here is a simple approach to generate gene-enriched plant genomic libraries called methylation filtration. This technique takes advantage of the fact that repetitive DNA is heavily methylated and genes are hypomethylated. Then, by simply using an Escherichia coli host strain harboring a wild-type modified cytosine restriction (McrBC) system, which cuts DNA containing methylcytosine, repetitive DNA is eliminated from these genomic libraries, while low copy DNA (i.e., genes) is recovered. To prevent cloning significant proportions of organelle DNA, a crude nuclear preparation must be performed prior to purifying genomic DNA. Adaptor-mediated cloning and DNA size fractionation are necessary for optimal results.

  18. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH).

    Science.gov (United States)

    Leveau, Johan H J; Gerards, Saskia; de Boer, Wietse; van Veen, Johannes A

    2004-09-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia coli of large-sized genomic DNA fragments of the mycophagous soil bacterium Collimonas fungivorans, and hybridized 768 library clones with the Collimonas-specific fluorescent probe CTE998-1015. Critical to the success of this approach (which we refer to as large-insert library FISH or LIL-FISH) was the ability to induce fosmid copy number, the exponential growth status of library clones in the FISH assay and the use of a simple pooling strategy to reduce the number of hybridizations. Twelve out of 768 E. coli clones were suspected to harbour and express Collimonas 16S rRNA genes based on their hybridization to CTE998-1015. This was confirmed by the finding that all 12 clones were also identified in an independent polymerase chain reaction-based screening of the same 768 clones using a primer set for the specific detection of Collimonas 16S ribosomal DNA (rDNA). Fosmids isolated from these clones were grouped by restriction analysis into two distinct contigs, confirming that C. fungivorans harbours at least two 16S rRNA genes. For one contig, representing 1-2% of the genome, the nucleotide sequence was determined, providing us with a narrow but informative view of Collimonas genome structure and content.

  19. Crotoxin: Structural Studies, Mechanism of Action and Cloning of Its gene

    Science.gov (United States)

    1989-12-01

    basic subunit clones and the acidic subunit clones that were partially sequenced, had multiple sequence differences. New cONA and genomic libraries from...were partially sequenced, had multiple sequence differences. New cDNA and genomic libraries from C. s. scutulatus are in being prepared and screened...unsuccessful in identifying any clones that contained basic subunit cDNA. Work is continuing with new cDNA and genomic libraries from . t , in collaboration

  20. Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing.

    Science.gov (United States)

    Yang, Tae-Jin; Yu, Yeisoo; Nah, Gyoungju; Atkins, Michael; Lee, Seunghee; Frisch, David A; Wing, Rod A

    2003-08-01

    Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.

  1. Isolation and characterization of rice cesium transporter genes from a rice-transporter-enriched yeast expression library.

    Science.gov (United States)

    Yamaki, Tomohiro; Otani, Masahiro; Ono, Kohei; Mimura, Takuro; Oda, Koshiro; Minamii, Takeshi; Matsumoto, Shingo; Matsuo, Yuzy; Kawamukai, Makoto; Akihiro, Takashi

    2017-08-01

    A considerable portion of agricultural land in central-east Japan has been contaminated by radioactive material, particularly radioactive Cs, due to the industrial accident at the Fukushima Daiichi nuclear power plant. Understanding the mechanism of absorption, translocation and accumulation of Cs(+) in plants will greatly assist in developing approaches to help reduce the radioactive contamination of agricultural products. At present, however, little is known regarding the Cs(+) transporters in rice. A transporter-enriched yeast expression library was constructed and the library was screened for Cs(+) transporter genes. The 1452 full length cDNAs encoding transporter genes were obtained from the Rice Genome Resource Center and 1358 clones of these transporter genes were successively subcloned into yeast expression vectors; which were then transferred into yeast. Using this library, both positive and negative selection screens can be performed, which have not been previously possible. The constructed library is an excellent tool for the isolation of novel transporter genes. This library was screened for clones that were sensitive to Cs(+) using a SD-Gal medium containing either 30 or 70 mM CsCl; resulting in the isolation of 13 Cs(+) sensitive clones. (137) Cs absorption experiments were conducted and confirmed that all of the identified clones were able to absorb (137) Cs. A total of 3 potassium transporters, 2 ABC transporters and 1 NRAMP transporter were among the 13 identified clones. © 2017 Scandinavian Plant Physiology Society.

  2. Analysis of a viral metagenomic library from 200 m depth in Monterey Bay, California constructed by direct shotgun cloning

    Directory of Open Access Journals (Sweden)

    Preston Christina M

    2011-06-01

    Full Text Available Abstract Background Viruses have a profound influence on both the ecology and evolution of marine plankton, but the genetic diversity of viral assemblages, particularly those in deeper ocean waters, remains poorly described. Here we report on the construction and analysis of a viral metagenome prepared from below the euphotic zone in a temperate, eutrophic bay of coastal California. Methods We purified viruses from approximately one cubic meter of seawater collected from 200m depth in Monterey Bay, CA. DNA was extracted from the virus fraction, sheared, and cloned with no prior amplification into a plasmid vector and propagated in E. coli to produce the MBv200m library. Random clones were sequenced by the Sanger method. Sequences were assembled then compared to sequences in GenBank and to other viral metagenomic libraries using BLAST analyses. Results Only 26% of the 881 sequences remaining after assembly had significant (E ≤ 0.001 BLAST hits to sequences in the GenBank nr database, with most being matches to bacteria (15% and viruses (8%. When BLAST analysis included environmental sequences, 74% of sequences in the MBv200m library had a significant match. Most of these hits (70% were to microbial metagenome sequences and only 0.7% were to sequences from viral metagenomes. Of the 121 sequences with a significant hit to a known virus, 94% matched bacteriophages (Families Podo-, Sipho-, and Myoviridae and 6% matched viruses of eukaryotes in the Family Phycodnaviridae (5 sequences or the Mimivirus (2 sequences. The largest percentages of hits to viral genes of known function were to those involved in DNA modification (25% or structural genes (17%. Based on reciprocal BLAST analyses, the MBv200m library appeared to be most similar to viral metagenomes from two other bays and least similar to a viral metagenome from the Arctic Ocean. Conclusions Direct cloning of DNA from diverse marine viruses was feasible and resulted in a distribution of virus

  3. The National Laboratory Gene Library Project

    Energy Technology Data Exchange (ETDEWEB)

    Deaven, L.L.; Van Dilla, M.A.

    1988-01-01

    The two National Laboratories at Livermore and Los Alamos have played a prominent role in the development and application of flow cytometry and sorting to chromosome classification and purification. Both laboratories began to receive numerous requests for specific human chromosomal types purified by flow sorting for gene library construction, but these requests were difficult to satisfy due to time and personnel constraints. The Department of Energy, through its Office of Health and Environmental Research, has a long-standing interest in the human genome in general and in the mutagenic and carcinogenic effects of energy-related environmental pollutants in particular. Hence, it was decided in 1983 to use the flow construct chromosome-specific gene libraries to be made available to the genetic research community. The National Laboratory Gene Library Project was envisioned as a practical way to deal with requests for sorted chromosomes, and also as a way to promote increased understanding of the human genome and the effects of mutagens and carcinogens on it. The strategy for the project was developed with the help of an advisory committee as well as suggestions and advice from many other geneticists. 4 refs., 2 tabs.

  4. Cloning of Rabbit HPRT Gene Using the Recombineering System

    Institute of Scientific and Technical Information of China (English)

    Jianjun SHI; Donghui CAI; Xuejin CHEN; Huizheng SHENG

    2007-01-01

    Hypoxanthine phosphoribosyltransferase (HPRT) plays an important role in the metabolic salvage of purines, and been used as an alternative pathway for mutant selection in many studies. To facilitate its application in rabbits, we have cloned the cDNA and genomic DNA of the rabbit HPRT gene using an approach that combines bioinformatics and recombineering methods. The cDNA is comprised of 1449 bp containing a coding sequence for a protein of 218 amino acids. The deduced amino acid sequence of the rabbit HPRT gene shares 98%, 97%, 98% and 94% identity with human, mouse, pig and cattle HPRT genes, respectively. Reverse transcription-polymerase chain reaction analysis showed that this gene is ubiquitously expressed in tissues of adult rabbit. The rabbit HPRT gene spans approximately 48 kb in length and consists of nine exons. The cloning of the rabbit HPRT gene shows the usefulness of the recombineering system in cloning genes of large size. This system may facilitate the subcloning of DNA from bacterial artificial chromosomes for cloning genes of large size or filling big gaps in genomic sequencing.

  5. Cloning of Omp1 Gene from Chlamydia trachomatis F Genotype

    Institute of Scientific and Technical Information of China (English)

    QI Manli(齐蔓莉); LIU Quanzhong(刘全中); JIAO Wenling(缴稳苓); TIAN Jingqun(田敬群); CHEN Jinying(陈锦英)

    2002-01-01

    Objectives: To directionally clone the omp1 gene fromChlamydia trachomatis (Ct) F genotype onto a plasmid vectorfor constructing a rudimentary DNA vaccine.Methods: The complete omp1 gene from genomic DNA of CtF genotype wild species was amplified with primers designedby computer. The recombinant gene was obtained byrestriction enzyme cutting, linking the gene with the plasmidvector in vitro, transforming the recombinant gene intobacteria, and extracting the DNA from the bacteria.Results: DNA extracted from the bacteria was composed ofthe omp1 gene and plasmid, which is identified by threemethods of singular restrictive enzyme cutting, doublerestrictive enzyme cutting and PCR.Conclusion: Cloning of the omp1 gene from the Ct Fgenotype means that a rudimentary DNA vaccine wassuccessfully constructed.

  6. Construction of a BAC library from cucumber (Cucumis sativus L.) and identification of linkage group specific clones

    Institute of Scientific and Technical Information of China (English)

    Yuan Guan; Qi Chen; Junsong Pan; Zheng Li; Huanle He; Aizhong Wu; Rentao Song; Run Cai

    2008-01-01

    A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each link-age group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen mark-ers gave at least two positive clones. As a result, 22 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.

  7. ISOLATION OF TUMOR DIFERENTIALLY EXPRESSED GENES BY MIXING PROBES LIBRARY SCREEN

    Institute of Scientific and Technical Information of China (English)

    余鹰; 朱诗国; 张必成; 周鸣; 李桂源; 沈守荣; 张晓梅

    2001-01-01

    Objective: This study was designed to clone candidate tumor suppressor genes down-expressed in Nasopharyngeal Carcinoma (NPC). Methods: Differentially expressed cDNA fragments (AF152605 and AF091517) were labeled by PCR, and Northern blot was used to confirmed transcript length of these genes. Skeleton muscle cDNA library was screened with PCR-labeled probe mixture. By sequencing the positive clones directly, three novel genes (Genbank accession number: AF179285, AF170307 and AF194971), with transcripts of 2.1 Kb, 1.1 Kb and 1.4 Kb respectively, were isolated successfully. Conclusions: Library screening using PCR-labeled probes mixture is an efficient method to get full-length cDNA from multi-cDNA fragment simultaneously and quickly.

  8. Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes.

    Directory of Open Access Journals (Sweden)

    Man Cheng

    Full Text Available Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3. The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx. Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N protein of SARS-associated coronavirus (SCoV were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.

  9. 大鲵皮肤cDNA文库ESTs分析及Dynll2基因的分离与表达%Construction of cDNA library of Andrias davidianus skin tissue and molecular cloning and expression analysis of Dynll 2 gene

    Institute of Scientific and Technical Information of China (English)

    王立新; 郑尧; 李锋刚; 李雯娟; 刘小林

    2011-01-01

    Dyneins are a group of evolutionarily highly conservative molecular proteins, which can be divided into two groups: cytoplasmic dyneins and axonemal dyneins. Cytoplasmic dynein probably moves along the microtubule essential for transporting cargo in eukaryotes. It is also probably involved in the movement of chromosomes and positioning the mitotic spindles for cell division. Dynein light chain 2, cytoplasmic, is a protein that in humans is encoded by the Dynll 2 gene. To clone dynein light chain, LC8-type 2 (Dynll 2) gene from A. davidianus, and to analyze the characteristics of the functional gene by bioinformatics analysis,the structure of Dynll 2 gene was isolated from skin cDNA library. A cDNA library of the skin tissue was constructed by using the isolated mRNA as the template during reverse transcription. The skin cDNA library of A. davidianus were detected and sequenced by picking clones randomly. It was confirmed that the titer of the skin cDNA library of A. davidianus was 1. 50 x 106 cfu ,the recombination rate was 94.8% ,and the PCR results showed that the average size of inserts segment was about 1 000 bp. A total of 343 ESTs from the library were sequenced and made alignment with sequences in GenBank database. Moreover,at least 214 clones (E-value < 10 -6) derived from these identified clones were categorized into nine categories. Immune-related genes accounted for 31. 3% of the largest distribution, metabolism genes (14.0% ),cytoskeleton genes( 13.0% )and signaling pathway-related genes( 12.6% ) took up a larger gene distribution though. It revealed that skin of A. davidianus made intense activities in the form of secretions and took part in metabolism reactions, according to their physiology function in the area of immunity, breathing, and osmotic pressure equilibrium. In order to investigate the contribution of Dynll 2 to motor protein in A. davidianus, molecular cloning, analyzing cDNA sequence and expression analysis of Dynll 2 gene from A

  10. Cloning and Sequence Analysis of the gp41 Gene of Clanis bilineata Nuclear Polyhedrosis Virus

    Institute of Scientific and Technical Information of China (English)

    ZHU Shan-ying; WANG Wen-bing; ZHU Jiang

    2006-01-01

    Clanis bilineata Nucleo Polyhedro Virus (CbNPV) was purified from Clanis bilineata larva. To obtain the molecular information of the virus, the genomic DNA of CbNPV was extracted, and a DNA fragment library of the virus was constructed using shotgun. The positive clones were then sequenced and analyzed. An open-reading frame (ORF) that has high identity with the gp41 gene of most NPVs was found in the library. The gp41 gene of CbNPV is 933 base pair long and encodes a protein of 310 amino acids. The result of the amino acid sequence analysis showed that the CbNPV gp41 has 53-61 and 56-73% identities with Group Ⅰ and Ⅱ NPVs gp41 proteins, respectively. The result indicates that the isolated CbNPV is a novel baculovirus, and the CbNPV shares a much closer relationship with Group Ⅱ NPVs.

  11. IDENTIFICATION OF ACTIVE BACTERIAL COMMUNITIES IN A MODEL DRINKING WATER BIOFILM SYSTEM USING 16S RRNA-BASED CLONE LIBRARIES

    Science.gov (United States)

    Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rDNA clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, a...

  12. Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha.

    Science.gov (United States)

    Okada, S; Fujisawa, M; Sone, T; Nakayama, S; Nishiyama, R; Takenaka, M; Yamaoka, S; Sakaida, M; Kono, K; Takahama, M; Yamato, K T; Fukuzawa, H; Brennicke, A; Ohyama, K

    2000-11-01

    Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.

  13. Cloning, structural organization, and chromosomal mapping of the human phenol sulfotransferase STP2 gene

    Energy Technology Data Exchange (ETDEWEB)

    Gaedigk, A.; Beatty, B.G.; Grant, D.M. [Hospital for Sick Children, Toronto, Ontario (Canada)

    1997-03-01

    Phenol- and monoamine-metabolizing sulfotransferases (STP and STM, respectively) are members of a superfamily of enzymes that add sulfate to a variety of xenobiotics and endobiotics containing hydroxyl or amino functional groups. To characterize related sulfotransferase genes further, we used extra-long PCR (XL-PCR) to generate three distinct sizes of amplification products from human genomic DNA or from genomic phage library clones, each of which contained sulfotransferase gene sequences. One of the PCR fragments contained a new sulfotransferase gene, STP2, corresponding to a recently published cDNA clone that encodes a sulfotransferase with catalytic specificity distinct from that of the previously described STP1 and STM. Additional upstream sequence information was obtained using a second STP2-specific XL-PCR-based approach. The STP2 gene is composed of eight exons and seven introns, with exon sizes ranging from 95 to 181 bp. Protein-coding exon lengths and locations of the splice junctions were identical to those in both the STM gene and an STP2 gene published independently by another group recently. The STP2 gene maps to a chromosomal location (16p11.2-p1.2) that is the same as that previously determined for both STP1 and STM. The characterization of the STP2 gene provides further insight into the organization, regulation, and multiplicity of the sulfotransferase supergene family. 27 refs., 3 figs.

  14. Screening and cloning of differentially expressed genes in placentas from patients of pregnancy-induced hypertension by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    尹国武; 姜锋; 李东红; 姚元庆

    2003-01-01

    Suppresssion subtractive hybridization (SSH) was preformed to compare gene expression profiles of PIH patients and normal pregnancy placentas. The subtractive cDNA library of PIH placenta was set up and screedned. Differential cDNAs were cloned, and sequenced by T 7 primer methodology. One hundred and three differential cDNAs were isolated by SSH. Sequencing and BLAST analysis showed 90 inserts shared more than 95% homolog with sequences in the GenBank/EMBL database. We identified 36 putative genes including pregnancy-specific glycoprotein gene (BC005924), serine protease inhibitor gene(BC012868), VEGFR-1 gene(AF063657, etc.

  15. Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes

    Energy Technology Data Exchange (ETDEWEB)

    Ono, M.

    1986-06-01

    By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identified as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent.

  16. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    Science.gov (United States)

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury.

  17. Construction of cDNA Library from NPC Tissue and Screening of Antigenic Genes

    Institute of Scientific and Technical Information of China (English)

    Jun Shu; Xiaojuan He; Guancheng Li

    2006-01-01

    To construct cDNA library of nasopharyngeal carcinoma (NPC) and obtain the NPC associated or specific antigens from it, we used a powerful new method to identify the antigens eliciting humoral immune response, which is SEREX (serological identification of antigen by recombinant cDNA expression library). Autologous serum of NPC patient was used to screen the reactive clones in the human NPC tissue cDNA library consisted of 3.64×106 recombinants. The 23 exact positive clones were subcloned to monoclonality and the size of cDNA inserts was identified by PCR. Then the nucleotide sequence of cDNA inserts was determined, and the sequence alignments were performed with BLAST software on GenBank database. They represented 16 different antigens. A detailed sequence analysis showed that 10 of 16 genes were high homologous to genes known in GenBank, such as RPL31,S100 A2, MT2A, etc. However, there were also 6 genes with low homology to genes in GenBank. Furthermore, 3 of 6 genes may be novel genes. The associations of these genes to NPC and the roles that they played in the occurrence and development of NPC should be further revealed.

  18. Molecular cloning and expression of a new gene, GON-SJTU1 in the rat testis

    Directory of Open Access Journals (Sweden)

    Tian Geng G

    2010-05-01

    Full Text Available Abstract Background Spermatogenesis is a complex process involving cell development, differentiation and apoptosis. This process is governed by a series of genes whose expressions are highly regulated. Male infertility can be attributed to multiple genetic defects or alterations that are related to spermatogenesis. The discovery, cloning and further functional study of genes related to spermatogenesis is of great importance to the elucidation of the molecular mechanism of spermatogenesis. It is also physiologically and pathologically significant to the therapy of male infertility. Methods GON-SJTU1 was identified and cloned from rat testis by cDNA library screening and 3'-and 5'-RACE. The products of GON-SJTU1 were assessed by Northern and Western blotting. The expression of GON-SJTU1 was also examined by In situ hybridization and immunohistochemistry. Results Here we identified and cloned a new gene, GON-SJTU1, with the biological process of spermatogenesis. GON-SJTU1 is highly expressed in the testis from day 1 to 15 and then decreased, suggesting that GON-SJTU1 might be a time-related gene and involved in the early stage of spermatogenesis. And the expression of GON-SJTU1 in the testis occurred in some male germ cells, particularly in gonocytes and spermatogonial stem cells. Conclusion GON-SJTU1 may play a role in the biological process of spermatogenesis.

  19. Molecular cloning of the human excision repair gene ERCC-6.

    NARCIS (Netherlands)

    C. Troelstra (Christine); H. Odijk (Hanny); J. de Wit (Jan); A. Westerveld (Andries); L.H. Thompson; D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1990-01-01

    textabstractThe UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5

  20. Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing.

    Science.gov (United States)

    Díez, B; Pedrós-Alió, C; Massana, R

    2001-07-01

    Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.

  1. Recent Achievement in Gene Cloning and Functional Genomics in Soybean

    Directory of Open Access Journals (Sweden)

    Zhengjun Xia

    2013-01-01

    Full Text Available Soybean is a model plant for photoperiodism as well as for symbiotic nitrogen fixation. However, a rather low efficiency in soybean transformation hampers functional analysis of genes isolated from soybean. In comparison, rapid development and progress in flowering time and photoperiodic response have been achieved in Arabidopsis and rice. As the soybean genomic information has been released since 2008, gene cloning and functional genomic studies have been revived as indicated by successfully characterizing genes involved in maturity and nematode resistance. Here, we review some major achievements in the cloning of some important genes and some specific features at genetic or genomic levels revealed by the analysis of functional genomics of soybean.

  2. Gene cloning: exploring cotton functional genomics and genetic improvement

    Institute of Scientific and Technical Information of China (English)

    Diqiu LIU; Xianlong ZHANG

    2008-01-01

    Cotton is the most important natural fiber plant in the world. The genetic improvement of the quality of the cotton fiber and agricultural productivity is imperative under the situation of increasing consumption and rapid development of textile technology. Recently, the study of cotton molecular biology has progressed greatly. A lot of specifically or preferentially expressed cotton fiber genes were cloned and analyzed. On the other hand, identification of stress response genes expressed in cotton was performed by other research groups. The major stress factors were studied including the wilt pathogens Verticillium dahliae, Fusarium oxy-sporum f. sp. vasinfectum, bacterial blight, root-knot nematode, drought, and salt stress. What is more, a few genes related to the biosynthesis of gossypol, other sesquiterpene phytoalexins and the major seed oil fatty acids were isolated from cotton. In the present review, we focused on the major advances in cotton gene cloning and expression profiling in the recent years.

  3. Cloning-free regulated monitoring of reporter and gene expression

    Directory of Open Access Journals (Sweden)

    Demirkaya Omer

    2009-03-01

    Full Text Available Abstract Background The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations. Results In this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes. Conclusion The simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile.

  4. 1株中度嗜盐菌质粒基因组文库的构建及2个膜蛋白基因的克隆和序列分析%Construction of Plasmid Genomic Library of One Moderately Halophilic Bacteria & Cloning, Sequence Analysis of Two Membrane Protein Genes

    Institute of Scientific and Technical Information of China (English)

    朱文华; 董冠群; 滕长财; 侯娟; 吕彦; 孙业盈

    2012-01-01

    中度嗜盐菌是生活在高盐环境中的微生物,以1株从蜢子虾酱中分离的中度嗜盐菌盐脱氮枝芽胞杆菌(Virgibacillus halodenitrificans)为研究对象,通过酶切,筛选DNA片段然后连接pUC19载体并转化大肠埃希菌,构建了该菌株的质粒基因组文库,推测可获得5×103个阳性克隆,覆盖约15 Mb的遗传信息.随机选取部分克隆进行测序,在测序结果中筛选到了2个具有完整ORF的膜蛋白基因分别为编码Na+H+逆向转运蛋白的NhaC基因,编码一离子通道蛋白的MscS基因,GenBank登陆号分别为JX849200、JX849202.其中NhaC基因ORF全长为1 446 bp,编码481个氨基酸,包括10个跨膜结构域;MscS基因的全长ORF为822 bp,编码273个氨基酸的蛋白质,包括3个跨膜区,与其他菌种的序列比对表明第3个跨膜区保守性最强.%Moderately halophilic bacteria are microorganism living in high salt environment. A moderately halophilic bacteria Virgibacillus halodenitrijicans was separated from a shrimp paste as research object. Plasmid genomic library of the strain was constructed. DNA fragments were selected after genomic DNA digestion by restriction enzyme, and then were linked with pUC19 vector and transformed into E. coli to construct the plasmid genomic library of the strain. It was speculated that about 5 × 103 positive clones were obtained, covering about 15 Mb of genetic information. Part of the clones was selected to carry out sequencing. From the sequencing results two membrane protein genes possessing complete ORF respectively encoding NhaC gene of Na+ H + reverse transporter protein, MscS genes of encoding an ion channel protein were screened. The CenBank accession numbers of the two genes were JX849200, JX849202 respectively. Among them the NhaC gene was 1 446 bp length ORF, encoded 481 amino acids, including 10 transmem-brane domains. MscS gene had 822 bp length ORF, encoded a protein of 273 amino acids, including 3 transmembrane domains. The

  5. Cloning and sequencing of the bovine gastrin gene

    DEFF Research Database (Denmark)

    Lund, T; Rehfeld, J F; Olsen, Jørgen

    1989-01-01

    In order to deduce the primary structure of bovine preprogastrin we therefore sequenced a gastrin DNA clone isolated from a bovine liver cosmid library. Bovine preprogastrin comprises 104 amino acids and consists of a signal peptide, a 37 amino acid spacer-sequence, the gastrin-34 sequence followed...... by an amidation-site (Gly-Arg-Arg), and a C-terminal nonapeptide. Comparison with human, porcine, and rat cDNA sequences revealed extensive homology in the coding region as well as in short noncoding structures....

  6. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  7. Gene Cloning of Iranian Leishmania major Mannose-1-Phosphate Guanyltransferase

    Directory of Open Access Journals (Sweden)

    R Salehi

    2009-07-01

    Full Text Available "nBackground: Leishmania is an obligatory intracellular protozoan parasite, which infects human be­ings when infected sand fly vector takes a blood meal.  Most efforts are towards designing an effective vaccine to prevent leishmaniasis. In this way, development of candidate antigen for vaccine has spe­cial im­portant. In this study, we cloned mannose-1-phosphate guanyltransferase gene of Iranian L .major in pET32a expression vector. "nMethods: Primers based on L. major mannose-1-phosphate guanyltransferase sequence gene was de­signed and synthesized. DNA of Leishmania promastigotes was extracted and PCR reaction was done. PCR product was cloned into pTZ57R and sub cloned into pET32a expression vector. "nResults: Recombinant plasmid containing 1140 bp as L. major mannose-1-phosphate guanyltrans­ferase gene was extracted and confirmed by restriction analysis. PCR product was sequenced and de­posited to GenBank. There were some differences in amino acid sequences between Iranian L. major mannose-1-phosphate guanyltransferase and others previously accepted in GenBank "nConclusion: We amplified and cloned Iranian L. major mannose-1-phosphate guanyltransferase successfully.

  8. Cloning of low-temperature induced gene from Morus mongolica CK ...

    African Journals Online (AJOL)

    SERVER

    2008-03-04

    Mar 4, 2008 ... gene WAP25 was then cloned by means of RT-PCR technique. The cloned gene has ..... clearing concentrated ions caused by cell dehydration when plants .... Canadian journal of botany-revue canadienne de botanique. 49.

  9. Construction of genomic libraries of Cryptosporidium parvum and identification of antigen-encoding genes.

    Science.gov (United States)

    Dykstra, C C; Blagburn, B L; Tidwell, R R

    1991-01-01

    Genomic libraries have been constructed from bovine C. parvum DNA in the lambda ZAP and lambda DASH vectors. Based on an estimated genome size of 2 x 10(4) kilobases (kb), each recombinant library contains greater than 10 genomic equivalents. The average recombinant size for the lambda ZAP library is 2.1 kb and for the lambda DASH library is 14 kb. We have identified genes to major antigens recognized by hyperimmune bovine antiserum. These recombinants are currently being purified and characterized. Limited DNA sequence analysis of random C. parvum clones confirms suggestions that the genome is quite AT-rich. The DNA sequence of random lambda ZAP fusion proteins has identified a potential ATPase, a structural protein and a DNA-binding protein.

  10. Cloning and expression of Neisseria meningitides luxS gene

    Institute of Scientific and Technical Information of China (English)

    Bahram Kazemi; Nazila Baghalzadeh-Mohammadi; Reza Hadavi; Mojgan Bandehpour; Elham Ghayour; Majid Moghbeli; Kazem Parivar

    2008-01-01

    Neisseria meningitides is a Gram-negative bacterium which is an important causative agent of septicemia and meningitis.Numerous pathogenic bacteria contain luxS,which is required for autoinducer-2 production.Neis-seria meningitides contains a functional copy of luxS that is necessary for full meningococcal virulence.Neisse-ria meningitides DNA was extracted and its luxS gene was amplified by nested PCR.PCR product was purified and cloned in to pQE-30 expression vector.Recombinant plasmid was transformed and mass cultured.luxS was expressed in E.coli and confirmed by western blot analysis.In this study,N.meningitides luxS was amplified, cloned and expressed successfully.The sequencing of PCR product confirmed that amplified gene was luxS. Gene was expressed and observed in SDS-PAGE.Protein was reacted by his tag monoclonal antibody through western blot analysis.

  11. Cloning and expression of the rat homologue of the Huntington disease gene

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, I.; Epplen, J.T.; Riess, O. [Ruhr-Univ. Bochum (Germany)] [and others

    1994-09-01

    Huntington`s disease (HD) is an autosomal dominant neurodegenerative disorder which is manifested usually in adult life. The age of onset is variable and leads to progressive symptoms including involuntary choreatic movements and various cognitive and psychiatric disturbances. Recently, a gene (IT15) was cloned containing a (CAG){sub n} repeat which is elongated and unstable in HD patients. IT15 is widely expressed in human tissues but unrelated to any known deduced protein sequence. To further investigate the HD gene, 15 rat cDNA libraries were screened. 24 clones have been identified covering the Huntingtin gene. Comparison of the Huntingtin gene between human and rat revealed homologies between 80% and 87% at the DNA level and about 90% at the protein level. These analyses will help to define biologically important sequence regions, e.g., via evolutionary conservation. One clone contains the (CAG){sub n} repeat which consists of eight triplets compared to seven triplets in the mouse and a median of 17 in human. As in humans there are two transcripts arising from differential 3{prime}-polyadenylation. In the 3{prime}UTR a stretch of about 280 bp is exchanged for a 250 bp fragment with no homology in rodents and man. The cDNA clones are currently used to study Huntingtin gene expression during development in rodent tissues. RNA in situ hybridization of embryonic sections shows predominant signals in all neuronal tissues. In contrast to previously published data Huntingtin mRNA expression in testis is increased in spermatocytes vs. spermatogonia.

  12. Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

    Institute of Scientific and Technical Information of China (English)

    徐军望; 冯德江; 李旭刚; 常团结; 朱祯

    2002-01-01

    The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.

  13. Cloning and Analysis of the Promoter Region of Rat uPA Gene

    Institute of Scientific and Technical Information of China (English)

    Yan LIU; Jin-wen XIONG; Li-gang CHEN; Yong-hong TIAN; Cheng-liang XIONG

    2007-01-01

    Objective To clone and analyze the promoter sequence of rat urokinase plasminogen activator protein gene.Methods The genomic DNA was extracted from rat testicular tissue. According to urokinase plasminogen activator, the gene sense primer and antisense primer of uPA gene were designed and synthesized, then Touch-Down PCR were performed. After proper purification, the PCR product was sequenced, analyzed with the promoter prediction software and compared with the DNA sequence of rattuas urokinase plasminogen activator.Results The cloned uPA gene was about 1 572 bp in length, which contained a full open-reading frame with 21 bp in length exons, and the upper region of transcriptional start was 1 551 bp in length which was eucaryon transcriptional control area.The 5' UTR had a promoter region including a non-responsive TATA-box. Not only the GC-box binding region was found in this gene, but also active protein 1 (AP1) and SP1 were seen in other regions.Conclusion A 1 572 bp uPA gene fragment (GenBank accession No. X65651) was obtained from rat genomic DNA library, containing eucaryon transcriptional control area with a promoter region, non-conspicuous TATA-box, GC-box and an extron. A non-responsive TATA-box is located at the upper -30 region.

  14. Human genomic library screened with 17-base oligonucleotide probes yields a novel interferon gene.

    OpenAIRE

    Torczynski, R M; Fuke, M; Bollon, A P

    1984-01-01

    A method is presented that has permitted a human genomic library to be screened for low-copy genes using 17-base synthetic oligonucleotides as probes. Parallel screening with two different 17-base probes permitted the unambiguous identification of clones containing interferon-alpha (IFN-alpha) genes. The isolated human IFN-alpha genes were sequenced, and one appears to be IFN-alpha L; the other is one not previously described, which we have designated IFN-alpha WA. The IFN-alpha WA sequence d...

  15. Cloning of the quail PIWI gene and characterization of PIWI binding to small RNAs.

    Directory of Open Access Journals (Sweden)

    Rong Chen

    Full Text Available The PIWI protein regulates gene expression at the epigenetic and post-transcriptional level with a variety of endogenous small non-coding RNAs. In poultry, the biological function of the PIWI protein and PIWI binding to small RNAs had not been determined. The present study cloned and analyzed the sequences of the PIWIL1 protein. We also characterized PIWIL1 binding to small RNAs from adult quail testis, where the PIWIL1 protein is specifically expressed. Small RNAs showed a strong peak at 24-27 nt in the testicular RNA library, mapped primarily to repeat sequences and were similar to rasiRNAs. MicroRNAs (miRNAs were abundant in the ovarian RNA library at a peak of 22 nt.

  16. Construction of an American mink Bacterial Artificial Chromosome (BAC library and sequencing candidate genes important for the fur industry

    Directory of Open Access Journals (Sweden)

    Christensen Knud

    2011-07-01

    Full Text Available Abstract Background Bacterial artificial chromosome (BAC libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. Results Here, we report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison. The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs, representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library. These included candidate genes for coat coloring, hair growth and length, coarseness, and some receptors potentially involved in viral diseases in mink. The extensive screening yielded positive results for 19 of these genes. Thirty-five clones corresponding to 19 genes were sequenced using 454 Roche, and large contigs (184 kb in average were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes. Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80% of BACs were obtained. An excess of 2 Mb has been analyzed, thus giving a snapshot of the mink genome. Conclusions The availability of the CHORI-321 American mink BAC library will aid in identification of genes and genomic regions of interest. We have demonstrated how the library can be used to identify specific genes of interest, develop genetic markers, and for BAC end sequencing and deep sequencing of selected clones. To our knowledge, this is the

  17. Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

    DEFF Research Database (Denmark)

    Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael;

    2011-01-01

    consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library...

  18. cDNA Cloning and Sequence Analysis of Rice Sbe1 and Sbe3 Genes

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiu-hua; LIU Qiao-quan; WU Hsin-kan; WANG Zong-yang; GU Ming-hong

    2004-01-01

    Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes Sbe1 and Sbe3 encoding SBE I and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA library derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned Sbe1 and Sbe3 cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of Sbe3 was the same as that of sbe3 (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference,which resulted in changes of two deduced amino acids between the cloned Sbe1 cDNA and the reported sbe1 (Genbank Accession No. D11082). The cloned Sbe1 and Sbe3 cDNAs make it possible to improve rice starch quality through genetic engineering

  19. Cloning and cell cycle-dependent expression of DNA replication gene dnaC from Caulobacter crescentus.

    OpenAIRE

    1990-01-01

    Chromosome replication in the asymmetrically dividing bacteria Caulobacter crescentus is discontinuous with the new, motile swarmer cell undergoing an obligatory presynthetic gap period (G1 period) of 60 min before the initiation of DNA synthesis and stalk formation. To examine the regulation of the cell division cycle at the molecular level, we have cloned the DNA chain elongation gene dnaC from a genomic DNA library constructed in cosmid vector pLAFR1-7. To ensure that the cloned sequence c...

  20. Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

    Science.gov (United States)

    Dokarry, Melissa; Laurendon, Caroline; O'Maille, Paul E

    2012-01-01

    Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of

  1. Cloning of Integral Mature Peptide Gene of Human GDF-5

    Institute of Scientific and Technical Information of China (English)

    王万山; 顾为望; 王启伟; 朴仲贤; 朴英杰

    2004-01-01

    Summary: The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the sequence of human GDF-5 in Genbank.

  2. Molecular cloning of Taenia taeniaeformis oncosphere antigen genes.

    Science.gov (United States)

    Cougle, W G; Lightowlers, M W; Bogh, H O; Rickard, M D; Johnson, K S

    1991-03-01

    Infection of mice with the cestode Taenia taeniaeformis exhibits several important features common to other cestode infections, including the ability to vaccinate with crude antigen mixtures. Partial purification of the protective oncosphere antigens has been reported with a cutout from deoxycholate (DOC) acrylamide gels; this cutout was called fraction II (FII), and comprises approximately 10% of total DOC-soluble oncosphere antigen. Western blots of DOC gels probed with anti-FII antisera revealed a series of 3-5 discrete bands within the FII region. Further fractionation of the FII antigens on DOC gels was impractical due to limitations in supply of oncospheres, so a cDNA library was constructed from 150 ng of oncosphere mRNA and screened with alpha-FII antisera. Two distinct clone families were identified, oncA and oncB. Antibodies affinity-purified on either of two representative members, oncA1 and oncB1, recognised all the FII bands. Individual FII bands excised from a DOC gel resolved into an overlapping series of molecules when re-run on SDS-PAGE, indicating that each FII band consisted of several polypeptides of differing molecular weight. Immunoprecipitates resolved on SDS-PAGE revealed that alpha-FII recognised 3 major oncosphere antigens, of 62, 34 and 25 kDa; antisera against oncB precipitated both the 34- and 25-kDa antigens, whereas alpha-oncA antisera precipitated the 62-kDa antigen. We conclude that oncA and oncB encode the major antigens in the FII complex. The 62-kDa antigen encoded by oncA1 was the only common antigen precipitated by anti-FII and two other antisera raised against different protective extracts, suggesting that it may be a protective component in all three. Southern blot results indicate that oncA and oncB are distinct genes present at low copy number in the genome. Evidence is also presented suggesting that some cestode mRNAs, including oncA, may use variant polyadenylation signals.

  3. New vectors in fission yeast: application for cloning the his2 gene

    DEFF Research Database (Denmark)

    Weilguny, D; Praetorius, M; Carr, Alan;

    1991-01-01

    We describe a new Escherichia coli vector (pON5) that allows positive selection for recombinant clones. In this plasmid, the bla gene from pBR322 is permanently active, whereas the neo gene from transposon Tn5 is repressed by the cI-encoded lambda repressor. When DNA is inserted into the Bc...... of transforming Sc. pombe ura4 strains, as well as ura 3 strains of the distantly related budding yeast Saccharomyces cerevisiae. We have used pON163 for the construction of two fission yeast genomic libraries. From these gene banks clones were isolated that were able to complement fission yeast his2 mutants....../I or HindIII restriction sites situated within the cI gene, the neo gene becomes transcribed from the lambda pR promoter. We have also made a Schizosaccharomyces pombe derivative of pON5 (= pON163) by introducing the fission yeast ars1 and ura4+ sequences. We show that this plasmid is capable...

  4. A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes

    OpenAIRE

    Jonathan L Schmid-Burgk; Tobias Schmidt; Vera Kaiser; Klara Höning; Veit Hornung

    2013-01-01

    Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DN...

  5. 基于16S rRNA基因克隆文库技术分析广西富钟水牛瘤胃产甲烷菌组成及多样性%Compositon and Diversity of Ruminal Methanogens in Guangxi Fuzhong Buffaloes:an Analysis of Using 16S rRNA Gene Cloning Library Technique

    Institute of Scientific and Technical Information of China (English)

    杨承剑; 梁辛; 韦升菊; 李舒露; 梁贤威; 邹彩霞; 杨炳壮; 李丽莉

    2014-01-01

    本研究旨在利用16S rRNA基因克隆库技术分析广西富钟水牛瘤胃产甲烷菌组成及多样性。选取3头体况基本一致的健康雌性富钟水牛作为试验动物,采用机械破壁法提取瘤胃内容物总DNA,采用产甲烷菌引物Met86F/Met1340R扩增16S rRNA基因,构建16S rRNA基因克隆文库。结果表明,本试验共获得93个非嵌合体16S rRNA序列,按照97%的相似性划分为39个分类操作单元( OTU)。其中,60个序列(15个OTU)与已培养细菌16S rRNA序列相似性≥97%,占总序列的64.5%;32个序列(23个OTU)与已培养菌16S rRNA 序列相似性处于90%~(<97%);仅有1个序列与Methanomassiliicoccus luminyensis相似性<90%。系统发育树分析表明,98.9%的序列均属于甲烷杆菌目( Methanobacteriales),部分序列与Methanobacteriales中任何已知相似序列都相隔较远,它们可能代表Methanobacteriales中新的属或种。由上述结果可见,富钟水牛瘤胃产甲烷菌以Methanobacteriales为优势菌群,其中有许多未知的产甲烷菌需进一步分离培养并对其功能进行分析。%The objective of this study was to analysis the composition and the diversity of ruminal methanogens in Guangxi Fuzhong buffaloes by using 16S rRNA gene cloning library technique. Three healthy female Fuzhong buffaloes with similar body condition were used in this study. Total DNA of ruminal contents was ex-tracted by bead-beating method. Primers of Met86F/Met1340R of methanogens were used to amplify 16S rRNA gene for the construction of 16S rRNA gene clone library. The results showed that 93 chimera-free 16S rRNA sequences were obtained in the present study. Based the 97% similarity, these sequences were assigned to 39 operational taxonomic units ( OTUs) . Among those, sixty sequences showed ≥97% sequence similarity to known species (15 OTUs, occupied 64.5% of total sequences), thirty two sequences had sequence similari-ty to known species in the range of 90% to <97% ( 23 OTUs

  6. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    Science.gov (United States)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  7. A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening

    Directory of Open Access Journals (Sweden)

    Deal Karin R

    2010-12-01

    Full Text Available Abstract Background A five-dimensional (5-D clone pooling strategy for screening of bacterial artificial chromosome (BAC clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones. Results A new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~2× genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on Aegilops tauschii chromosome 2D and Ae. tauschii contig maps. Clones in single contigs were successfully assigned to 48 (87% specific SNP markers on the map with 91% precision. Conclusion A new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml.

  8. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.

    2004-01-01

    To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU minitra...

  9. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.;

    2004-01-01

    To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU...

  10. Cloning, sequencing and expression of the gene encoding the extracellular metalloprotease of Aeromonas caviae.

    Science.gov (United States)

    Kawakami, K; Toma, C; Honma, Y

    2000-01-01

    A gene (apk) encoding the extracellular protease of Aeromonas caviae Ae6 has been cloned and sequenced. For cloning the gene, the DNA genomic library was screened using skim milk LB agar. One clone harboring plasmid pKK3 was selected for sequencing. Nucleotide sequencing of the 3.5 kb region of pKK3 revealed a single open reading frame (ORF) of 1,785 bp encoding 595 amino acids. The deduced polypeptide contained a putative 16-amino acid signal peptide followed by a large propeptide. The N-terminal amino acid sequence of purified recombinant protein (APK) was consistent with the DNA sequence. This result suggested a mature protein of 412 amino acids with a molecular mass of 44 kDa. However, the molecular mass of purified recombinant APK revealed 34 kDa by SDS-PAGE, suggesting that further processing at the C-terminal region took place. The 2 motifs of zinc binding sites deduced are highly conserved in the APK as well as in other zinc metalloproteases including Vibrio proteolyticus neutral protease, Emp V from Vibrio vulnificus, HA/P from Vibrio cholerae, and Pseudomonas aeruginosa elastase. Proteolytic activity was inhibited by EDTA, Zincov, 1,10-phenanthroline and tetraethylenepentamine while unaffected by the other inhibitors tested. The protease showed maximum activity at pH 7.0 and was inactivated by heating at 80 C for 15 min. These results together suggest that APK belongs to the thermolysin family of metalloendopeptidases.

  11. Microbial diversity in uranium mining-impacted soils as revealed by high-density 16S microarray and clone library.

    Science.gov (United States)

    Rastogi, Gurdeep; Osman, Shariff; Vaishampayan, Parag A; Andersen, Gary L; Stetler, Larry D; Sani, Rajesh K

    2010-01-01

    Microbial diversity was characterized in mining-impacted soils collected from two abandoned uranium mine sites, the Edgemont and the North Cave Hills, South Dakota, using a high-density 16S microarray (PhyloChip) and clone libraries. Characterization of the elemental compositions of soils by X-ray fluorescence spectroscopy revealed higher metal contamination including uranium at the Edgemont than at the North Cave Hills mine site. Microarray data demonstrated extensive phylogenetic diversity in soils and confirmed nearly all clone-detected taxonomic levels. Additionally, the microarray exhibited greater diversity than clone libraries at each taxonomic level at both the mine sites. Interestingly, the PhyloChip detected the largest number of taxa in Proteobacteria phylum for both the mine sites. However, clone libraries detected Acidobacteria and Bacteroidetes as the most numerically abundant phyla in the Edgemont and North Cave Hills mine sites, respectively. Several 16S rDNA signatures found in both the microarrays and clone libraries displayed sequence similarities with yet-uncultured bacteria representing a hitherto unidentified diversity. Results from this study demonstrated that highly diverse microbial populations were present in these uranium mine sites. Diversity indices indicated that microbial communities at the North Cave Hills mine site were much more diverse than those at the Edgemont mine site.

  12. Cloning of thienamycin biosynthetase genes from Streptomyces cattleya.

    Science.gov (United States)

    Li, R; Wang, Y; Zeng, Y

    1993-01-01

    A mutant Y3 blocked in the thienamycin biosynthetic pathway was obtained from thienamycin producing strain Streptomyces cattleya by NTG treatment. Preliminary cloning system has been established on the basis of studies on the conditions for protoplast formation, regeneration, as well as DNA transformation for Y3 mutant strain. The shotgun cloning was carried out from S. cattleya using pIJ680 as a vector and the Y3 mutant as a host. The transformant No. 12 produced a thienamycin-like substance identified by paper chromatography and HPLC analysis. A recombinant plasmid p6BC12, which has molecular size of 9.8kb and an insert of 4.5kb, could be recovered. Southern hybridization confirmed that the transformant No. 12 harbors the recombinant plasmid p6BC12. The intermediate accumulated by Y3 mutant was identified as a polypeptide. The product of transformant containing p6BC12 could turn this polypeptide to a bioactivity substance in vitro. This gene can hybridizate with S. lipmanii IPNS gene. We presume that a cyclase gene from S. cattleya was cloned according to the function of the expression product.

  13. [Cloning of pigeon invariant chain (Ii) gene by RACE].

    Science.gov (United States)

    Liu, Gang; Zhong, Da-Lian; Liu, Xue-Lan; Yu, Wei-Yi

    2008-01-01

    In order to compare the structure and function of pigeon invariant chain (pIi) gene with other avian's, pIi gene was cloned using a method of RACE (Rapid Amplification of cDNA Ends). Firstly, according to high conservative nucleotide sequence of homologous fragment in avian invariant chain (Ii) gene, a pair of degenerated primer was designed, and a special DNA fragment was gained from pigeon spleen cell RNA by PCR. Then based on the sequence of gained DNA fragment, some new primers were designed, and the 3'terminal and the 5'terminal of pIi gene were cloned by RACE respectively. Finally a complete cDNA of pIi was to extend with newly designed primer by PCR. The product was identified by electrophresis and sequence analysis. The results of sequencing indicate that pIi gene is 1,050 bp in length (GenBank No. AY904337), which includes an open reading frame of 633 bp encoding a precursor protein with 211 amino acid residues. In comparison with the nucleotide sequences of other species' Ii genes, pIi is similar to chicken's, showing an overall identity of 82.8 with chicken and over 52.0 with human and other mammalian animals. In addition, some amino acid residues in Ii molecule manifest extremely conservative among animals, which suggests that they could have an important biological function.

  14. Positional cloning of disease genes on chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Doggett, N. [Los Alamos National Lab., NM (United States); Bruening, M. [Leiden Univ. (Netherlands); Callen, D. [Adelaide Women`s and Children`s Hospital, North Adelaide, South Australia (Australia); Gardiner, M. [University Coll., London (United Kingdom); Lerner, T. [Massachusetts General Hospital, Boston, MA (United States)

    1996-04-01

    The project seeks to elucidate the molecular basis of an important genetic disease (Batten`s disease) by molecular cloning of the affected gene by utilizing an overlapping clone map of chromosome 16. Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a recessively inherited neurodegenerative disorder of childhood characterized by progressive loss of vision, seizures, and psychomoter disturbances. The Batten disease gene was genetically mapped to the chromosome region 16p 12.1 in close linkage with the genetic markers D16S299 and D16S298. Exon amplification of a cosmid containing D16S298 yielded a candidate gene that was disrupted by a 1 kb genomic deletion in all patients containing the most common haplotype for the disease. Two separate deletions and a point mutation altering a splice site in three unrelated families have confirmed the gene as the Batten disease gene. The disease gene encodes a novel 438 amino acid membrane binding protein of unknown function.

  15. Molecular cloning and characterization of the human beta-like globin gene cluster.

    Science.gov (United States)

    Fritsch, E F; Lawn, R M; Maniatis, T

    1980-04-01

    The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

  16. Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

    Institute of Scientific and Technical Information of China (English)

    HE Cong-fen; MA You-zhi; XIN Zhi-yong; XU Qiong-fang; LI Lian-cheng

    2004-01-01

    The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) was identified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes were microisolated and collected. After two rounds of PCR amplification, the PCR products were ranged from 150 - 3 000 bp,with predominant fragments at about 200 - 2 000 bp. Using Ag.intermediumgenomic DNA as a probe, Southern blotting analysis confirmed the products originated from Ag. intermediumgenome. The products were purified, ligated to pUC18 and then transformed into competence E.coli DH5α to produce a 2Ai-2 chromosome DNA library. The microcloning experiments produced approximately 5×105 clones, the size range of the cloned inserts was 200- 1 500 bp, with an average of 580bp. Using Ag. intermediumgenomic DNA as a probe, dot blotting results showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences in the library. Four Ag. intermedium clones were screened from the library by RFLP, and three clones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitive sequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitive DNA sequence.

  17. Molecular Cloning of Human Gene(s) Directing the Synthesis of Nervous System Cholinesterases

    Science.gov (United States)

    1987-09-01

    Report No. 4 If MOLECULAR CLONING OF O HUMAN GENE(S) DIRECTING qTHE SYNTHESIS OF NERVOUS SYSTEM CHOLINESTERASES cc Annual/Final Report 0 N November...62734A I734A875 IAl 451 MOLECULAR CLONING OF HUMAN GEME(S) DIRECTING THE SYNTHESIS OF NERVOUS SYSTEM CHOLINESTERASE 12. PERSONAL AUTHOR(S) Hermona Soreq...important roles in regulating the pace and mode of function of particular types of synapses. For example, molecular cloning of the nicotinic (44-46) and the

  18. Soil clone library analyses to evaluate specificity and selectivity of PCR primers targeting fungal 18S rDNA for denaturing-gradient gel electrophoresis (DGGE).

    Science.gov (United States)

    Takada Hoshino, Yuko; Morimoto, Sho

    2010-01-01

    We evaluated the fungal specificity and detection bias of four fungal 18S rRNA gene (18S rDNA) primer sets for denaturing-gradient gel electrophoresis (DGGE). We constructed and compared clone libraries amplified from upland and paddy field soils with each primer set (1, NS1/GCFung; 2, FF390/FR1-GC; 3, NS1/FR1-GC; and 4, NS1/EF3 for the first PCR and NS1/FR1-GC for the second PCR). Primer set 4 (for nested PCR) showed the highest specificity for fungi but biased specific sequences. Sets 1, 2, and 3 (for single PCR) amplified non-fungal eukaryotic sequences (from 7 to 16% for upland soil and from 20 to 31% for paddy field soil) and produced libraries with similar distributions of fungal 18S rDNA sequences at both the phylum and the class level. Set 2 tended to amplify more diverse fungal sequences, maintaining higher specificity for fungi. In addition, clone analyses revealed differences among primer sets in the frequency of chimeras. In upland field soil, the libraries amplified with primer sets 3 and 4, which targeted long fragments, contained many chimeric 18S rDNA sequences (18% and 48%, respectively), while the libraries obtained with sets 1 and 2, which targeted short fragments, contained fewer chimeras (5% and 10%, respectively).

  19. Cloning and Clone Analysis of GRA1 Gene from Local Isolate Toxoplasma gondii Tachyzoite

    Directory of Open Access Journals (Sweden)

    Didik T Subekti

    2008-03-01

    Full Text Available The GRA1 gene of Toxoplasma gondii encoding protein called GRA1 protein. GRA1 protein known to be immunogenic and essentialy involved in modification of parasitophorus vacoule which has role in immune evasion and virulency of organism. The local isolate of T. gondii is successfuly isolated and known as highly pathogenic isolate similarly as its RH strain. Unfortunately, the homology sequence of GRA1 gene between those isolate still unknown. The purpose of the research are to clone the GRA1 gene and to analyze the homology from pathogenic T. gondii isolate and RH strain. Tachyzoite of T. gondii was grown in mice peritoneum by intraperitoneal injection. Then, total mRNA was isolated and purified. cDNA was synthesized from mRNA and then amplified using F1 dan R1 primers to get clone of GRA1 from local isolate. Homology analysis was perform using several bioinformatic softwares. The result showed that cDNA of GRA1 from local isolate has 84% homologs with RH strain of T.gondii. However, when subsequently editing performed to parts of suspected non coding sequence of cDNA GRA1 to get CDS of GRA1, the homology was increase to 100% compare to CDS of GRA1 of RH strain.

  20. Cloning,sequencing and phylogenic analysis of duck prion gene

    Institute of Scientific and Technical Information of China (English)

    WANG Qigui; ZHANG Lei; HU Xiaoxiang; FAN Baoliang; LI Ning; LI Hui; WU Changxin

    2004-01-01

    Duck prion gene was cloned and sequenced. Similar to mammalian prion protein (PrP), duck prion is encoded by a single exon of a single copy in genome, which was confirmed by Southern blot analysis. All of the structural features of mammalian PrP were also identified in the duck PrP. Compared with mammalian PrP, it exhibited a 30 % of general similarity. When compared with chicken PrP, it showed a higher homology of 97%. A phylogenetic tree was constructed to trace evolution of prion gene in animals.

  1. Cloning of the Protective Antigen Gene of Bacillus anthracis

    Science.gov (United States)

    1983-09-01

    of the complicated precedents of duplicate toxin genes in chro- muumm mosomall and plasmid DNA of B. thuringiensis (Schnepf and Whitely, 1981; Klier...OiL V4. 34. S-W7. SW 1v 99 CwI 0193 by MT 0 009-7483/06O-002.00/0 mU"- - 1*;)-0Cloning of the Protective Antigen Gene OCT 19 MI L Sof Bacillus ...Sumnler uncertain, it is probably caused by other Bacillus antigens, 4 t which may include LF and EF. PA produced from recom- A The - "w t of a

  2. Production of cloned pigs with targeted attenuation of gene expression.

    Directory of Open Access Journals (Sweden)

    Vilceu Bordignon

    Full Text Available The objective of this study was to demonstrate that RNA interference (RNAi and somatic cell nuclear transfer (SCNT technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE, a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45-82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA expression vector under the control of RNA polymerase III (U6 promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.

  3. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5)

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, H.D.; Tucker, W.Q.J.; Hort, Y.; Martinson, M.E.; Mayo, G.; Clutterbuck, E.J.; Sanderson, C.J.; Young, I.G.

    1987-10-01

    The human eosinophil differentiation factor (EDF) gene was cloned from a genomic library in lambda phage EMBL3A by using a murine EDF cDNA clone as a probe. The DNA sequence of a 3.2-kilobase BamHI fragment spanning the gene was determined. The gene contains three introns. The predicted amino acid sequence of 134 amino acids is identical with that recently reported for human interleukin 5 but shows no significant homology with other known hemopoietic growth regulators. The amino acid sequence shows strong homology (approx. 70% identity) with that of murine EDF. Recombinant human EDF, expressed from the human EDF gene after transfection into monkey COS cells, stimulated the production of eosinophils and eosinophil colonies from normal human bone marrow but had no effect on the production of neutrophils or mononuclear cells (monocytes and lymphoid cells). The apparent specificity of human EDF for the eosinophil lineage in myeloid hemopoiesis contrasts with the properties of human interleukin 3 and granulocyte/macrophage and granulocyte colony-stimulating factors but is directly analogous to the biological properties of murine EDF. Human EDF therefore represents a distinct hemopoietic growth factor that could play a central role in the regulation of eosinophilia.

  4. Targeted oligonucleotide-mediated microsatellite identification (TOMMI from large-insert library clones

    Directory of Open Access Journals (Sweden)

    Ren Jun

    2005-11-01

    Full Text Available Abstract Background In the last few years, microsatellites have become the most popular molecular marker system and have intensively been applied in genome mapping, biodiversity and phylogeny studies of livestock. Compared to single nucleotide polymorphism (SNP as another popular marker system, microsatellites reveal obvious advantages. They are multi-allelic, possibly more polymorphic and cheaper to genotype. Calculations showed that a multi-allelic marker system always has more power to detect Linkage Disequilibrium (LD than does a di-allelic marker system 1. Traditional isolation methods using partial genomic libraries are time-consuming and cost-intensive. In order to directly generate microsatellites from large-insert libraries a sequencing approach with repeat-containing oligonucleotides is introduced. Results Seventeen porcine microsatellite markers were isolated from eleven PAC clones by targeted oligonucleotide-mediated microsatellite identification (TOMMI, an improved efficient and rapid flanking sequence-based approach for the isolation of STS-markers. With the application of TOMMI, an average of 1.55 (CA/GT microsatellites per PAC clone was identified. The number of alleles, allele size distribution, polymorphism information content (PIC, average heterozygosity (HT, and effective allele number (NE for the STS-markers were calculated using a sampling of 336 unrelated animals representing fifteen pig breeds (nine European and six Chinese breeds. Sixteen of the microsatellite markers proved to be polymorphic (2 to 22 alleles in this heterogeneous sampling. Most of the publicly available (porcine microsatellite amplicons range from approximately 80 bp to 200 bp. Here, we attempted to utilize as much sequence information as possible to develop STS-markers with larger amplicons. Indeed, fourteen of the seventeen STS-marker amplicons have minimal allele sizes of at least 200 bp. Thus, most of the generated STS-markers can easily be

  5. Identification of genes and pathways related to phenol degradation in metagenomic libraries from petroleum refinery wastewater.

    Directory of Open Access Journals (Sweden)

    Cynthia C Silva

    Full Text Available Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

  6. Identification of genes and pathways related to phenol degradation in metagenomic libraries from petroleum refinery wastewater.

    Science.gov (United States)

    Silva, Cynthia C; Hayden, Helen; Sawbridge, Tim; Mele, Pauline; De Paula, Sérgio O; Silva, Lívia C F; Vidigal, Pedro M P; Vicentini, Renato; Sousa, Maíra P; Torres, Ana Paula R; Santiago, Vânia M J; Oliveira, Valéria M

    2013-01-01

    Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

  7. Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

    Directory of Open Access Journals (Sweden)

    Kudrna David

    2011-03-01

    Full Text Available Abstract Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1 digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb to 157 Kb (Eg_Ba, very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×, contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae

  8. Cloning of tomato (Lycopersicon esculentum Mill.) arginine decarboxylase gene and its expression during fruit ripening.

    Science.gov (United States)

    Rastogi, R; Dulson, J; Rothstein, S J

    1993-11-01

    Arginine decarboxylase (ADC) is the first enzyme in one of the two pathways of putrescine biosynthesis in plants. The genes encoding ADC have previously been cloned from oat and Escherichia coli. Degenerate oligonucleotides corresponding to two conserved regions of ADC were used as primers in polymerase chain reaction amplification of tomato (Lycopersicon esculentum Mill.) genomic DNA, and a 1.05-kb fragment was obtained. This genomic DNA fragment encodes an open reading frame of 350 amino acids showing about 50% identity with the oat ADC protein. Using this fragment as a probe, we isolated several partial ADC cDNA clones from a tomato pericarp cDNA library. The 5' end of the coding region was subsequently obtained from a genomic clone containing the entire ADC gene. The tomato ADC gene contains an open reading frame encoding a polypeptide of 502 amino acids and a predicted molecular mass of about 55 kD. The predicted amino acid sequence exhibits 47 and 38% identify with oat and E. coli ADCs, respectively. Gel blot hybridization experiments show that, in tomato, ADC is encoded by a single gene and is expressed as a transcript of approximately 2.2 kb in the fruit pericarp and leaf tissues. During fruit ripening the amount of ADC transcript appeared to peak at the breaker stage. No significant differences were seen when steady-state ADC mRNA levels were compared between normal versus long-keeping Alcobaca (alc) fruit, although alc fruit contain elevated putrescine levels and ADC activity at the ripe stage. The lack of correlation between ADC activity and steady-state mRNA levels in alc fruit suggests a translational and/or posttranslational regulation of ADC gene expression during tomato fruit ripening.

  9. Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli.

    Science.gov (United States)

    Marillia, Elizabeth-France; MacPherson, Jim M.; Tsang, Edward W. T.; Van Audenhove, Katrien; Keller, Wilf A.; GrootWassink, Jan W. D.

    2001-10-01

    A genomic clone encoding a thiohydroximate S-glucosyltransferase (S-GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S-GT gene were present in B. napus. The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli, and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S-GT activity detected from the recombinant protein confirmed that this clone was indeed a S-glucosyltransferase.

  10. Molecular cloning and chromosomal localization of the ADH7 gene encoding human class IV ({sigma}) ADH

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Hirokazu; Baraona, E.; Lieber, C.S. [Mount Sinai School of Medicine, Bronx, NY (United States)

    1996-01-15

    The ADH7 gene encoding human Class IV ({sigma}) alcohol dehydrogenase (ADH) was cloned from a Caucasian genomic DNA library and characterized. It has nine exons and eight introns that span about 22 kb, and its intron insertion is identical to that of the other ADH genes (ADH1 to ADH5). The nucleotide sequences of the exons encoding 374 amino acids are identical to the previously reported cDNA sequence of {sigma} ADH. Fluorescence in situ hybridization analysis showed that ADH7 is located on human chromosome 4q23-q24, close to the ADH cluster locus (4q21-q25). These data are consistent with the view that Class IV ADH is a member of the ADH family and is phylogenetically close to the other ADHs. 15 refs., 2 figs., 1 tab.

  11. Human estrogen sulfotransferase gene (STE): Cloning, structure, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Aksoy, I.A.; Weinshilboum, M. [Mayo Foundation, Rochester, MI (United States)] [and others

    1995-09-01

    Sulfation is an important pathway in the metabolism of estrogens. We recently cloned a human liver estrogen sulfotransferase (EST) cDNA. We have now determined the structure and chromosomal localization of the EST gene, STE, as a step toward molecular genetic studies of the regulation of EST in humans. STE spans approximately 20 kb and consists of 8 exons, ranging in length from 95 to 181 bp. The locations of most exon-intron splice junctions within STE are identical to those found in a human phenol ST (PST) gene, STM, and in a rat PST gene. In addition, the locations of five STE introns are also conserved in the human dehydroepiandrosterone (DBEA) ST gene, STD. The 5{prime} flanking region of STE contains one CCAAT and two TATA sequences. The location of one of the TATA box elements is in excellent agreement with the site of transcription initiation as determined by 5{prime}-rapid amplification of cDNA ends. STE was mapped to human chromosome 4q13.1 by fluorescence in situ hybridization. Cloning and structural characterization of STE will now make it possible to study potential molecular genetic mechanisms involved in the regulation of EST in human tissues. 50 refs., 6 figs., 1 tab.

  12. An "instant gene bank" method for heterologous gene cloning: complementation of two Aspergillus nidulans mutants with Gaeumannomyces graminis DNA.

    Science.gov (United States)

    Bowyer, P; Osbourn, A E; Daniels, M J

    1994-02-01

    We present a novel technique for gene cloning by complementation of mutations in Aspergillus nidulans with DNA from a heterologous organism, Gaeumannomyces graminis. This technique bypasses the time-consuming and difficult construction of gene libraries, making it both rapid and simple. The method relies on recombination between a fungal replicating vector pHELP1 and linear G. graminis genomic DNA during co-transformation. We were able to complement two out of seven A. nidulans mutants tested and to rescue transforming DNA from both in Escherichia coli. Complementation of the A. nidulans argB mutation resulted from integration of 8-10 kb segments of G. graminis DNA into pHELP1. The complementation of the A. nidulans pyrG mutation resulted from a complex rearrangement. Complementing DNA was shown to originate from G. graminis, and was capable of retransforming the original mutants to give the expected phenotype.

  13. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

    Science.gov (United States)

    Theaker, Sarah M.; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J.; Cole, David K.; Peakman, Mark; Sewell, Andrew K.; Dolton, Garry

    2016-01-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. PMID:26826277

  14. Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers

    Energy Technology Data Exchange (ETDEWEB)

    Kandpal, R.P.; Kandpal, G.; Weissman, S.M. (Yale Univ. School of Medicine, New Haven, CT (United States))

    1994-01-04

    The authors describe a simple and rapid method for constructing small-insert genomic libraries highly enriched for dimeric, trimeric, and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo I-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotde. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)[sub n] microsatellites with this approach contained clones with inserts containing CA repeats. They have also used this protocol for enrichment of (CAG)[sub n] and (AGAT)[sub n] sequence repeats and for Not I jumping clones. They have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.

  15. Tripeptidase Gene (pepT) of Lactococcus lactis : Molecular Cloning and Nucleotide Sequencing of pepT and Construction of a Chromosomal Deletion Mutant

    NARCIS (Netherlands)

    Mierau, Igor; Haandrikman, Alfred J.; Velterop, Odilia; Tan, Paris S.T.; Leenhouts, Kees L.; Konings, Wilhelmus; Venema, Gerhardus; Kok, Jan

    1994-01-01

    The gene encoding a tripeptidase (pepT) of lactococcus lactis subsp. cremoris (formerly subsp. lactis) MG1363 was cloned from a genomic library in pUC19 and subsequently sequenced. The tripeptidase of L. lactis was shown to be homologous to PepT of Salmonella typhimurium with 47.4% identity in the d

  16. Cloning and Expression of Nano Body Gene against Enterotoxin B of Staphylococcus Aureus

    Directory of Open Access Journals (Sweden)

    Zahra Tavassoli

    2017-02-01

    Full Text Available Background & Objectives: Staphylococcus aureus bacteria causes many different diseases by secretion of various enterotoxins. Therefore, it is necessary to develop ways that facilitate the detection of enterotoxins. Nowadays, immunochemical methods which are based on monoclonal antibody technology are used. The heavy chain antibodies that are called VHH or Nano body were found in blood serum of the Camelidae family. The unique properties of this antibody such as their binding to small molecules like toxins make them attractive candidates for the development of immunodiagnostic tests. The present study was done to achieve a VHH molecules against Staphylococcus enterotoxin B. Materials & Methods: Freighting phage library for isolate private Nano bodies against enterotoxin B was done in previous works. Next, pCANTAB 5E vector that consists VHH, extracted from E.coli bacteria strain xl1blue, and after doing PCR process with relative primers, sub cloning in pET21a(+ as an expression vector with cut sites NdeI and XhoI was done. Transformation in E.coli bacteria strain BL21(DE3 was done. Then, the cells effected with IPTG and producing time, and other terms were optimized. Finally, the expression of the protein with SDS-PAGE and western blot techniques was evaluated. Result: For proving cloning of nano body gene in pET21a (+ vector, nucleotide sequence of gene was analyzed, and transforming to E.coli bacteria strain BL21(DE3 was successful. After inspiration, active protein in cell was seen by SDS-PAGE technique and proved by western blot. Conclusion: cloning, sub cloning, and nonabody expression were surveyed in this research. Production of this protein can help to develop new therapeutic methods and produce vaccine against enterotoxin B of Staphylococcus aureus

  17. Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments.

    Science.gov (United States)

    Santangelo, G M; Tornow, J; Moldave, K

    1986-01-01

    We have developed a novel efficient method, carrier-facilitated insertion, to insert small (150-600 bp) DNA fragments into plasmid vectors. This method employs a carrier segment of vector DNA to circumvent the difficulties in ligating two fragments together to generate a recombinant circle efficiently. We have used carrier-facilitated insertion to construct three genomic libraries of random (DNase I-generated) fragments from the Saccharomyces cerevisiae genome. One of these was an expression library, and the other two were promoter-cloning libraries. 87-90% of the Escherichia coli colonies in each library contained recombinant plasmids, and less than 3% of the recombinants contained more than one insert. Detection of open reading frames among the inserts in the expression library was accomplished by testing for beta-galactosidase activity. This methodology, unencumbered by the intrinsic disproportionality of cDNA libraries, can be used to identify and clone DNA that codes for a specific antigenic determinant. When used in combination with a method to detect and isolate random constitutive, repressible and inducible yeast promoters, these libraries should permit a comprehensive analysis of the yeast genome and its expression.

  18. Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences

    NARCIS (Netherlands)

    Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Jong, S. de; Kline, E.; Stephen, J.R.; Laanbroek, H.J.

    2001-01-01

    A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a m

  19. Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

    NARCIS (Netherlands)

    Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Jong, de S.; Kline, E.; Stephen, J.R.; Laanbroek, H.J.

    2001-01-01

    A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a m

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

    Science.gov (United States)

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

    2011-04-01

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

  1. CLONING AND SEQUENCING OF MATURED FRAGMENT OF HUMAN NEVER GROWTH FACTOR GENE

    Institute of Scientific and Technical Information of China (English)

    马巍; 吴玲; 王德利; 刘淼; 任惠民; 杨广笑; 王全颖

    2003-01-01

    Objective Molecular cloning and sequencing of the human matured fragment of human nerve growth factor(NGF) gene. Methods Extracting the human genomic DNA from the white blood cells as templates, the gene of NGF was cloned by using PCR and T-vector cloning method. Screening the positive clones and identified by the restriction enzymes, and then the cloned amplified fragment was sequenced and analyzed. Results DNA sequence comparison the cloned gene of NGF with the GenBank (V01511) sequence demonstrated that both of sequences were identical, 354bp length. Conclusion Cloning the NGF gene from the human genomic DNA has paved the way for further study on gene therapy of nerve system injury.

  2. Cloning, expression, and polymorphism of the porcine calpain10 gene

    Institute of Scientific and Technical Information of China (English)

    Xiuqin Yang; Di Liu; Hao Yu; Lijuan Guo; Hui Liu

    2008-01-01

    Calpains are calcium-regulated protcases involved in cellular functions that include muscle proteolysis both ante- and postmortem. This study was designed to clone the complete coding sequence of the porcine calpain10 gene, CAPN10, to analyze its expression characteristics and to investigate its polymorphism. Two isoforms of the CAPN10 gene, CAPN10A and CAPN10B, were obtained by reverse transcriptionpolymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods combined with in silico cloning. RT-PCR results indicated that CAPN10 mRNA was ubiquitously expressed in all tissues examined and, with increasing age,the expression level increased in muscles at six different growth points. In the same tissues, the expression level of CAPN10A was higher than that of CAPN10B. In addition,three single nucleotide polymorphisms were detected by the PCR-single-stranded conformational polymorphism method and by comparing the sequences of Chinese Min pigs with those of Yorkshire pigs. C527T mutation was a missense mutation and led to transforming Pro into Leu at the 176th amino acid. The results of the current study provided basic molecular information for further study of the function of the porcine CAPN10 gene.

  3. Cloning and Identification of Methionine Synthase Gene from Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Lan HUANG; Dong-Yang LI; Shao-Xiao WANG; Shi-Ming ZHANG; Jun-Hui CHEN; Xiang-Fu WU

    2005-01-01

    Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5' and 3' regions were further cloned by 5'- and 3'-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.

  4. Rapid restriction enzyme-free cloning of PCR products: a high-throughput method applicable for library construction.

    Science.gov (United States)

    Chaudhary, Vijay K; Shrivastava, Nimisha; Verma, Vaishali; Das, Shilpi; Kaur, Charanpreet; Grover, Payal; Gupta, Amita

    2014-01-01

    Herein, we describe a novel cloning strategy for PCR-amplified DNA which employs the type IIs restriction endonuclease BsaI to create a linearized vector with four base-long 5'-overhangs, and T4 DNA polymerase treatment of the insert in presence of a single dNTP to create vector-compatible four base-long overhangs. Notably, the insert preparation does not require any restriction enzyme treatment. The BsaI sites in the vector are oriented in such a manner that upon digestion with BsaI, a stuffer sequence along with both BsaI recognition sequences is removed. The sequence of the four base-long overhangs produced by BsaI cleavage were designed to be non-palindromic, non-compatible to each other. Therefore, only ligation of an insert carrying compatible ends allows directional cloning of the insert to the vector to generate a recombinant without recreating the BsaI sites. We also developed rapid protocols for insert preparation and cloning, by which the entire process from PCR to transformation can be completed in 6-8 h and DNA fragments ranging in size from 200 to 2200 bp can be cloned with equal efficiencies. One protocol uses a single tube for insert preparation if amplification is performed using polymerases with low 3'-exonuclease activity. The other protocol is compatible with any thermostable polymerase, including those with high 3'-exonuclease activity, and does not significantly increase the time required for cloning. The suitability of this method for high-throughput cloning was demonstrated by cloning batches of 24 PCR products with nearly 100% efficiency. The cloning strategy is also suitable for high efficiency cloning and was used to construct large libraries comprising more than 108 clones/µg vector. Additionally, based on this strategy, a variety of vectors were constructed for the expression of proteins in E. coli, enabling large number of different clones to be rapidly generated.

  5. Construction of differentially expressed genes library of bighead carp (Aristichthys nobilis) exposed to microcystin-lr using ssh and expression profile of related genes.

    Science.gov (United States)

    Cui, Zhihui; Zhang, Kaiyue; Qu, Xiancheng; Liu, Qigen

    2011-12-01

    Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria (blue-green algae). There are more than 70 MCs variants of which the most common and widely studied is MC-LR. We screened the hepatocellular differentially expressed genes against MC-LR in the bighead carp (Aristichthys nobilis). Suppression subtractive hybridization was used to construct the forward subtracted and reverse subtracted cDNA libraries, and one hundred and thirty two positive clones (seventy one in forward library and sixty one in reverse library) were randomly selected and sequenced. Finally, fifty five reliable sequences from the forward subtracted library were used in a homology search by BLASTn and BLASTx, as were 57 reliable sequences from the reverse subtracted library. Furthermore, eight analyzed sequences from the forward subtracted cDNA library and seven from the reverse subtracted library were found to be non-homologous sequences. The screening identified genes induced by MC-LR in both libraries that are involved in various processes, such as energy metabolism, immunity, and apoptosis. Some are cytoskeleton- and transportation-related genes, while signal transduction-related genes were also found. Significant genes, such as the apoptosis-related gene p53 and the proto-oncogene c-myc, are involved in inhibition of the MC-LR response in the reverse subtracted library. In addition, several immune-related genes, which play an important role in antioxidation and detoxification of MC-LR, were characterized and identified in both of the subtracted libraries. The study provides the basic data to further identify the genes and molecular mechanism of detoxification of microcystins.

  6. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica

    Institute of Scientific and Technical Information of China (English)

    Zheng Min; Wu Yi-jun; Cai Wei-min; Weng Hong-lei; Liu Rong-hua

    2005-01-01

    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosomajaponicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes.Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library.Amplification of the library was carried out with transformation of DH5α. The amplified library contained more than 400 positive bacterial clones, which were then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. The subtracted cDNA library of differentially expressed genes of HSCs was constructed successfully,the library is efficient and lays foundation for screening and cloning new and specific genes of schistosomiasis.

  7. Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica*

    OpenAIRE

    2005-01-01

    To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Ampli...

  8. Cloning

    Science.gov (United States)

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  9. Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs.

    Science.gov (United States)

    Chumakov, I M; Zabarovsky, E R; Prassolov, V S; Mett, V L; Kisselev, L L

    1982-01-01

    A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage lambda Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.

  10. The cloning, genomic organization and tissue expression profile of the human DLG5 gene

    Directory of Open Access Journals (Sweden)

    Gibbs Richard A

    2002-02-01

    Full Text Available Abstract Background Familial atrial fibrillation, an autosomal dominant disease, was previously mapped to chromosome 10q22. One of the genes mapped to the 10q22 region is DLG5, a member of the MAGUKs (Membrane Associated Gyanylate Kinase family which mediates intracellular signaling. Only a partial cDNA was available for DLG5. To exclude potential disease inducing mutations, it was necessary to obtain a complete cDNA and genomic sequence of the gene. Methods The Northern Blot analysis performed using 3' UTR of this gene indicated the transcript size to be about 7.2 KB. Using race technique and library screening the entire cDNA was cloned. This gene was evaluated by sequencing the coding region and splice functions in normal and affected family members with familial atrial fibrillation. Furthermore, haploid cell lines from affected patients were generated and analyzed for deletions that may have been missed by PCR. Results We identified two distinct alternately spliced transcripts of this gene. The genomic sequence of the DLG5 gene spanned 79 KB with 32 exons and was shown to have ubiquitous human tissue expression including placenta, heart, skeletal muscle, liver and pancreas. Conclusions The entire cDNA of DLG5 was identified, sequenced and its genomic organization determined.

  11. Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota

    Directory of Open Access Journals (Sweden)

    Chiara Devirgiliis

    2014-01-01

    Full Text Available Lactic acid bacteria (LAB represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.

  12. Cloning of type 8 capsule genes and analysis of gene clusters for the production of different capsular polysaccharides in Staphylococcus aureus.

    Science.gov (United States)

    Sau, S; Lee, C Y

    1996-04-01

    Eleven serotypes of capsular polysaccharide from Staphylococcus aureus have been reported. We have previously cloned a cluster of type 1 capsule (cap1) genes responsible for type 1 capsular polysaccharide biosynthesis in S. aureus M. To clone the type 8 capsule (cap8) genes, a plasmid library of type 8 strain Becker was screened with a labelled DNA fragment containing the cap1 genes under low-stringency conditions. One recombinant plasmid containing a 14-kb insert was chosen for further study and found to complement 14 of the 18 type 8 capsule-negative (Cap8-) mutants used in the study. Additional library screening, subcloning, and complementation experiments showed that all of the 18 Cap8- mutants were complemented by DNA fragments derived from a 20.5-kb contiguous region of the Becker chromosome. The mutants were mapped into six complementation groups, indicating that the cap8 genes are clustered. By Southern hybridization analyses under high-stringency conditions, we found that DNA fragments containing the cap8 gene cluster show extensive homology with all 17 strains tested, including type 1 strains. By further Southern analyses and cloning of the cap8-related homolog from strain M, we show that strain M carries an additional capsule gene cluster different from the cap1 gene cluster. In addition, by using DNA fragments containing different regions of the cap8 gene cluster as probes to hybridize DNA from different strains, we found that the central region of the cap8 gene cluster hybridizes only to DNAs from certain strains tested whereas the flanking regions hybridize to DNAs of all strains tested. Thus, the cap8 gene clusters and its closely related homologs are likely to have organizations similar to those of the encapsulation genes of other bacterial systems.

  13. Cloning and characterization of nanos gene in silkworm Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    Guoli Zhao; Keping Chen; Qin Yao; Weihua Wang

    2008-01-01

    Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Droso phila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5'-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mori and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5. 38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee (Apis mellifera). This gene has been registered in the GenBank under the accession number EF647589. One encoding se quence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.

  14. Cloning and analysis of the antagonistic related genes of Enterobacter cloacae B8

    Institute of Scientific and Technical Information of China (English)

    YU Xuping; ZHU Junli; YAO Xunping; HE Shicheng; HUANG Haining; CHEN Weiliang; LI Debao

    2004-01-01

    To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kanr gene on Tn5,an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF,from one of the mutant strains B8F. The 735 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM,admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid.

  15. Construction of a 7-fold BAC library and cytogenetic mapping of 10 genes in the giant panda (Ailuropoda melanoleuca

    Directory of Open Access Journals (Sweden)

    Zhang Ying

    2006-11-01

    Full Text Available Abstract Background The giant panda, one of the most primitive carnivores, is an endangered animal. Although it has been the subject of many interesting studies during recent years, little is known about its genome. In order to promote research on this genome, a bacterial artificial chromosome (BAC library of the giant panda was constructed in this study. Results This BAC library contains 198,844 clones with an average insert size of 108 kb, which represents approximately seven equivalents of the giant panda haploid genome. Screening the library with 15 genes and 8 microsatellite markers demonstrates that it is representative and has good genome coverage. Furthermore, ten BAC clones harbouring AGXT, GHR, FSHR, IRBP, SOX14, TTR, BDNF, NT-4, LH and ZFX1 were mapped to 8 pairs of giant panda chromosomes by fluorescence in situ hybridization (FISH. Conclusion This is the first large-insert genomic DNA library for the giant panda, and will contribute to understanding this endangered species in the areas of genome sequencing, physical mapping, gene cloning and comparative genomic studies. We also identified the physical locations of ten genes on their relative chromosomes by FISH, providing a preliminary framework for further development of a high resolution cytogenetic map of the giant panda.

  16. Differential cloning of novel intestine-specific genes whose expression is altered under conditions of villus atrophy.

    Science.gov (United States)

    Hodin, R A; Meng, S; Shei, A

    1995-07-01

    Atrophy of the small intestinal villi occurs in a variety of disease states and is associated with diarrhea, malabsorption, and impaired barrier function. We have previously demonstrated that villus atrophy is associated with an increase in lactase and a decrease in intestinal alkaline phosphatase gene expression. Given these changes in enterocyte phenotype with villus atrophy, we speculated that there may be other intestine-specific genes whose expression is altered as a function of epithelial growth state. We have employed two molecular techniques in order to identify and clone complementary DNAs (cDNA) which are differentially expressed in atrophic compared to normal small intestinal mucosa. In differential cDNA library (+/-) screening, duplicate filters of a normal jejunal cDNA library are hybridized with radiolabeled cDNA probes from either atrophic or control tissues. Comparisons of the intensities of hybridized clones allows for the identification of differentially expressed gene products. In the mRNA differential display system, RT-PCR is used to randomly amplify mRNA species. Similar to cDNA library screening, comparisons of radiolabeled bands on a polyacrylamide sequencing gel allow for the identification of differentially expressed genes. Using these methods, we have identified a novel cDNA, called D9, which appears to be expressed exclusively in the intestinal mucosa. Northern analyses have confirmed that the expression of the D9 mRNA is dramatically decreased under conditions of villus atrophy, suggesting an underlying relationship with epithelial growth state. DNA sequence analysis (GenBank) reveals no identity to previously cloned genes.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Cloning and Characterization of Gene Promoters from Bacillus pumilus

    Institute of Scientific and Technical Information of China (English)

    Pan Jiao(潘皎); Zhang Yizheng

    2004-01-01

    DNA fragments obtained from Sau3AI partially digested total DNA of Bacillus pumilus UN31-C-42 are first inserted into BamHI site of pSUPV4, a promoter-probe vector. The recombinant DNA molecules are transformed into Escherichia coli cells and eight-three Kanr clones (named pSUBp1- pSUBp83) are obtained. The inserted fragments in pSUBp53, pSUBp57, pSUBp21, which showed high level of kanamycin - resistance, are sequenced and analyzed, respectively. These fragments contain some conserved sequences of prokaryotic gene promoters, such as TATAAT and TTGACA box. The promoter fragment Bp53 could efficiently promote the alkaline protease gene of B.pumilus expression not only in E.coli but also in B.subtilis cells.

  18. Molecular Cloning of MSRG-11 Gene Related to Apoptosis of Mouse Spermatogenic Cells

    Institute of Scientific and Technical Information of China (English)

    Yun DENG; Dong-Song NIE; Jian WANG; Xiao-Jun TAN; Zhao-Yan NIE; Hong-Mei YANG; Liang-Sha HU; Guang-Xiu LU

    2005-01-01

    Beginning with a new contig of the expressed sequence tags (Mm.63892) obtained by comparing testis libraries with other tissue and cell line libraries using the digital differential display program,we cloned a new gene which is related to the apoptosis of mouse spermatogenic cells using the Genscan program and polymerase chain reaction (PCR) technology. The sequence data have been submitted to the GenBank database under accession number AY747687. The full cDNA length is 1074 bp, and the gene with7 exons and 6 introns is located in mouse chromosome 1 H5. The protein is recognized as a new member of calmodulin (CaM) binding protein family because the sequence contains three short calmodulin-binding motifs containing conserved Ile and Gln residues (IQ motif) and is considered to play a critical role in interactions of IQ motif-containing proteins with CaM proteins. The putative protein encoded by this gene has 192 amino acid residues with a theoretical molecular mass of 23.7 kDa and a calculated isoelectric point of 9.71. The sequence shares no significant homology with any known protein in databases. RT-PCR and Northern blot analyses revealed that 1.3 kb MSRG-11 transcript was strongly expressed in adult mouse testis but weakly expressed in the spleen and thymus. The MSRG-11 gene was expressed at various levels, faintly at two weeks postpartum and strongly from three weeks postpartum in adult testes. The green fluorescence produced by pEGFP-C2/MSRG-11 was detected in the cytoplasm of COS7 cells 24 h post-transfection. The pcDNA3. 1(-)/MSRG-11 plasmid was constructed and introduced into COS7 cells using Lipofectamine 2000transfection reagent (Invitrogen, Carlsbad, USA). MSRG-11 can accelerate COS7 cell apoptosis, which suggests that this gene may play an important role in the development of mouse testes and is a candidate gene of testis-specific apoptosis. Based on these observations, it was considered that we cloned a new gene which probably accelerates

  19. [Cloning and expression of Streptococcus salivarius urease gene in Escherichia coli].

    Science.gov (United States)

    Wang, Yan; Feng, Xi-ping; Xie, You-hua; Tao, Dan-ying; Luan, Xiao-ling

    2010-08-01

    To clone Streptococcus salivarius (Ss) 57. I urease gene, which can express ureolytic activity in Escherichia coli (Ec) without adding extra nickel ions. Urease gene was cloned by polymerase chain reaction in three separate parts. The three separate plasmids were digested by specific restriction enzymes and ligated together. The expression of the complete urease gene in Ec was detected by phenol red assay and pH analysis. Urease gene of Ss 57.I was eventually cloned and proved correct. Urease activity of the obtained clone was positive in Ec. Without adding extra NiCl(2), the recombinant Ec could hydrolyze urea to produce ammonia, resulting in the increase of pH value. The clone of Ss urease gene obtained in this study could express ureolytic activity in Ec without adding extra nickel ions. The current clone can be used to construct ureolytic effector strain used in replacement therapy in caries prevention.

  20. Insight into screening immunoglobulin gene combinatorial libraries in a phage display vector: a tale of two antibodies.

    Science.gov (United States)

    Kakinuma, A; Portolano, S; Chazenbalk, G; Rapoport, B; McLachlan, S M

    1997-01-01

    Combinatorial libraries of immunoglobulin genes in "phage display" vectors are a powerful tool for obtaining antigen-specific antibody fragments. To date, this approach has been used to isolate abundant, but not rare, human autoantibodies of IgG class. We have compared the relative efficiencies of panning pComb3 libraries made from intrathyroidal plasma cells for abundant human autoantibodies to thyroid peroxidase (TPO) and rare autoantibodies to the thyrotropin receptor (TSHR). TPO-specific Fab were readily obtained from a library using three different forms of recombinant antigen, (i) purified TPO, (ii) impure TPO in culture medium and, (iii) TPO expressed on the surface of CHO cells. In contrast, TSHR-specific Fab were not isolated. This was the case despite repeated pannings of six libraries from three optimal patients (IgG/kappa and IgG/lambda libraries for each patient). Both purified recombinant TSHR and CHO cells expressing TSHR on their surface were used. Library enrichment was observed on some screenings. However, Fab expressed by individual clones or from enriched libraries were not specific as determined by (i) binding to purified, radio-labeled antigen, (ii) FACS analysis of TSHR on intact CHO cells and, (iii) inhibition of radiolabeled TSH binding. Remarkably, in screening for both TPO- and TSHR-specific Fab, neither library enrichment nor the retention of cDNA inserts of the correct size correlated with obtaining Fab with the antigenic specificity sought. Indeed, excellent enrichment could be observed with conditioned medium from untransfected cells. Our data suggest that the key to isolating rare antibodies from phage display libraries is not the creation of vast libraries of greater diversity or even the development of more stable vectors. Rather, success in this endeavor appears to require reducing the "noise" of non-specific clones in a moderately sized library.

  1. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    WANG; Xin; (王鑫); WANG; Duncheng; (王敦成); CHEN; Xing; (陈兴),; HU; Meiru; (胡美茹); WANG; Jian'an; (王建安); LI; Yan; (黎燕); GUO; Ning; (郭宁); SHEN; Beifen; (沈倍奋)

    2001-01-01

    Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

  2. cDNA cloning and function analysis of two novel erythroid differentiation related genes

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Our previous studies showed that some nuclear proteins that wereexpressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

  3. Cloning and tissue expression characterization of the chicken APOB gene.

    Science.gov (United States)

    Zhang, Sen; Shi, Hui; Li, Hui

    2007-01-01

    Apolipoprotein B (APOB) serves an essential role in the assembly and secretion of triglyceride-rich lipoproteins and lipids transport. This study was designed to clone the full-length cDNA of the chicken APOB gene, to characterize the expression profile, and investigate the differential expression between layer and broiler of the chicken APOB gene. The full-length cDNA sequence (14,150-bp) that contained a 13,896-bp ORF encoding 4,631 amino acids was obtained by RT-PCR, RACE, and bioinformatics analysis. qReal-Time PCR analysis showed that the chicken APOB gene was highly expressed in kidney, liver, and intestine. The results of differential expression showed that the APOB gene was more highly expressed in intestine and kidney in Bai'er layer than in broiler, but there was no significant difference in liver between the two breeds. The results of this study provided basic molecular information for studying the role of APOB in the energy transportation in avian species.

  4. [Cloning and characterization of CMO gene from Atriplex hortensis].

    Science.gov (United States)

    Shen, Y G; Du, B X; Zhang, J S; Chen, S Y

    2001-01-01

    Glycine betaine is a widespread osmopretectant existed in many organisms. In higher plant, glycine betaine is synthesized via a two-sep oxidation reaction: choline-->betaine aldehyde-->glycine betaine. The first step, also the speed-limiting step, is catalyzed by choline monooxygenase(CMO). Choosing halophyte Atriplex hortensis as material, we constructed a salt stress-induced cDNA library, and isolated a 1.77 kb length cDNA clone with spinach CMO cDNA as probe. The sequencing result showed a complete Open Reading Frame encoding a 438-amino-acid polypeptide which was 81% and 72% identified to CMO sequences of spinach and sugar beet in amino acid homology respectively. Compared with the CMO from spinach and sugar beet, the AhCMO had one conserved Rieske-Type [2Fe-2S] cluster-binding region and one conserved mononuclear Fe-binding motif. The expression pattern of AhCMO under salt stress was also stuied, the transcriptional level of AhCMO raised about three folds after the plant was treated with brine for 4 days. The AhCMO was then transfered into tobacco(Nictiana tabacum var. Xanthi) with 35S promotor and seven transgenic plants were certified by northern blot, these plants displayed some salt- and drought-stress tolerance when grew well on MS medium contained 1.2% NaCl or 10% PEG while the control was stagnated under the same cndition.

  5. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene ...

  6. Detection and isolation of selected genes of interest from metagenomic libraries by a DNA microarray approach.

    Science.gov (United States)

    Pathak, Gopal P; Gärtner, Wolfgang

    2010-01-01

    A DNA microarray-based approach is described for screening metagenomic libraries for the presence of selected genes. The protocol is exemplified for the identification of flavin-binding, blue-light-sensitive biological photoreceptors (BL), based on a homology search in already sequenced, annotated genomes. The microarray carried 149 different 54-mer oligonucleotides, derived from consensus sequences of BL photoreceptors. The array could readily identify targets carrying 4% sequence mismatch, and allowed unambiguous identification of a positive cosmid clone of as little as 10 ng against a background of 25 μg of cosmid DNA. The protocol allows screening up to 1,200 library clones in concentrations as low as ca. 20 ng, each with a ca. 40 kb insert size readily in a single batch. Calibration and control conditions are outlined. This protocol, when applied to the thermophilic fraction of a soil sample, yielded the identification and functional characterization of a novel, BL-encoding gene that showed a 58% similarity to a known, BL-encoding gene from Kineococcus radiotolerans SRS30216 (similarity values refer to the respective LOV domains).

  7. Screening of a xylanase clone from a fosmid library of rumen microbiota in Hu sheep.

    Science.gov (United States)

    Wang, Jiakun; Sun, Zhongyuan; Zhou, Yan; Wang, Qian; Ye, Jun'an; Chen, Zhenming; Liu, Jianxin

    2012-01-01

    The glycosyl hydrolase family 11, which is responsible for carbohydrate metabolism, was identified in the open reading frame (ORF) 6 of a xylanase positive clone from a fosmid library of rumen microbiota of Hu sheep. A BLASTP search of GenBank revealed that ORF6 encoded a 355-amino acid putative endoxylanase, having 61% similarity (e(-73)) to endo-1,4-β-xylanase of Fibrobacter succinogenes S85 (YP_003250510.1). Predicted with the SWISS-MODEL, there were two separate β-sandwich clusters linked with a high serine containing linker in ORF6. The N-terminal β-sandwich is a novel endoxylanase of the glycosyl hydrolase family 11 with a specific activity of 1150.00 U/mg. The optimal pH and temperature for this enzyme were shown to be pH 5.0 and 50°C, respectively. The C-terminal helped increase the stability of the xylanase but decreased the activity to some degree. The C-terminal β-sandwich could bind avicel, but no conserved domain could be found. It may be a novel carbohydrate-binding module.

  8. Morquio A syndrome: Cloning, sequence, and structure of the human N-acetylgalactosamine 6-sulfatase (GALNS) gene

    Energy Technology Data Exchange (ETDEWEB)

    Morris, C.P.; Guo, Xiao-Hui; Apostolou, S. [Adelaide Children`s Hospital, North Adelaide (Australia)] [and others

    1994-08-01

    Deficiency of the lysosomal enzyme, N-acetylgalactosamine 6-sulfatase (GALNS;EC 3.1.6.4), results in the storage of the glycosaminoglycans, keratan sulfate and chrondroitin 6-sulfate, which leads to the lysosomal storage disorder Morquio A syndrome. Four overlapping genomic clones derived from a chromosome 16-specific gridded cosmid library containing the entire GALNS gene were isolated. The structure of the gene and the sequence of the exon/intron boundaries and the 5{prime} promoter region were determined. The GALNS gene is split into 14 exons spanning approximately 40 kb. The potential promoter for GALNS lacks a TATA box but contains GC box consensus sequences, consistent with its role as a housekeeping gene. The GALNS gene contains an Alu repeat in intron 5 and a VNTR-like sequence in intron 6. 12 refs., 3 figs., 1 tab.

  9. Cloning and molecular evolution research of porcine GAD65 gene

    Institute of Scientific and Technical Information of China (English)

    YU Hao; SONG Yuefen; LI Li; LIU Di

    2007-01-01

    Glutamate decarboxylase (GAD) has been found in animal and higher plant tissues as well as in yeasts and microorganisms.In animals the enzyme plays an important role in central nervous system activity because the enzyme substrate glutamic acid is a mediator of excitation process and the product, gamma-aminobutyric acid, is the most important mediator of inhibition process in the central nervous system. GAD65 is one form of the glutamate decarboxylases (GAD), GAD65 has been identified as a major autoantigen in type 1 diabetes, so the GAD65 gene of porcine was cloned by RT-PCR method to construct phylogenetic tree, the homology of 13glutamate decarboxylases (GAD) of different origin was analyzed by multiple alignment.

  10. Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L.

    Indian Academy of Sciences (India)

    Meng-Jun Li; Ai-Qin Li; Han Xia; Chuan-Zhi Zhao; Chang-Sheng Li; Shu-Bo Wan; Yu-Ping Bi; Xing-Jun Wang

    2009-06-01

    The cultivated peanut is a valuable source of dietary oil and ranks fifth among the world oil crops. Plant fatty acid biosynthesis is catalysed by type II fatty acid synthase (FAS) in plastids and mitochondria. By constructing a full-length cDNA library derived from immature peanut seeds and homology-based cloning, candidate genes of acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, -ketoacyl-ACP synthase (I, II, III), -ketoacyl-ACP reductase, -hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were isolated. Sequence alignments revealed that primary structures of type II FAS enzymes were highly conserved in higher plants and the catalytic residues were strictly conserved in Escherichia coli and higher plants. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 1 in KASII, KASIII and HD to 5 in ENR. The number of single-nucleotide polymorphisms (SNPs) was quite different in each gene. Peanut type II FAS genes were predicted to target plastids except ACP2 and ACP3. The results suggested that peanut may contain two type II FAS systems in plastids and mitochondria. The type II FAS enzymes in higher plants may have similar functions as those in E. coli.

  11. Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L.

    Science.gov (United States)

    Li, Meng-Jun; Li, Ai-Qin; Xia, Han; Zhao, Chuan-Zhi; Li, Chang-Sheng; Wan, Shu-Bo; Bi, Yu-Ping; Wang, Xing-Jun

    2009-06-01

    The cultivated peanut is a valuable source of dietary oil and ranks fifth among the world oil crops. Plant fatty acid biosynthesis is catalysed by type II fatty acid synthase (FAS) in plastids and mitochondria. By constructing a full-length cDNA library derived from immature peanut seeds and homology-based cloning, candidate genes of acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, beta-ketoacyl-ACP synthase (I, II, III), beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were isolated. Sequence alignments revealed that primary structures of type II FAS enzymes were highly conserved in higher plants and the catalytic residues were strictly conserved in Escherichia coli and higher plants. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 1 in KASII, KASIII and HD to 5 in ENR. The number of single-nucleotide polymorphisms (SNPs) was quite different in each gene. Peanut type II FAS genes were predicted to target plastids except ACP2 and ACP3. The results suggested that peanut may contain two type II FAS systems in plastids and mitochondria. The type II FAS enzymes in higher plants may have similar functions as those in E. coli.

  12. Cloning, expression, and purification of a new antimicrobial peptide gene from Musca domestica larva.

    Science.gov (United States)

    Pei, Zhihua; Sun, Xiaoning; Tang, Yan; Wang, Kai; Gao, Yunhang; Ma, Hongxia

    2014-10-01

    Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram- bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.

  13. Cloning and identification of measles virus receptor gene from marmoset cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The measles virus (MV) strains with mutated hemagglutinin gene (ha) lost the capacity to infect its sensitive host cells (Vero cells), but it may infect the marmoset B-lymphoblastoid cell line B95a. From above, we can presume that there is a novel cellular receptor for those measles virus strains on B95a cell s. Using the yeast two-hybrid system, we screened and cloned a novel gene--bip (B-lympho- blastoid interaction protein of marmoset) from B95a cell cDNA library, which encoded a protein interacting with measles virus hemagglutinin protein (Ha). The bip cDNA was 1540 base pairs in length and contained a unique open rea ding frame (ORF) of 1011 base pairs encoding a transmembrane protein of 337 amino acid residues. The primary structure of amino acids residue is predicted that the Bip comprised a hydrophobic transmembrane domain and a hydrophobic leader region. The researches about the deletion mutants showed that the deletion of tran smembrane domain in Bip did not affect the interaction between Bip and Ha protei ns. Expression of bip in measles virus non-permissive cell line--CHO (Chinese hamster ovary) cells was performed to prove that CHO/Bip can be infected by meas les virus and then turned to the MV permissive cells. We concluded that the bip gene is a novel measles virus receptor gene in marmoset B-lymphoblastoid cells.

  14. Cloning and identification of NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A

    Institute of Scientific and Technical Information of China (English)

    Qian Yang; Jun Cheng; Yan Liu; Yuan Hong; Jian-Jun Wang; Shu-Lin Zhang

    2004-01-01

    AIM: To clone, identify and study new NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus nonstructural protein 5A.METHODS: On the basis of subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus, the coding sequence of new gene and its spliced variant were obtained by bioinformatics method. Polymerase chain reaction (PCR)was conducted to amplify NS5ATP2 gene.RESUJLTS: The coding sequence of a new gene and its spliced variant were cloned and identified successfully.CONCLUSION: A new gene has been recognized as the new target transactivated by HCV NS5A protein. These results brought some new clues for studying the biological functions of new genes and pathogenesis of the viral proteins.

  15. Cloning and Functional Characterization of SAD Genes in Potato

    Science.gov (United States)

    Li, Fei; Bian, Chun Song; Xu, Jian Fei; Pang, Wan fu; Liu, Jie; Duan, Shao Guang; Lei, Zun-Guo; Jiwan, Palta; Jin, Li-Ping

    2015-01-01

    Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato. PMID:25825911

  16. Cloning and Expression Profiles of Myf5 Gene of Yak

    Directory of Open Access Journals (Sweden)

    Yaqiu Lin

    2015-01-01

    Full Text Available To reveal the sequence characteristic and expression pattern of Myf5 gene in Jiulong yaks (Bos grunniens, a full-length cDNA of Myf5 was cloned from yak muscle tisssue by RT-PCR. The cDNA obtained was 821bp nucleotide (nt long with an ORF of 768 bp which encoding 255 amino acids. Compared with cattle, sheep, pig, horse, human, pygmy chimpanzee, mouse, rat and dog, the homology of amino acid sequences were higher (89-9%, but lower in Zebrafish (60%. SQ RT-PCR analysis showed that Myf5 gene expression was observed only in longissimus muscle, but not be detected in heart, liver, kidney, spleen and adipose tissues. The expression level of Myf5 gene in longissium muscle of 0.5 and over 9 years old yaks was significantly higher than those of 3.5-5.5 years old yaks (p<0.05. These results suggest that Myf5 may play an important role in the regulation of muscle growth and development of yak.

  17. Cloning the human gene for macrophage migration inhibitory factor (MIF)

    Energy Technology Data Exchange (ETDEWEB)

    Paralkar, V.; Wistow, G. (National Institutes of Health, Bethesda, MD (United States))

    1994-01-01

    Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine. However, recent work strongly suggests a wider role for MIF beyond the immune system. It is expressed specifically in the differentiating cells of the immunologically privileged eye lens and brain, is a delayed early response gene in fibroblasts, and is expressed in many tissues. Here, the authors report the structure of the remarkably small gene for human MIF that has three exons separated by introns of only 189 and 95 bp and covers less than 1 kb. The cloned sequence also includes 1 kb of 5[prime] flanking region. Primer extension and 5[prime] rapid amplification of cDNA ends (RACE) of human brain RNA both indicate the presence of a single transcription start site in a TATA-less promoter. Northern blot analysis shows a single size of MIF mRNA (about 800 nt) in all human tissues examined. In contrast to previous reports, they find no evidence for multiple genes for MIF in the human genome. 20 refs., 3 figs.

  18. Clone DB: an integrated NCBI resource for clone-associated data.

    Science.gov (United States)

    Schneider, Valerie A; Chen, Hsiu-Chuan; Clausen, Cliff; Meric, Peter A; Zhou, Zhigang; Bouk, Nathan; Husain, Nora; Maglott, Donna R; Church, Deanna M

    2013-01-01

    The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents.

  19. A highly efficient molecular cloning platform that utilises a small bacterial toxin gene.

    Science.gov (United States)

    Mok, Wendy W K; Li, Yingfu

    2013-04-15

    Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective.

  20. Partial Cloning and Nucleotide Sequencing of Glutamate Decarboxylase Gene Isoform 65 from Human Brain

    Directory of Open Access Journals (Sweden)

    Abolghasem Esmaeili

    2015-04-01

    Conclusion: Because obtaining fresh human brain is difficult and amount of mRNA is low, it may not be easy to clone full length of human gad gene. The approach described in this paper may be useful in cloning of other genes for which the corresponding mRNA is present at low levels.

  1. Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

    DEFF Research Database (Denmark)

    Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael

    2011-01-01

    of genes of interest in small genomics projects. The BAC end sequences described in this paper have been deposited in the GenBank data library [HN339419-HN341884, HN604664-HN604702]. The 454 produced contigs derived from selected clones are deposited with reference numbers [GenBank: JF288166-JF288183 &JF......BACKGROUND: Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. RESULTS: Here, we...

  2. Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

    Directory of Open Access Journals (Sweden)

    Bharti Arvind K

    2008-12-01

    Full Text Available Abstract Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR and methylation spanning linker libraries (MSLL. These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig, while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%. These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of

  3. Cloning, Sequencing and Characterization of 3-Hydroxybuty- rate Dehydrogenase Encoding Gene (bdh A) in Bradyrhizobium japonicum USDA110 Strain

    Institute of Scientific and Technical Information of China (English)

    DAI Mei-xue; WU Bo; BAI Xue-liang; ZHANG Cheng-gang; MA Qing-sheng; Charles Trevor C

    2002-01-01

    The current study describes the molecular characterization of a clone which can restore the ability of bdhA mutant strains NGRPA2 and Rm11107 to utilize 3-hydroxybutyrate as a sole carbon source (Hbu+). This clone was screened out by complementation experiment from Bradyrhizobium japonicum USDA110 genomic library, and the presence of bdhA gene in the clone was verified by Bdh assay and Southern blot analysis. Furthermore, the entire sequence of bdhA gene was sequenced and the sequence was deposited in GenBank database under the accession number AY077581. bdhA gene comprises 789 base pairs and encodes Bdh with 262 amino acid of MW 27.59 kDa. Interposon ΩKm was inserted into the bdh A ORF at EcoR I site and the bdhA mutant was constructed in B .japonicum by homologous recombination. Plant assay result did not show obvious effects of mutation of bdhA gene on nodulation and nitrogen-fixation.

  4. Location and cloning of the herpes simplex virus type 2 thymidine kinase gene.

    OpenAIRE

    McDougall, J K; Masse, T H; Galloway, D A

    1980-01-01

    The herpes simplex virus type 2 thymidine kinase gene has been mapped to a position colinear with the herpes simplex virus type 1 thymidine kinase gene and cloned within a 4.0-kilobase fragment in pBR 322.

  5. Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

    Institute of Scientific and Technical Information of China (English)

    Li Liang; Yan-Qing Ding; Xin Li; Guang-Zhi Yang; Jun Xiao; Li-Chun Lu; Jin-Hua Zhang

    2004-01-01

    AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization.METHODS: Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH)was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the bluewhite screening system to establish cDNA library.RESULTS: After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively.CONCLUSIONS: A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.

  6. Systematic mining of salt-tolerant genes in halophyte-Zoysia matrella through cDNA expression library screening.

    Science.gov (United States)

    Chen, Yu; Zong, Junqin; Tan, Zhiqun; Li, Lanlan; Hu, Baoyun; Chen, Chuanming; Chen, Jingbo; Liu, Jianxiu

    2015-04-01

    Though a large number of salt-tolerant genes were identified from Glycophyte in previous study, genes involved in salt-tolerance of halophyte were scarcely studied. In this report, an important halophyte turfgrass, Zoysia matrella, was used for systematic excavation of salt-tolerant genes using full-length cDNA expression library in yeast. Adopting the Gateway-compatible vector system, a high quality entry library was constructed, containing 3 × 10(6) clones with an average inserted fragments length of 1.64 kb representing a 100% full-length rate. The yeast expression library was screened in a salt-sensitive yeast mutant. The screening yielded dozens of salt-tolerant clones harboring 16 candidate salt-tolerant genes. Under salt-stress condition, these 16 genes exhibited different transcription levels. According to the results, we concluded that the salt-tolerance of Z. matrella might result from known genes involved in ion regulation, osmotic adjustment, as well as unknown pathway associated with protein folding and modification, RNA metabolism, and mitochondrial membrane translocase, etc. In addition, these results shall provide new insight for the future researches with respect to salt-tolerance.

  7. Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

    Directory of Open Access Journals (Sweden)

    Sakaki Yoshiyuki

    2007-12-01

    Full Text Available Abstract Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs. Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome.

  8. Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

    Science.gov (United States)

    Chaudhary, Vijay K.; Das, Shilpi; Kaur, Charanpreet; Grover, Payal; Gupta, Amita

    2014-01-01

    Herein, we describe a novel cloning strategy for PCR-amplified DNA which employs the type IIs restriction endonuclease BsaI to create a linearized vector with four base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in presence of a single dNTP to create vector-compatible four base-long overhangs. Notably, the insert preparation does not require any restriction enzyme treatment. The BsaI sites in the vector are oriented in such a manner that upon digestion with BsaI, a stuffer sequence along with both BsaI recognition sequences is removed. The sequence of the four base-long overhangs produced by BsaI cleavage were designed to be non-palindromic, non-compatible to each other. Therefore, only ligation of an insert carrying compatible ends allows directional cloning of the insert to the vector to generate a recombinant without recreating the BsaI sites. We also developed rapid protocols for insert preparation and cloning, by which the entire process from PCR to transformation can be completed in 6–8 h and DNA fragments ranging in size from 200 to 2200 bp can be cloned with equal efficiencies. One protocol uses a single tube for insert preparation if amplification is performed using polymerases with low 3′-exonuclease activity. The other protocol is compatible with any thermostable polymerase, including those with high 3′-exonuclease activity, and does not significantly increase the time required for cloning. The suitability of this method for high-throughput cloning was demonstrated by cloning batches of 24 PCR products with nearly 100% efficiency. The cloning strategy is also suitable for high efficiency cloning and was used to construct large libraries comprising more than 108 clones/µg vector. Additionally, based on this strategy, a variety of vectors were constructed for the expression of proteins in E. coli, enabling large number of different clones to be rapidly generated. PMID:25360695

  9. Cloning and Expression Characteristics of the Pig Stra8 Gene

    Directory of Open Access Journals (Sweden)

    Xiaoyan Wang

    2014-07-01

    Full Text Available Stra8 (Stimulated by Retinoic Acid 8 is considered a meiotic gatekeeper gene. Using reverse transcriptase PCR and rapid amplification of cDNA ends (RACE, the complete sequence of the pig Stra8 gene was cloned. Bioinformatics analyses of this sequence were performed. Using semi-quantitative methods, the expression characteristics of Stra8 in Testis, cauda epididymis, body epididymis, caput epididymis, seminal vesicles, prostate gland, Cowper’s gland, heart, liver, spleen, lung, kidney, stomach, hypothalamus, pituitary gland, cerebrum, cerebellum, and hippocampus of adult Meishan boar and sow tissues were examined. The expression pattern in the testis of 2-, 30-, 60-, 90-, and 150-day old Meishan boars were analyzed using real-time PCR. We constructed a eukaryotic expression vector for the Stra8 gene and used it to transfect NIH-3T3 cells and third generation pig spermatogonial stem cells (SSCs cultured in vitro. Testes weight and sperm count in the cauda epididymis were evaluated at various time points. The results showed that the length of the pig Stra8 gene cDNA was 1444 bp encoding 366 amino acids with one typical helix-loop-helix (HLH domain. It is testes-specific expression. Expression was first detected in boar testis starting at day 2, and its expression significantly (p < 0.05 increased with age and body weight. When NIH-3T3 cells and pig SSCs were transfected with the eukaryotic expression vector EGFP (enhanced green fluorescent protein-N1-pStra8, it was expressed in the cytoplasm of NIH-3T3 cells. However, in SSCs, Stra8 was expressed predominantly in cytoplasm and few in nucleus. Our data suggest that perhaps Stra8 acts as a transcription factor to initiate meiosis in young boar.

  10. Cloning of two genes encoding Rab7 in Paramecium.

    Science.gov (United States)

    Surmacz, Liliana; Wiejak, Jolanta; Wyroba, Elzbieta

    2006-01-01

    Rab7 is a small GTPase that plays a crucial role in the regulation of transport from early to late endosomes and lysosomes, phagosome maturation and in lysosomal biogenesis in mammalian cells. It contains conserved and unique sequence elements that mediate its function. Two Rab7 genes, Rab7a (703 bp) and Rab7b (707 bp) were identified in the unicellular eukaryote Paramecium by PCR amplification. They contain three short introns of different lengths (28-32 bp) and sequence located at identical positions in both genes. The presence of two Rab7 genes in the Paramecium genome was confirmed by Southern hybridization analysis performed with six different restriction enzymes. Expression of both genes was assessed by Northern blot and RT-PCR. Two transcripts of 1.8 and 2.2 kb were identified by hybridization analysis. The cloned complementary DNAs, both of 618 nucleotides in length, encode polypeptides of 206 amino acids that are 97.6% identical and differ in their C-termini. The predicted protein sequences of Rab7a and Rab7b contain all characteristic domains essential for Rab function: the effector domain (YRATVGADF) and four GTP-binding consensus sequences (GDSGVGKT, WDTAGQ, NKLD, SAK) as well as the prenylation motif (-CC) at the C-terminus indispensable for Rab binding to the membrane. Similarity searches revealed 81.6-82.1% homology of Paramecium Rab7 isoforms to human Rab7 and a lack of an insert typical for the Kinetoplastida - the species that appeared earlier in evolution. Paramecium is the first free-living lower eukaryote in which homologues of Rab7 have been identified that exhibit features similar to those of mammalian Rab7.

  11. Molecular cloning and analysis of the Catsper1 gene promoter.

    Science.gov (United States)

    Mata-Rocha, Minerva; Alvarado-Cuevas, Edith; Hernández-Sánchez, Javier; Cerecedo, Doris; Felix, Ricardo; Hernández-Reyes, Adriana; Tesoro-Cruz, Emiliano; Oviedo, Norma

    2013-05-01

    CatSper channels are essential for hyperactivity of sperm flagellum, progesterone-mediated chemotaxis and oocyte fertilization. Catsper genes are exclusively expressed in the testis during spermatogenesis, but the function and regulation of the corresponding promoter regions are unknown. Here, we report the cloning and characterization of the promoter regions in the human and murine Catsper1 genes. These promoter regions were identified and isolated from genomic DNA, and transcriptional activities were tested in vitro after transfection into human embryonic kidney 293, mouse Sertoli cells 1 and GC-1spg cell lines as well as by injecting plasmids directly into mouse testes. Although the human and murine Catsper1 promoters lacked a TATA box, a well-conserved CRE site was identified. Both sequences may be considered as TATAless promoters because their transcriptional activity was not affected after deletion of TATA box-like sites. Several transcription initiation sites were revealed by RNA ligase-mediated rapid amplification of the cDNA 5'-ends. We also found that the immediate upstream region and the first exon in the human CATSPER1 gene negatively regulate transcriptional activity. In the murine Catsper1 promoter, binding sites for transcription factors SRY, SOX9 and CREB were protected by the presence of nuclear testis proteins in DNAse degradation assays. Likewise, the mouse Catsper1 promoter exhibited transcriptional activity in both orientations and displayed significant expression levels in mouse testis in vivo, whereas the suppression of transcription signals in the promoter resulted in low expression levels. This study, thus, represents the first identification of the transcriptional control regions in the genes encoding the human and murine CatSper channels.

  12. Prevalence of Adhesion and Regulation of Biofilm-Related Genes in Different Clones of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Salman Sahab Atshan

    2012-01-01

    Full Text Available Clinical information about genotypically different clones of biofilm-producing Staphylococcus aureus is largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs in biofilm formation. The study used 60 different types of spa and determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA, a microtiter plate assay (MPA, polymerase chain reaction (PCR, and reverse transcriptase polymerase chain reaction (RT-PCR. Clones belonging to the same spa type were found to have similar properties in adheringto thepolystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes. icaADBC genes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM and icaADBC was confirmed by RT-PCR.

  13. Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries.

    Science.gov (United States)

    Bouhajja, E; McGuire, M; Liles, M R; Bataille, G; Agathos, S N; George, I F

    2017-01-01

    The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic approach. High molecular weight environmental DNA from contaminated sediments was extracted, purified, and cloned into fosmid and BAC vectors and transformed into Escherichia coli. The fosmid library was screened by hybridization with a PCR amplicon of the α-subunit of the toluene 4-monooxygenase gene to identify genes and pathways encoding toluene degradation. Fourteen clones were recovered from the fosmid library, among which 13 were highly divergent from known tmoA genes and several had the closest relatives among Acinetobacter species. The BAC library was transferred to the heterologous hosts Cupriavidus metallidurans (phylum Proteobacteria) and Edaphobacter aggregans (phylum Acidobacteria). The resulting libraries were screened for expression of toluene degradation in the non-degradative hosts. From expression in C. metallidurans, three novel toluene monooxygenase-encoding operons were identified that were located on IncP1 plasmids. The E. aggregans-hosted BAC library led to the isolation of a cloned genetic locus putatively derived from an Acidobacteria taxon that contained genes involved in aerobic and anaerobic toluene degradation. These data suggest the important role of plasmids in the spread of toluene degradative capacity and indicate putative novel tmoA genes present in this hydrocarbon-polluted environment.

  14. [Cloning and prokaryotic expression of transcriptional co-activator gene of Clonorchis sinensis and functional analysis of the expressed protein].

    Science.gov (United States)

    Zhang, Yong-li; Yu, Xin-bing; Wu, De; Wu, Zhong-dao; Bi, Hui-xiang

    2005-02-28

    To construct prokaryotic recombinant plasmids of transcriptional co-activator (TC) gene of Clonorchis sinensis, express and purify the recombinant protein and analyze its biological function. A pair of primers was designed according to the known sequence of TC gene. The TC gene fragment was amplified by PCR. After purification and digestion with BamH I and Sal I, the TC gene was connected to the prokaryotic expression vectors, pGEX-4T-1 and pET30a(+). By cloning target gene into these vectors, pGEX-4T-1 and pET30a(+), prokaryotic recombinant plasmids of TC gene were constructed and transferred into E. coli BL21. The positive expressed recombinants were detected by SDS-PAGE and Western blotting. Immobilized metal (Ni2+) chelation affinity chromatography was used to purify His-TC produced by the expression of the recombinant protein pET30a(+)-TC. The recombinant plasmids, pGEX-4T-1-TC and pET30a(+)-TC, were constructed successfully. SDS-PAGE testified that the molecular weight of the recombinant protein was correct. Western blot analysis of GST-TC recombinant protein testified that the recombinant protein could be recognized by immunized rabbit serum, which means the protein is GST-immune active and the clone can express recombinant Clonorchis sinensis antigen. After affinity chromatography of the pET-TC protein, there was only one protein band with expected size on the SDS-PAGE gel. The TC gene was screened from cDNA library of adult Clonorchis sinensis, cloned, expressed and purified. The purified protein of TC gene will be of importance for further research on the biological function of the gene.

  15. Cloning, expression and characterization of phycoerythrin gene from Ceramium boydenn.

    Science.gov (United States)

    Zhang, Xiaowen; Zhao, Fangqing; Qin, Song; Yan, Binlun

    2006-04-01

    Phycobiliproteins function as a major light harvesting protein-pigment complex in the cyanobacteria and the eukaryotic algae. Phycoerythrin (PE) is a kind of phycobiliproteins, widely located in all rhodophytes, some species of cyanobacteria and cryptophytes, and different ecotypes of Prochlorococcus populations. PeBA encoding beta and alpha subunits of PE from Ceramium boydenn was cloned and sequenced in this research. A peBA specific PCR primer was synthesized, based on the peBA gene conserved sequences. The beta subunit encoding gene (peB) contained an open reading frame of 534 bp, while the alpha subunit (peA) was 495 bp. Recombinant expression plasmid pET-peAB was constructed and expressed in Escherichia coli BL21. The molecular weight of expressive product of peB and peA was about 23.3 and 18.2 KD, respectively. Results of codon usage analysis show that G + C content is heterogeneous among different groups of PE and spacers have dramatically lower G + C contents than coding regions. Also there is a high variance in G + C content among sequences at the third position sites. It is also found in this paper that several sequence regions, which might reflect functional or structural requirements of the PE organization, and several residues known for their functional importance are conserved in almost all the sequences.

  16. Cloning and Expression of Osteonectin Gene from Rats

    Institute of Scientific and Technical Information of China (English)

    ZHOU Lingde; YUAN Lin; YAN Yuhua; LI Shipu

    2006-01-01

    Total cellular RNA was extracted from the osteoblast cells of newborn rats' calvarial bones, and the cDNA containing open-reading frame of osteonectin was amplified by reverse transcription polymerase chain reaction (RT-PCR). The obtained product was named On. The On fragment was inserted into pBT-T vector. Then, the On was subcloned, in-frame fused to 3'-end of the GST gene of the prokaryotic expression vector pGEX-KG, and the resulting recombinant plasmid was transformed into E. coli BL21 (DE3) pLysS competent cells. A 60 kD fusion protein was expressed after IPTG induction. The On fragment was sequenced, and the sequencing result shows that it shares 99.8% homology with the sequence published in GenBank. The On SDS-PAGE analysis exhibits that the On was expressed with the GST gene. There is 10% fused protein in the total E.coli proteins, and the fusion protein is a soluble protein. These experimental results imply that On from Wistar rats was cloned successfully and expressed efficiently.

  17. Cloning and sequence analysis of the Antheraea pernyi nucleopolyhedrovirus gp64 gene

    Indian Academy of Sciences (India)

    Wenbing Wang; Shanying Zhu; Liqun Wang; Feng Yu; Weide Shen

    2005-12-01

    Frequent outbreaks of the purulence disease of Chinese oak silkworm are reported in Middle and Northeast China. The disease is produced by the pathogen Antheraea pernyi nucleopolyhedrovirus (AnpeNPV). To obtain molecular information of the virus, the polyhedra of AnpeNPV were purified and characterized. The genomic DNA of AnpeNPV was extracted and digested with HindIII. The genome size of AnpeNPV is estimated at 128 kb. Based on the analysis of DNA fragments digested with HindIII, 23 fragments were bigger than 564 bp. A genomic library was generated using HindIII and the positive clones were sequenced and analysed. The gp64 gene, encoding the baculovirus envelope protein GP64, was found in an insert. The nucleotide sequence analysis indicated that the AnpeNPV gp64 gene consists of a 1530 nucleotide open reading frame (ORF), encoding a protein of 509 amino acids. Of the eight gp64 homologues, the AnpeNPV gp64 ORF shared the most sequence similarity with the gp64 gene of Anticarsia gemmatalis NPV, but not Bombyx mori NPV. The upstream region of the AnpeNPV gp64 ORF encoded the conserved transcriptional elements for early and late stage of the viral infection cycle. These results indicated that AnpeNPV belongs to group I NPV and was far removed in molecular phylogeny from the BmNPV.

  18. A new approach for molecular cloning in cyanobacteria: cloning of an anacystis nidulans met gene using a Tn 907-induced mutant

    NARCIS (Netherlands)

    Tandeau de Marsac, N.; Borrias, W.E.; Kuhlemeijer, C.J.; Castets, A.M.; Arkel, G.A. van; Hondel, C.A.M.J.J. van den

    A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn 901 mutant was isolated. Chromosomal DNA fragments were cloned in the

  19. A new approach for molecular cloning in cyanobacteria: cloning of an anacystis nidulans met gene using a Tn 907-induced mutant

    NARCIS (Netherlands)

    Tandeau de Marsac, N.; Borrias, W.E.; Kuhlemeijer, C.J.; Castets, A.M.; Arkel, G.A. van; Hondel, C.A.M.J.J. van den

    1982-01-01

    A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn 901 mutant was isolated. Chromosomal DNA fragments were cloned in the

  20. Identification of novel lipolytic genes and gene families by screening of metagenomic libraries derived from soil samples of the German Biodiversity Exploratories.

    Science.gov (United States)

    Nacke, Heiko; Will, Christiane; Herzog, Sarah; Nowka, Boris; Engelhaupt, Martin; Daniel, Rolf

    2011-10-01

    Microbial metagenomes derived from soils are rich sources for the discovery of novel genes and biocatalysts. Fourteen environmental plasmid and seven fosmid libraries obtained from 10 German forest soils (A horizons) and six grassland soils (A and B horizons) were screened for genes conferring lipolytic activity. The libraries comprised approximately 29.3 Gb of cloned soil DNA. Partial activity-based screening of the constructed libraries resulted in the identification of 37 unique lipolytic clones. The amino acid sequences of the 37 corresponding lipolytic gene products shared 29-90% identity to other lipolytic enzymes, which were mainly uncharacterized or derived from uncultured microorganisms. Multiple sequence alignments and phylogenetic tree analysis revealed that 35 of the predicted proteins were new members of known families of lipolytic enzymes. The remaining two gene products represent two putatively new families. In addition, sequence analysis indicated that two genes encode true lipases, whereas the other genes encode esterases. The determination of substrate specificity and chain-length selectivity using different triacylglycerides and p-nitrophenyl esters of fatty acids as substrates supported the classification of the esterases.

  1. Whitefly (Bemisia tabaci genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous cDNA libraries

    Directory of Open Access Journals (Sweden)

    Czosnek Henryk

    2006-04-01

    Full Text Available Abstract Background The past three decades have witnessed a dramatic increase in interest in the whitefly Bemisia tabaci, owing to its nature as a taxonomically cryptic species, the damage it causes to a large number of herbaceous plants because of its specialized feeding in the phloem, and to its ability to serve as a vector of plant viruses. Among the most important plant viruses to be transmitted by B. tabaci are those in the genus Begomovirus (family, Geminiviridae. Surprisingly, little is known about the genome of this whitefly. The haploid genome size for male B. tabaci has been estimated to be approximately one billion bp by flow cytometry analysis, about five times the size of the fruitfly Drosophila melanogaster. The genes involved in whitefly development, in host range plasticity, and in begomovirus vector specificity and competency, are unknown. Results To address this general shortage of genomic sequence information, we have constructed three cDNA libraries from non-viruliferous whiteflies (eggs, immature instars, and adults and two from adult insects that fed on tomato plants infected by two geminiviruses: Tomato yellow leaf curl virus (TYLCV and Tomato mottle virus (ToMoV. In total, the sequence of 18,976 clones was determined. After quality control, and removal of 5,542 clones of mitochondrial origin 9,110 sequences remained which included 3,843 singletons and 1,017 contigs. Comparisons with public databases indicated that the libraries contained genes involved in cellular and developmental processes. In addition, approximately 1,000 bases aligned with the genome of the B. tabaci endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from the egg and instar libraries. Apart from the mitochondrial sequences, the longest and most abundant sequence encodes vitellogenin, which originated from whitefly adult libraries, indicating that much of the gene expression in this insect is directed toward the production

  2. Cloning,sequencing and function of sanA,a gene involved in nikkomycin biosynthesis of Streptomyces ansochromogenes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Several genetically stable mutants blocked in nikkomycin biosynthesis were obtained after the slightly germinated spores of Streptomyces ansochromogenes,a nikkomycin producer,were treated with ultra violet radiation.One of the mutants is the same in morpholotical differentiation as the wild type strain and is designated as NBB19.A DNA library was constructed using plasmid pIJ702 as cloning vector,NBB19 as cloning recipient.A 6 kb DNA fragment which can genetically complement NBB19 was cloned when screening the library for antifungal activity.Sequence analysis showed that the 3 kb Bgl II-Sal I fragment contains one complete ORF (ORF1) and one partial ORF (ORF2).ORF1 is designated as sanA.sanA is 1 365 bp,encoding a protein consisting of 454 amino acid residues.Database searching indicated that sanA is homologous to the hypothetical methyltransferase in Pyrococcus horikoshii with 25% identities and 41% positives.Disruptant of sanA lost the ability to synthesize nikkomycin.It indicated that sanA is a novel gene which is essential for nikkomycin biosynthesis.

  3. Analysis of SSH library of rice variety Aganni reveals candidate gall midge resistance genes.

    Science.gov (United States)

    Divya, Dhanasekar; Singh, Y Tunginba; Nair, Suresh; Bentur, J S

    2016-03-01

    The Asian rice gall midge, Orseolia oryzae, is a serious insect pest causing extensive yield loss. Interaction between the gall midge and rice genotypes is known to be on a gene-for-gene basis. Here, we report molecular basis of HR- (hypersensitive reaction-negative) type of resistance in Aganni (an indica rice variety possessing gall midge resistance gene Gm8) through the construction and analysis of a suppressive subtraction hybridization (SSH) cDNA library. In all, 2,800 positive clones were sequenced and analyzed. The high-quality ESTs were assembled into 448 non-redundant gene sequences. Homology search with the NCBI databases, using BlastX and BlastN, revealed that 73% of the clones showed homology to genes with known function and majority of ESTs belonged to the gene ontology category 'biological process'. Validation of 27 putative candidate gall midge resistance genes through real-time PCR, following gall midge infestation, in contrasting parents and their derived pre-NILs (near isogenic lines) revealed induction of specific genes related to defense and metabolism. Interestingly, four genes, belonging to families of leucine-rich repeat (LRR), heat shock protein (HSP), pathogenesis related protein (PR), and NAC domain-containing protein, implicated in conferring HR+ type of resistance, were found to be up-regulated in Aganni. Two of the reactive oxygen intermediates (ROI)-scavenging-enzyme-coding genes Cytosolic Ascorbate Peroxidase1, 2 (OsAPx1 and OsAPx2) were found up-regulated in Aganni in incompatible interaction possibly suppressing HR. We suggest that Aganni has a deviant form of inducible, salicylic acid (SA)-mediated resistance but without HR.

  4. Mammalian cDNA Library from the NIH Mammalian Gene Collection (MGC) | Office of Cancer Genomics

    Science.gov (United States)

    The MGC provides the research community full-length clones for most of the defined (as of 2006) human and mouse genes, along with selected clones of cow and rat genes. Clones were designed to allow easy transfer of the ORF sequences into nearly any type of expression vector. MGC provides protein ‘expression-ready’ clones for each of the included human genes. MGC is part of the ORFeome Collaboration (OC).

  5. [The analysis of Bacillus thuringiensis vegetative insecticical protein gene cloning and expression].

    Science.gov (United States)

    Cai, Qi-Liang; Liu, Zi-Duo; Sun, Ming; Wei, Fang; Yu, Zi-Niu

    2002-09-01

    Three kinds of Bacillus thuringiensis serotype-subsp. Leesis(H33) strain YBT-833, subsp. Aizawai(H7) strain YBT-1416 and subsp. Kurstaki(H3ab) strain YBT-1535, which were isolated by our lab, are chosen as original strain to clone vegetative insecticidal protein gene. Southern hybridization showed that vip genes are all localized at roughly 4-5 kb size-fractionated XbaI fragments of total DNA from YBT-833, YBT-1416 and YBT-1535. Three subgenomic libraries containing the vip gene fragment, were constructed with pUC19 as vector. Then, three vegetative insecticidal protein gene vip83, vip14 and vip15 are obtained from the libraries through the methods of colony-blot-in-situ screening and enzyme-cut detection. Comparision of DNA sequence made out that only vip83 gene exist five different base pairs with known vip genes. Because the sequences of vip14 and vip15 are the same, two of the three genes, vip83 and vip14, were subcloned to shuttle vehicle pHT315 to get recombinant plasmids pBMB8901 and pBMB8902 in turn. The plasmids were separately transformed into vip Bt. receptors BMB171 and 4Q7 to obtain four engineered strains BMB8901-171, BMB8902-171, BMB8901-4Q7 and BMB8902-4Q7. SDS-PAGE results indicated that all recombinant strains express 88 kD vegetative insecticidal protein. Bioassay also showed that the proteins of genes vip83 and vip14 both have certain toxicity to Lepidopteran insect larvae such as Heliochis armigera, Spodotera exigua and Plutella xylostella. While the toxicity of vip protein from four engineered strains to Plutella xylostellas are highest, whose LC50 value is 28.6, 31.6, 45.4 and 37.6 microL/mL respectively. This study will contributed to construct high efficacy and wide spectrum engineered strains on theory and reality.

  6. Comparison of bacterial community in aqueous and oil phases of water-flooded petroleum reservoirs using pyrosequencing and clone library approaches.

    Science.gov (United States)

    Wang, Li-Ying; Ke, Wen-Ji; Sun, Xiao-Bo; Liu, Jin-Feng; Gu, Ji-Dong; Mu, Bo-Zhong

    2014-05-01

    Bacterial communities in both aqueous and oil phases of water-flooded petroleum reservoirs were characterized by molecular analysis of bacterial 16S rRNA genes obtained from Shengli Oil Field using DNA pyrosequencing and gene clone library approaches. Metagenomic DNA was extracted from the aqueous and oil phases and subjected to polymerase chain reaction amplification with primers targeting the bacterial 16S rRNA genes. The analysis by these two methods showed that there was a large difference in bacterial diversity between the aqueous and oil phases of the reservoir fluids, especially in the reservoirs with lower water cut. At a high phylogenetic level, the predominant bacteria detected by these two approaches were identical. However, pyrosequencing allowed the detection of more rare bacterial species than the clone library method. Statistical analysis showed that the diversity of the bacterial community of the aqueous phase was lower than that of the oil phase. Phylogenetic analysis indicated that the vast majority of sequences detected in the water phase were from members of the genus Arcobacter within the Epsilonproteobacteria, which is capable of degrading the intermediates of hydrocarbon degradation such as acetate. The oil phase of reservoir fluid samples was dominated by members of the genus Pseudomonas within the Gammaproteobacteria and the genus Sphingomonas within the Alphaproteobacteria, which have the ability to degrade crude oil through adherence to hydrocarbons under aerobic conditions. In addition, many anaerobes that could degrade the component of crude oil were also found in the oil phase of reservoir fluids, mainly in the reservoir with lower water cut. These were represented by Desulfovibrio spp., Thermodesulfovibrio spp., Thermodesulforhabdus spp., Thermotoga spp., and Thermoanaerobacterium spp. This research suggested that simultaneous analysis of DNA extracted from both aqueous and oil phases can facilitate a better understanding of the

  7. Expression of cloned genes of transgenic microorganisms introduced into man-made ecosystems

    Science.gov (United States)

    Maksimova, E. E.; Popova, L. Yu.

    Modeling of transgenic microorganism introduction into small man-made ecosystems can help forecast changes in expression of cloned genes under different conditions of existence. Introduction of the E. coli Z905/pPHL7 strain containing a plasmid with luminescent system genes of luminous bacteria led to changes in cell and colony morphology, reduction in metabolic activity of cells, and, as a result, a lower level of expression of cloned gene. A low concentration of nutrients has been shown to favor greatly the phenotypic change of cells of the recombinant strain. Expression of cloned genes changed due to: a lower concentration of plasmid DNA, a change in regulation of cloned genes, and a change in cells of biosynthesis of substrates needed for expression of luminescent genes. The conducted investigations can provide a basis for the use of marker transgenic microorganisms in closed ecosystems of different types.

  8. Genomic libraries: I. Construction and screening of fosmid genomic libraries.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Large insert genome libraries have been a core resource required to sequence genomes, analyze haplotypes, and aid gene discovery. While next generation sequencing technologies are revolutionizing the field of genomics, traditional genome libraries will still be required for accurate genome assembly. Their utility is also being extended to functional studies for understanding DNA regulatory elements. Here, we present a detailed method for constructing genomic fosmid libraries, testing for common contaminants, gridding the library to nylon membranes, then hybridizing the library membranes with a radiolabeled probe to identify corresponding genomic clones. While this chapter focuses on fosmid libraries, many of these steps can also be applied to bacterial artificial chromosome libraries.

  9. Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

    Science.gov (United States)

    Liu, X; Gorovsky, M A

    1996-01-01

    A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced. PMID:8760889

  10. 16S ribosomal DNA clone libraries to reveal bacterial diversity in anaerobic reactor-degraded tetrabromobisphenol A.

    Science.gov (United States)

    Peng, Xingxing; Zhang, Zaili; Zhao, Ziling; Jia, Xiaoshan

    2012-05-01

    Microorganisms able to rapidly degrade tetrabromobisphenol A (TBBPA) were domesticated in an anaerobic reactor and added to gradually increased concentrations of TBBPA. After 240 days of domestication, the degradation rate reached 96.0% in cultivated batch experiments lasting 20 days. The optimum cultivating temperature and pH were 30°C and 7.0. The bacterial community's composition and diversity in the reactor was studied by comparative analysis with 16S ribosomal DNA clone libraries. Amplified rDNA restriction analysis of 200 clones from the library indicate that the rDNA richness was high (Coverage C 99.5%) and that evenness was not high (Shannon-Weaver index 2.42). Phylogenetic analysis of 63 bacterial sequences from the reactor libraries demonstrated the presence of Betaproteobacteria (33.1%), Gammaproteobacteria (18.7%), Bacteroidetes (13.9%), Firmicutes (11.4%), Chloroflexi (3.6%), Actinobacteria (0.6%), the candidate division TM7 (4.2%) and other unknown, uncultured bacterial groups (14.5%). Comamonas, Achromobacter, Pseudomonas and Flavobacterium were the dominant types.

  11. Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Although the centromeres of some plants have been investigated previously, our knowledge of the wheat centromere is still very limited. To understand the structure and function of the wheat centromere, we used two centromeric repeats (RCS1 and CCS1-5ab) to obtain some centromere-associated bacterial artificial chromosome (BAC) clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat (Triticum boeoticum Boiss.; 2n=2x=14) BAC library. Southern hybridization results indicated that, of the 32 candidates,there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization (FISH) analysis indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromeric regions in hexaploid wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or D-genome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific (or rich) sequences dispersing on chromosome arms in the BAC clone TbBAC5. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation in the period before metaphase.

  12. Construction of chimeric antibodies: cloning of immunoglobulin genes including their promoter regions by PCR.

    Science.gov (United States)

    Mocikat, R; Kütemeier, G; Harloff, C

    1992-03-01

    In the production of recombinant antibodies, it is necessary to have an immunoglobulin gene promoter for driving the expression of the antibody genes. Here we describe a simple PCR method that allows cloning of the immunoglobulin genes together with their own promoters despite the fact that the sequence of the upstream part of the gene is unknown.

  13. Cloning, Characterization and Primary Function Study of a Novel Gene, Cymgl, Related to Family 2 Cystatins

    Institute of Scientific and Technical Information of China (English)

    Yang XIANG; Dong-Song NIE; Jian WANG; Xiao-Jun TAN; Yun DENG; Shu-Wei LUO; Guang-Xiu LU

    2005-01-01

    Cystatins are cysteine proteinase inhibitors. We found two expression sequence tags (ESTs),CA463109 and AV042522, from a mouse testis library using Digital differential display (DDD). By electrical hybridization, a novel gene, Cymgl (GenBank accession No. AY600990), which has a full length of 0.78 kb,and contains four exons and three introns, was cloned from a mouse testis cDNA library. The gene is located in the 2G3 area of chromosome 2. The full cDNA encompasses the entire open reading frame, encoding 141amino acid residues. The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the CRES subfamily, which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract. CYMG1 has a 44% (48/108) identity with mouse CRES and 30% (42/140)identity with mouse cystatin C. Northern blot analysis showed that the Cymgl is specifically expressed in adult mouse testes. Cell location studies showed that the GFP-tagged CYMG1 protein was localized in the cytoplasm of HeLa cells. Immunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium, spermatocytes, round spermatids, elongating spermatids and spermatozoa. RT-PCR results also showed that Cymgl was expressed in mouse testes and spermatogonium. The Cymgl expression level varied in different developmental stages: it was low 1 week postpartum, steadily increased 2 to 5 weeks postpartum, and was highest 7 weeks postpartum. The expression level at 5 weeks postpartum was maintained during 13 to 57 weeks postpartum. The Cymgl expression level in the testes over different developmental stages correlates with the mouse spermatogenesis and sexual maturation process. All these indicate that Cymgl might play an important role in mouse spermatogenesis and sexual maturation.

  14. Zebrafish Cx35: cloning and characterization of a gap junction gene highly expressed in the retina.

    Science.gov (United States)

    McLachlan, Elizabeth; White, Thomas W; Ugonabo, Chioma; Olson, Carl; Nagy, James I; Valdimarsson, Gunnar

    2003-09-15

    The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos.

  15. Molecular cloning and polymorphism of major histocompatibility complex class I genes from grass carp (Ctenophayngodon idellus)

    Institute of Scientific and Technical Information of China (English)

    XIA Chun; XU Guangxian; LIN Changyou; HU Tuanjun; YAN Ruoqian; George F GAO

    2004-01-01

    In order to clarify the molecular sequences,allelic polymorphism and the tertiary structure of grass carp (Ctenophayngodon idellus) MHC class I,and to further study their relationship with disease resistances,grass carp MHC class I gene (Ctid-MHC I) was cloned from a cDNA library and the allelic polymorphism in the population was investigated.The results showed that most of the variations exist in the peptide-binding domain (PBD) and high polymorphism was identified in the Ctid-MHC I allelic genes from 12 individuals.Based on the genetic distance,Ctid-MHC class I can be classified into 6 types (from Ctid-MHC I-UA to Ctid-MHC I-UF) which were subdivided into 9 lineages (from A to I).Comparison of the Ctid-MHC I among animals and humans showed that the key amino acids of the peptide binding sites are conserved.Analysis of the tertiary structure of the PBD between Grass carp and human crystallographic data of HLA-A2,the variation with insertion or deletion was found in eight regions (A~H).The phylogenetic tree of MHC class I indicates the evolution of MHC class I among grass carp,fish,amphibian,birds,higher vertebrates and humans.

  16. Cloning and sequence characteristics of the genomic gene of a rice metallothionein

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Northern blot analysis showed that a metallothionein gene, ricMT, is expressed strongly in the stem of rice with an expression level that could be more than 100-fold stronger than in leaf blades. The results suggest that the 5'upstream region flanking the coding sequence of the ricMT may contain a fairly strong promoter. To elucidate its regulation and promoter structure, the genomic clones of ricMT were screened out from a rice genomic library and a fragment of about 4 084 bp was sequenced. The fragment included a 5'upstream region of ca. 2 970 bp, a transcription region of ca. 690 bp and a 3'downstream region of ca. 420 bp. Computer analysis of the sequence homology showed that the 5'upstream region included a putative TATA box, a putative CAAT box, and a typical metal-responsive element TGCGCGCG. The results will promote further understanding of the mechanisms of gene regulation and metal response of plant metallothionein proteins.

  17. A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes.

    Science.gov (United States)

    Schmid-Burgk, Jonathan L; Schmidt, Tobias; Kaiser, Vera; Höning, Klara; Hornung, Veit

    2013-01-01

    Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.

  18. Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum.

    Science.gov (United States)

    Wankhede, Dhammaprakash Pandhari; Biswas, Dipul Kumar; Rajkumar, Subramani; Sinha, Alok Krishna

    2013-12-01

    Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

  19. Specific genetic modifications of domestic animals by gene targeting and animal cloning.

    Science.gov (United States)

    Wang, Bin; Zhou, Jiangfeng

    2003-11-13

    The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined.

  20. Cloning of HPV16 E2 Gene from a Biopsied Cervical Cancer Sample

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To clone HPV16 E2 gene from a biopsied cervical cancer sampleMaterials & Methods HPV 1 6 E2 gene was amplified from specimen derived from aHPV 16 positive patient, then cloned and sequenced. Results The full-length of HPV 16 E2 gene was successfully cloned. In comparisonwith the prototype accepted by GenBank, six point mutations in HPV 1 6 E2 nucleotideacid sequence were identified. Of them, three were missense, and one was in the over-lapping E4 gene and was synonymous to E4.Conclusion HPV 16 E2 gene was successfully cloned, and some nucleotide acids in itssequence were different from the prototype.

  1. Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

    Science.gov (United States)

    Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

    2009-01-01

    We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students…

  2. Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

    Science.gov (United States)

    Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

    2001-01-01

    This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

  3. Gene enrichment in plant genomic shotgun libraries.

    Science.gov (United States)

    Rabinowicz, Pablo D; McCombie, W Richard; Martienssen, Robert A

    2003-04-01

    The Arabidopsis genome (about 130 Mbp) has been completely sequenced; whereas a draft sequence of the rice genome (about 430 Mbp) is now available and the sequencing of this genome will be completed in the near future. The much larger genomes of several important crop species, such as wheat (about 16,000 Mbp) or maize (about 2500 Mbp), may not be fully sequenced with current technology. Instead, sequencing-analysis strategies are being developed to obtain sequencing and mapping information selectively for the genic fraction (gene space) of complex plant genomes.

  4. Molecular cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite, Coptotermes formosanus.

    Science.gov (United States)

    Inoue, Tetsushi; Moriya, Shigeharu; Ohkuma, Moriya; Kudo, Toshiaki

    2005-04-11

    The endo-beta-1,4-glucanase gene was cloned from a cDNA library constructed from the mixed population of symbiotic protists in the hindgut of the lower termite, Coptotermes formosanus, using the lambda ZAP II vector. The recombinant phage library was screened for cellulolytic activity by the Congo red staining procedure. The nucleotide sequence comprised 941 nucleotides including a polyA tail sequence and showed high sequence similarity with endoglucanase genes belonging to glycosyl hydrolase family 5. Determination of the 5' end of the cellulase gene using the 5'RACE method showed that the full-length cDNA comprised a 921-bp ORF, encoding a putative 33,620 Da protein. The organismal source of this cellulase gene was identified using PCR with gene-specific primers and whole-cell in situ hybridization as the smallest symbiotic hypermastigote protist, Spirotrichonympha leidyi. The optimal pH and temperature of the cellulase heterologously expressed in Escherichia coli were 5.8-6.0 and 70 degrees C, respectively. The Km and Vmax values on carboxymethyl cellulose (CMC) substrate were 1.90 mg/ml and 148.2 units/mg protein, respectively.

  5. Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

    Science.gov (United States)

    Coyne, C J; McClendon, M T; Walling, J G; Timmerman-Vaughan, G M; Murray, S; Meksem, K; Lightfoot, D A; Shultz, J L; Keller, K E; Martin, R R; Inglis, D A; Rajesh, P N; McPhee, K E; Weeden, N F; Grusak, M A; Li, C-M; Storlie, E W

    2007-09-01

    Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

  6. Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.

  7. Cloning of Tumor Suppressor Genes in Breast Cancer

    Science.gov (United States)

    2003-05-01

    panels and genomic library screening , and the locus was scru- Mutagenesis. Mutation was introduced by using the Exsite Mu- tinized further. We constructed...cDNA library screening and completeness confirmed by using RNA ligase-mediated amplification of cDNA ends. PFAM anal- ER48 ysis of our sequence revealed

  8. Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning.

    Science.gov (United States)

    Sone, Takefumi; Yahata, Kazuhide; Sasaki, Yukari; Hotta, Junko; Kishine, Hiroe; Chesnut, Jonathan D; Imamoto, Fumio

    2008-09-10

    Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.

  9. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov;

    2008-01-01

    technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium...... with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. Conclusion: The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi....

  10. Genetic Alterations in Familial Breast Cancer: Mapping and Cloning Genes Other Than BRCAl

    Science.gov (United States)

    1997-09-01

    predispose to breast cancer . These mutations are always in the context of Cowden’s Syndrome, and do not appear in families with brest cancer in the...AD AWARD NUMBER DAMD17-94-J-4307 TITLE: Genetic Alterations in Familial Breast Cancer : Mapping and Cloning Genes Other Than BRCA1 PRINCIPAL...Aug97-) Genetic Alterations in Familial Breast Cancer : Mapping and Cloning Genes Other than BRCA1 6. AUTHOR{S) Mary-Clair King, Ph.D. 7

  11. Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

    OpenAIRE

    Jiang, Chunyan; Wang, Hougen; Kang, Qianjin; Jing LIU; Bai, Linquan

    2012-01-01

    Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211...

  12. Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library.

    Science.gov (United States)

    Rahumatullah, Anizah; Ahmad, Azimah; Noordin, Rahmah; Lim, Theam Soon

    2015-10-01

    Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.

  13. Cloning and analysis of an HMG gene from the lamprey Lampetra fluviatilis

    DEFF Research Database (Denmark)

    Sharman, A C; Hay-Schmidt, Anders; Holland, P W

    1997-01-01

    Evolution has shaped the organisation of vertebrate genomes, including the human genome. To shed further light on genome history, we have cloned and analysed an HMG gene from lamprey, representing one of the earliest vertebrate lineages. Genes of the HMG1/2 family encode chromosomal proteins...... that bind DNA in a non-sequence-specific manner, and have been implicated in a variety of cellular processes dependent on chromatin structure. They are characterised by two copies of a conserved motif, the HMG box, followed by an acidic C-terminal region. We report here the cloning of a cDNA clone from...

  14. CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene.

    Science.gov (United States)

    Rouvier, E; Luciani, M F; Mattéi, M G; Denizot, F; Golstein, P

    1993-06-15

    To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that we named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival.

  15. CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus Saimiri gene

    Energy Technology Data Exchange (ETDEWEB)

    Rouvier, E.; Luciani, M.F.; Golstein, P. (Centre d' Immunologie INSERM-CNRS de Marseille-Luminy, Marseille (France)); Mattei, M.G. (INSERM U242, Marseille (France)); Denizot, F. (Centre de Sequencage d' ADN, Marseille (France))

    1993-06-15

    To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that was named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival. 69 refs., 5 figs.

  16. Cloning, sequencing and expression of a xylanase gene from the maize pathogen Helminthosporium turcicum

    DEFF Research Database (Denmark)

    Degefu, Y.; Paulin, L.; Lübeck, Peter Stephensen

    2001-01-01

    A gene encoding an endoxylanase from the phytopathogenic fungus Helminthosporium turcicum Pass. was cloned and sequenced. The entire nucleotide sequence of a 1991 bp genomic fragment containing an endoxylanase gene was determined. The xylanase gene of 795 bp, interrupted by two introns of 52 and ...

  17. Cloning, sequence analysis, and characterization of the genes involved in isoprimeverose metabolism in Lactobacillus pentosus

    NARCIS (Netherlands)

    Chaillou, S.; Lokman, B.C.; Leer, R.J.; Posthuma, C.; Postma, P.W.; Pouwels, P.H.

    1998-01-01

    Two genes, xylP and xylQ, from the xylose regulon of Lactobacillus pentosus were cloned and sequenced. Together with the repressor gene of the regulon, xylR, the xylPQ genes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream of xylP. A putati

  18. CLONING, SEQUENCING, AND EXPRESSION OF BACILLUS-SUBTILIS GENES INVOLVED IN ATP-DEPENDENT NUCLEASE SYNTHESIS

    NARCIS (Netherlands)

    KOOISTRA, J; VENEMA, G

    1991-01-01

    The genes encoding the subunits of the Bacillus subtilis ATP-dependent nuclease (add genes) have been cloned. The genes were located on an 8.8-kb SalI-SmaI chromosomal DNA fragment. Transformants of a recBCD deletion mutant of Escherichia coli with plasmid pGV1 carrying this DNA fragment showed ATP-

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

    Science.gov (United States)

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

    2003-05-15

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

  20. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH{sub 2}-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH{sub 2}-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids.

  1. Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases.

    Science.gov (United States)

    Laurent, F; Bourdieu, C; Kaga, M; Chilmonczyk, S; Zgrzebski, G; Yvoré, P; Péry, P

    1993-12-01

    A lambda ZapII cDNA library was constructed using mRNA from Eimeria acervulina sporulated oocysts and screened with monoclonal antibodies raised against Eimeria tenella sporulated oocytes. Monoclonal antibody N3C8B12 identified a clone (6S2) potentially encoding an aspartyl proteinase since significant homology with cathepsin D, pepsin and renin proteinases was revealed by sequence comparisons. The 1500-bp cDNA fragment containing the coccidial gene was subcloned into pGEX-FA expression vector, leading to the production of an 80-kDa fusion protein (FA6S2) which was used to immunize rabbits. The anti-FA6S2 rabbit sera revealed a single 43-kDa protein present in Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria falciformis sporulated oocyst antigens. Indirect immunofluorescence and electron microscopy with mAb N3C8B12 localized the putative aspartyl proteinase in the refractile bodies of Eimeria tenella sporozoites.

  2. Product-induced gene expression, a product-responsive reporter assay used to screen metagenomic libraries for enzyme-encoding genes.

    Science.gov (United States)

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-11-01

    A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.

  3. The cloning and expression characterization of the centrosome protein genes family (centrin genes) in rat testis

    Institute of Scientific and Technical Information of China (English)

    SUN; Xiaodong(孙晓冬); GE; Yehua(葛晔华); MA; Jing(马静); YU; Zuoren(俞作仁); LI; Sai(李赛); WANG; Yongchao(王永潮); XUE; Shepu(薛社普); HAN; Daishu(韩代书)

    2002-01-01

    Centrins are members of the centrosome protein family, which is highly conserved during revolution. The homologous genes of centrin in many organisms had been cloned, but the sequences of the rat centrin genes were not reported yet in GenBank. We cloned the cDNA fragments of centrin-1, -2 and -3 from the rat testis by RT-PCR, and analyzed the homology of the deduced amino acid sequences. The expression characterization of centrin genes in rat spermatogenesis was carried out by semi-quantitative RT-PCR. The results show that the homology of the corresponding centrin proteins in human, mouse and rat is high. The expression of centrin-1 is testis-specific, spermatogenic cell-specific and developmental stage-related. Centrin-1 begins to be transcribed when the meiosis occurs, and its mRNA level reaches the peak in round spermatids. Centrin-2 and centrin-3 are highly expressed in spermatogonia and their mRNA level decreases markedly when meiosis occurs. These results suggest that centrin-1 may play roles in meiosis and spermiogenesis, and centrin-2 and centrin-3 may be related to mitosis.

  4. Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli

    Directory of Open Access Journals (Sweden)

    Projan Steve

    2003-09-01

    Full Text Available Abstract Background Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H, is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost in the amplified library. Results Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport. Conclusion Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the

  5. Tetranectin, a plasminogen kringle 4-binding protein. Cloning and gene expression pattern in human colon cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R

    1992-01-01

    BACKGROUND: Tetranectin is a recently discovered protein that binds to kringle 4 region of plasminogen (Clemmensen I, Petersen LC, Kluft C. Eur J Biochem 1986; 156:327. EXPERIMENTAL DESIGN: The mRNA encoding human tetranectin was cloned by using degenerate primers in a reverse transcriptase...... reaction followed by polymerase chain reaction amplification. The resulting polymerase chain reaction product was examined by DNA sequencing and subsequently used as probe for screening a human placental cDNA library. A full length cDNA clone (TET-1) was isolated, characterized, and used for Northern blot...

  6. Cloning of fish enzymes and other fish protein genes.

    Science.gov (United States)

    Macouzet, M; Simpson, B K; Lee, B H

    1999-01-01

    Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.

  7. Characterization of a Novel Amylolytic Enzyme Encoded by a Gene from a Soil-Derived Metagenomic Library

    Science.gov (United States)

    Yun, Jiae; Kang, Seowon; Park, Sulhee; Yoon, Hyunjin; Kim, Myo-Jeong; Heu, Sunggi; Ryu, Sangyeol

    2004-01-01

    It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC19 vector. The library was screened for amylase activity, and one clone from among approximately 30,000 recombinant Escherichia coli clones showed amylase activity. Sequencing of the clone revealed a novel amylolytic enzyme expressed from a novel gene. The putative amylase gene (amyM) was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of the AmyM protein were 42°C and pH 9.0; Ca2+ stabilized the activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high levels of maltose and hydrolyzed pullulan to panose. The enzyme showed a high transglycosylation activity, making α-(1, 4) linkages exclusively. The hydrolysis and transglycosylation properties of AmyM suggest that it has novel characteristics and can be regarded as an intermediate type of maltogenic amylase, α-amylase, and 4-α-glucanotransferase. PMID:15574921

  8. Cloning and structural and expressional characterization of BcpLH gene preferentially expressed in folding leaf of Chinese cabbage

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Vegetative growth of Chinese cabbage undergoes the four successive stages which are characterized with the definite types of juvenile,rosette,folding and head leaves.From shoot tips of Chinese cabbage at early folding stage,we constructed a cDNA library and screened the differentially expressed cDNA clones using the cDNAs derived from developing folding leaves and rosette leaves as probes.One complete length of cDNA clone is designated as BcpLH.Computer alignment matched BcpLH to the domains of double-stranded RNA binding (DBRM) and the homologous regions were recognized between BcpLH and human and mouse double-stranded RNA-binding protein TRBP.PCR expression analysis shows that during vegetative growth BcpLH gene was expressed preferentially in folding leaves at folding stage.Transcripts of BcpLH gene were increased when plants were sprayed with IAA.It is deduced that BcpLH gene may be related to initiation of folding leaf and leafy head and induced by auxin in the aspect of transcriptional expression.

  9. Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes

    Institute of Scientific and Technical Information of China (English)

    Xing Zhong; Yu-Liang Ran; Jin-Ning Lou; Dong Hu; Long Yu; Yu-Shan Zhang; Zhuan Zhou; Zhi-Hua Yang

    2004-01-01

    AIM: To gain tumor endothelium associated antigen genes from human liver cancer vascular endothelial cells (HLCVECs)cDNA expression library, so as to find some new possible targets for the diagnosis and therapy of liver tumor.METHODS: HLCVECs were isolated and purified from a fresh hepatocellular carcinoma tissue sample, and were cultured and proliferated in vitro. A cDNA expression library was constructed with the mRNA extracted from HLCVECs.Anti-sera were prepared from immunized BALB/c mice through subcutaneous injection with high dose of fixed HLCVECs, and were then tested for their specificity against HLCVECs and angiogenic effectsin vitro, such as inhibiting proliferation and inducing apoptosis of tumor endothelial cells, using immunocytochemistry, immunofiuorescence,cell cycle analysis and MTT assays, etc. The identified xenogeneic sera from immunized mice were employed to screen the library of HLCVECs by modified serological analyses of recombinant cDNA expression libraries (SEREX).The positive clones were sequenced and analyzed by bioinformatics.RESULTS: The primary cDNA library consisted of 2x106recombinants. Thirty-six positive clones were obtained from6×10s independent clones by immunoscreening. Bio-informatics analysis of cDNA sequences indicated that 36 positive clones represented 18 different genes. Among them, 3 were new genes previously unreported, 2 of which were hypothetical genes. The other L5 were already known ones. Series analysis of gene expression (SAGE) database showed that ERP70,GRP58, GAPDH, SSB, S100A6, BMP-6, DVS27, HSP70 and NAC alpha in these genes were associated with endothelium and angiogenesis, but their effects on HLCVECs were still unclear. GAPDH, S100A6, BMP-6 and hsp70 were identified by SEREX in other tumor cDNA expression libraries.CONCLUSION: By screening of HLCVECs cDNA expression library using sera from immunized mice with HLCVECs,the functional genes associated with tumor endothelium or angiogenesis were identified. The

  10. Recent Advances in Cloning and Characterization of Disease Resistance Genes in Rice

    Institute of Scientific and Technical Information of China (English)

    Liang-Ying Dai; Xiong-Lun Liu; Ying-Hui Xiao; Guo-Liang Wang

    2007-01-01

    Rice diseases caused by fungi, bacteria and viruses are one of the major constraints for sustainable rice (Oryza sativa L.) production worldwide. The use of resistant cultivars is considered the most economical and effective method to control rice diseases. In the last decade, a dozen resistance genes against the fungal pathogen Magnaporthe grisea and the bacterial pathogen Xanthomonas oryzae pv. oryzae have been cloned. Approximately half of them encode nuclear binding site (NBS) and leucine rich repeat (LRR)-containing proteins, the most common type of cloned plant resistance genes. Interestingly, four of them encode novel proteins which have not been identified in other plant species, suggesting that unique mechanisms might be involved in rice defense responses. This review summarizes the recent advances in cloning and characterization of disease resistance genes in rice and presents future perspectives for in-depth molecular analysis of the function and evolution of rice resistance genes and their interaction with avirulence genes in pathogens.

  11. Gene recovery microdissection (GRM) a process for producing chromosome region-specific libraries of expressed genes

    Energy Technology Data Exchange (ETDEWEB)

    Christian, A T; Coleman, M A; Tucker, J D

    2001-02-08

    Gene Recovery Microdissection (GRM) is a unique and cost-effective process for producing chromosome region-specific libraries of expressed genes. It accelerates the pace, reduces the cost, and extends the capabilities of functional genomic research, the means by which scientists will put to life-saving, life-enhancing use their knowledge of any plant or animal genome.

  12. Cloning of nod gene regions from mesquite rhizobia and bradyrhizobia and nucleotide sequence of the nodD gene from mesquite rhizobia.

    Science.gov (United States)

    Thomas, P M; Golly, K F; Virginia, R A; Zyskind, J W

    1995-09-01

    Nitrogen-fixing symbiosis between bacteria and the tree legume mesquite (Prosopis glandulosa) is important for the maintenance of many desert ecosystems. Genes essential for nodulation and for extending the host range to mesquite were isolated from cosmid libraries of Rhizobium (mesquite) sp. strain HW17b and Bradyrhizobium (mesquite) sp. strain HW10h and were shown to be closely linked. All of the cosmid clones of rhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite also supported nodulation of a Sym- mesquite strain. The cosmid clones of bradyrhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite were only able to confer nodulation ability in the Sym- mesquite strain if they also contained a nodD-hybridizing region. Subclones containing just the nodD genes of either genus did not extend the host range of Rhizobium (Parasponia) sp. to mesquite, indicating that the nodD gene is insufficient for mesquite nodulation. The nodD gene region is conserved among mesquite-nodulating rhizobia regardless of the soil depth from which they were collected, indicating descent from a common ancestor. In a tree of distance relationships, the NodD amino acid sequence from mesquite rhizobia clusters with homologs from symbionts that can infect both herbaceous and tree legumes, including Rhizobium tropici, Rhizobium leguminosarum bv; phaseoli, Rhizobium loti, and Bradyrhizobium japonicum.

  13. Cloning and analysis of a constitutive heat shock (cognate) protein 70 gene inducible by L-glutamine.

    Science.gov (United States)

    LéJohn, H B; Cameron, L E; Yang, B; MacBeath, G; Barker, D S; Williams, S A

    1994-02-11

    An intronless gene encoding a protein of 652 amino acid residues with an M(r) of 71,266, showing between 79% and 59% identity in nucleotide sequence with heat shock protein 70 (HSP 70) genes of Bremia lactucae (a parasitic Oomycete of lettuce) and a wide range of organisms that include humans, was isolated from the nonparasitic Oomycete Achlya klebsiana. While the gene appears to be constitutively expressed, L-glutamine augmented its expression particularly under conditions of nutritional stress. L-Glutamine enhanced the transcription of a 2.4-kilobase poly(A)+ RNA simultaneously in the same way as it elevated the cellular level of the HSP 70-like protein. A polyclonal antibody (affinity-purified) raised in rabbit against the purified monomeric (M(r) 120,000) form of an NAD-specific glutamate dehydrogenase (Yang, B., and LéJohn, H.B. (1994) J. Biol. Chem. 269, 4506-4512) immunoprecipitated the HSP 70-like protein, and it was used to study the kinetics of induction of this stress-related protein and the effect of proteinase inhibitors on its metabolism. By using as probes four partial length cDNA clones, nine overlapping DNA fragments of the organism's genome carrying the HSP 70-like protein gene were isolated from a genomic library. The nucleotide sequence of the gene, including its boundaries, was determined by using these genomic clones. The 5'-untranslated boundary of the gene displayed the classical nucleotide arrangement of heat shock elements as well as CCAAT and TATA box motifs. Within the coding region are the typical conserved amino acid heat shock protein signatures 1 and 2 at the predicted locations. By primer extension and S1 nuclease protection mapping system, we estimated that the gene is probably transcribed into a message of 2.2 kilobases.

  14. Cloning and expression of porcine SRPK1 gene

    African Journals Online (AJOL)

    Academic Journals

    2012-01-10

    Jan 10, 2012 ... cloned by real time polymerase chain reaction (RT-PCR), yet coding sequence ... phosphorylation of the RNA splicing factors with RS ... basic molecular information that is useful for the further .... of transcription, such as: HSF1, HSF2, Ik-1, IK-2, SRY, ... protein and human SRPK1 in the structure data base of.

  15. Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid

    NARCIS (Netherlands)

    Belkum, Marco J. van; Hayema, Bert Jan; Geis, Arnold; Kok, Jan; Venema, Gerard

    1989-01-01

    Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4

  16. [Construction of root library by SSH and preliminary analysis of genes responsible for phosphorus deficiency in maize].

    Science.gov (United States)

    Huang, Q; Gao, S B; Zhang, Z M; Lin, H J; Pan, G T; Yang, K C; Rong, T Z

    2010-12-01

    An elite maize inbred line with high tolerance to low phosphorus, 178, was studied for constructing root library and analyzing some genes closely related to phosphorus (P) deficiency using SSH and Semi-quantitative RT-PCR. As a result, 3648 preliminary clones were obtained for root library under stress of P deficiency. By DNA sequencing of 34 random clones, we obtained 23 unique EST sequences which are involved in functions of root cell structure, tolerance and defense, protein modification and composition, transcription regulation, metabolism, and other unknown aspects. Five representative genes were further analyzed for their expression models. The results suggested that the molecular mechanism to adapt P deficiency in maize, performed by multi-genes with different contributions, is similar to rice, Arabidopsis and soybean. The expression order of 5 low P tolerant genes in maize root was PAP, GCS, TOM, PDI and AIP. And it was considered preliminarily that physiological and biochemical changes were prior to morphologic changes in maize root and the essential tolerance to low P may be determined by extending absorption of P to wide soil range through adaption of root architecture and root secretions, which is the greatest difference between tolerant and sensitive maize varieties under low P stress.

  17. Differential differences in methylation status of putative imprinted genes among cloned swine genomes.

    Directory of Open Access Journals (Sweden)

    Chih-Jie Shen

    Full Text Available DNA methylation is a major epigenetic modification in the mammalian genome that regulates crucial aspects of gene function. Mammalian cloning by somatic cell nuclear transfer (SCNT often results in gestational or neonatal failure with only a small proportion of manipulated embryos producing live births. Many of the embryos that survive to term later succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Aberrant methylation patterns of imprinted genes in cloned cattle and mice have been elucidated, but few reports have analyzed the cloned pig genome. Four surviving cloned sows that were created by ear fibroblast nuclear transfer, each with a different life span and multiple organ defects, such as heart defects and bone growth delay, were used as epigenetic study materials. First, we identified four putative differential methylation regions (DMR of imprinted genes in the wild-type pig genome, including two maternally imprinted loci (INS and IGF2 and two paternally imprinted loci (H19 and IGF2R. Aberrant DNA methylation, either hypermethylation or hypomethylation, commonly appeared in H19 (45% of imprinted loci hypermethylated vs. 30% hypomethylated, IGF2 (40% vs. 0%, INS (50% vs. 5%, and IGF2R (15% vs. 45% in multiple tissues from these four cloned sows compared with wild-type pigs. Our data suggest that aberrant epigenetic modifications occur frequently in the genome of cloned swine. Even with successful production of cloned swine that avoid prenatal or postnatal death, the perturbation of methylation in imprinted genes still exists, which may be one of reason for their adult pathologies and short life. Understanding the aberrant pattern of gene imprinting would permit improvements in future cloning techniques.

  18. Aberrant epigenetic changes and gene expression in cloned cattle dying around birth

    Directory of Open Access Journals (Sweden)

    Zhao Dingsheng

    2008-02-01

    Full Text Available Abstract Background Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. To assess the extent of abnormal epigenetic modifications and gene expression in clones, we simultaneously examined DNA methylation, histone H4 acetylation and expression of six genes (β-actin, VEGF, oct4, TERT, H19 and Igf2 and a repetitive sequence (art2 in five organs (heart, liver, spleen, lung and kidney from two cloned cattle groups that had died at different stages. In the ED group (early death, n = 3, the cloned cattle died in the perinatal period. The cattle in the LD group (late death, n = 3 died after the perinatal period. Normally reproduced cattle served as a control group (n = 3. Results Aberrant DNA methylation, histone H4 acetylation and gene expression were observed in both cloned groups. The ED group showed relatively fewer severe DNA methylation abnormalities (p Conclusion Deaths of clones may be ascribed to abnormal expression of a very limited number of genes.

  19. Insertional mutagenesis in Neurospora crassa: cloning and molecular analysis of the preg+ gene controlling the activity of the transcriptional activator NUC-1.

    Science.gov (United States)

    Kang, S; Metzenberg, R L

    1993-02-01

    The transcriptional activator NUC-1 controls the transcription of the genes for phosphorus acquisition enzymes, and its activity is regulated by the negative regulatory factors, PREG and PGOV In this report, we describe the cloning and molecular analysis of the preg+ gene. In Neurospora crassa, as in higher eukaryotes, transformation frequently results in nonhomologous integration of transforming DNA. Insertion of transforming DNA into host genes mutates the gene and provides a molecular tag for cloning it. We obtained two mutants that have an insertion in the preg+ and pgov+ genes, respectively, among 2 x 10(5) transformants. The preg+ gene was cloned by screening a Neurospora genomic DNA library with DNA sequences flanking the transforming DNA of the rescued plasmid. Northern analysis showed that the transcription of the preg+ gene is not regulated by phosphate. The carboxy-terminal half of PREG shows strong homology with Saccharomyces cerevisiae PHO80 whose function is analogous to that of PREG. The pregc mutations are located in the well conserved residues which may directly interact with the residues in the regulatory domain of NUC-1.

  20. Gene cloning and function analysis of ABP9 protein which specifically binds to ABRE2 motif of maize Cat1 gene

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A cDNA library was constructed using mRNA extracted from 17 days post-pollination (dpp) maize embryos and was screened by employing a yeast one-hybrid system for proteins specifically interacting with ABRE2 motif of maize Cat1 gene. Three truncated overlapping positive clones designated ABP9 were obtained and the full-length cDNA was isolated by 5′ RACE. Searching the database revealed that ABP9 protein belongs to a bZIP-type transcription factor family. ABP9 protein specifically binds to ABRE2 motif and activates the expression of downstream reporter gene in yeast cells. Our results strongly suggest that the ABP9 protein functions as a transcription activator.

  1. Expression of chromatin modification genes in organs of cloned cattle that died within hours after birth

    Institute of Scientific and Technical Information of China (English)

    LI Shijie; LIAN Zhengxing; LI Dongjie; YU Shuyang; ZHANG Lei; DAI Yunping; LI Rong; FEI Jing; LI Ning

    2006-01-01

    Cloning by somatic nuclear transfer is an inefficient process in which many of the cloned animals died shortly after birth and displayed organ abnormalities. In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we have examined expression patterns of four genes that modified chromatin (DNMT1, PCAF,MeCP2 and EED) in six organs (heart, liver, spleen, lung, kidney and brain) of both neonatal death cloned bovines (n=9) and normal control calves produced by artificial insemination (AI) using real-time quantitative RT-PCR. The effect of the age of the fibroblast donor cell on the gene expression profiles was also investigated. Aberrant expressions of DNMT1 and PCAF were found in some studied tissues, but the expression of MeCP2 and EED had similar levels to those of the normal controls. The expression of DNMT1 showed a higher level in heart, liver and brain of both cloned bovines. A higher expression level of PCAF was seen in heart and liver of both cloned bovines, but a lower level was seen only in spleen of adult fibroblast (AF) cell-derived clones. Our results suggest that aberrant expression in gene that modified chromatins were found in cloned bovine tissues of neonatal death. Because DNMT1 and PCAF play an important role in DNA methylation and histone acetylation on nuclear chromatin respectively, and normal expression of DNMT1 and PCAF is needed for precious reprogramming of donor nuclear, the aberrant transcription patterns of DNMT1 and PCAF in these clones 5 contribute to the defects of organs reported in neonatal death of clones.

  2. Cloning, tissue expression pattern, and chromosome localization of human protein kinase Bγ gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Protein kinase B (PKB) is a member of the second messenger-regulated subfamily of protein kinases, and plays a key role in cell-cycle regulation, glucose uptake and promotion of cell differentiation. Evidence shows that PKB undergoes activation in some human tumors and is involved in Ras pathway, which implies that PKB can trigger a pathway to induce oncogenic transformation. A nucleotide sequence of mouse Pkb? was used as a probe to screen homolog in a human liver cDNA library. A fragment of 1998 bp containing a 1440 bp ORF encoding 479 amino acid residues was obtained. Then the 3'-terminal of this fragment was extended to 2788 bp by 'electronic walking' screening, and the extended fragment was confirmed by PCR amplification. The protein deduced by the gene had a high identity of 83% and 78% to the human PKBγ and γ, respectively, and was designated as human PKB?. Northern hybridization detected two equally expressed transcripts of 8.5 and 6.5 kb in length in all 16 human tissues tested, with the highest expression level in brain, and lower levels with variation in the other tissues. By RH mapping, the PKBγ was placed on chromosome 1q43, between markers D1S304 and D1S2693. It is a valuable clue for cloning the candidate genes related to prostate cancer; Arrhythmogenic Right Ventricular Dysplasia (ARVD); Chediak-Higashi, NK cell Deficiency (CHS); and Hypoparathyrodism with Short Stature, Mental Retardation and Seizures which have already been mapped in this chromosomal region.

  3. Cloning and expression of nucleocapsid protein gene of TGEV HB06 strain

    Institute of Scientific and Technical Information of China (English)

    FAN Jinghui; ZUO Yuzhu; ZHAO Yuelan; LI Tanqing; ZHANG Xiaobo

    2007-01-01

    The nucleocapsid protein gene of transmissible gastroenteritis virus,1 149 bp in length,was amplified by RT-PCR from isolated strain HB06 and cloned into pMD 18T.Sequence comparison with other transmissible gastroenteritis virus (TGEV) strains selected from the Gene Bank revealed that the homology of N gene complete sequence shares more than 97% in nucleotide.N gene was cloned into BamHI and EcoRI multiple cloning sites of the prokaryotic expression vector pET 20 b,and named pETN.After being induced by isopropyl-β-D-thiogalactopyranoside (IPTG),the recombinant nucleocapsid protein was expressed.The result of SDS-PAGE and Western-blot showed that the recombinant nucleocapsid protein was 47 kDa and had strong positive reactions with TGEV-specific antibody.

  4. Cloning and expression of the HpaI restriction-modification genes.

    OpenAIRE

    Ito, H; Shimato, H; Sadaoka, A; Kotani, H; Kimizuka, F; Kato, I

    1992-01-01

    The genes from Haemophilus parainfluenzae encoding the HpaI restriction-modification system were cloned and expressed in Escherichia coli. From the DNA sequence, we predicted the HpaI endonuclease (R.HpaI) to have 254 amino acid residues (Mr 29,630) and the HpaI methyltransferase (M.HpaI) to have 314 amino acid residues (37,390). The R.HpaI and M.HpaI genes overlapped by 16 base pairs on the chromosomal DNA. The genes had the same orientation. The clone, named E. coli HB101-HPA2, overproduced...

  5. Integrase-directed recovery of functional genes from genomic libraries.

    Science.gov (United States)

    Rowe-Magnus, Dean A

    2009-09-01

    Large population sizes, rapid growth and 3.8 billion years of evolution firmly establish microorganisms as a major source of the planet's biological and genetic diversity. However, up to 99% of the microorganisms in a given environment cannot be cultured. Culture-independent methods that directly access the genetic potential of an environmental sample can unveil new proteins with diverse functions, but the sequencing of random DNA can generate enormous amounts of extraneous data. Integrons are recombination systems that accumulate open reading frames (gene cassettes), many of which code for functional proteins with enormous adaptive potential. Some integrons harbor hundreds of gene cassettes and evidence suggests that the gene cassette pool may be limitless in size. Accessing this genetic pool has been hampered since sequence-based techniques, such as hybridization or PCR, often recover only partial genes or a small subset of those present in the sample. Here, a three-plasmid genetic strategy for the sequence-independent recovery of gene cassettes from genomic libraries is described and its use by retrieving functional gene cassettes from the chromosomal integron of Vibrio vulnificus ATCC 27562 is demonstrated. By manipulating the natural activity of integrons, we can gain access to the caches of functional genes amassed by these structures.

  6. Cloning and functional analysis of the genes involved in signal transduction in tomato Cf-4-Avr4 pathosystem

    Institute of Scientific and Technical Information of China (English)

    LIU Qing; FENG Dongxin; WANG Xiaowu; DU Yongchen

    2007-01-01

    Hypersensitive response(HR)is one of the most efficient and common resistance mechanisms in plants.Cloning signaling genes are very important to elucidate the resistance mechanisms.A gene in tomato homologous to several resistance proteins in plant was involved in HR and named as RGL(Resistance Gene Like).RGL protein was used as a bait to screen interacting protein(s)from tomato cDNA library through the yeast two-hybrid system.Two interacting proteins were found,which were called as RGLIP-I and RGLIP2(RGL Interacting Protein),respectively.RGLIP-1 is a protein of 291 amino acids with significant homology with thylakoid lumen protein.RGLIP-2 is a protein of 248 amino acids with significant homology with transducin protein.Virus-Induced Gene Silencing(VIGS)of the two genes results in a partial and complete suppression of Avr4induced HR,which indicates that both genes are involved in hypersensitive response.

  7. Cloning of a gene localized and expressed at the ecdysteroid regulated puff 74EF in salivary glands of Drosophila larvae.

    Science.gov (United States)

    Möritz, T; Edström, J E; Pongs, O

    1984-02-01

    The puffing cycle of salivary gland chromosomes of Drosophila larvae, which initiates the developmental path to pupariation, is induced by ecdysteroid hormone. Its action leads to prominent puffs at loci 2B5, 74EF and 75B. Fragments of the 74EF puff of the D. melanogaster 3L chromosome were microdissected from salivary gland squashes. EcoRI-digested DNA of these fragments was cloned into lambda phage. Clones were screened with puff stage-specific cDNA probes. Thirteen out of 650 clones hybridized preferentially with puff stage 4-specific cDNA. The prominent early puffs at 74EF and 75B are most active between puff stage 4 and 6. Therefore, one of the 13 lambda phages was chosen for further analysis. It was used to isolate 24 kb of Drosophila DNA from genomic libraries. The DNA hybridized in situ to locus 74F. The 74F DNA coded for a transcript, which was made in salivary glands, but not in fat body of third instar larvae. It accumulated in K(C) cells in response to ecdysteroid treatment. The polyadenylated transcript size was 2.7 kb as judged by Nothern blot analysis. The transcription start site of the 74F gene has been mapped. Sequences upstream of the transcription site contain several sequence elements common to other eucaryotic genes, including potential Z-DNA forming sequences. Also, there is sequence homology to upstream sequences, which have been involved in the regulation of transcription of the salivary gland glue protein 4 gene.

  8. Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque

    Science.gov (United States)

    : Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

  9. Microarray analysis identifies a common set of cellular genes modulated by different HCV replicon clones

    OpenAIRE

    Gerosolimo Germano; Dallapiccola Bruno; Bruni Roberto; Ferraris Alessandro; Tataseo Paola; Tritarelli Elena; Marcantonio Cinzia; Ciccaglione Anna; Costantino Angela; Rapicetta Maria

    2008-01-01

    Abstract Background Hepatitis C virus (HCV) RNA synthesis and protein expression affect cell homeostasis by modulation of gene expression. The impact of HCV replication on global cell transcription has not been fully evaluated. Thus, we analysed the expression profiles of different clones of human hepatoma-derived Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system). Results First, we compared the expression profile of HCV replicon clone 21-5 ...

  10. Cloning and Sequence Analysis of Capsid Protein Gene of Iridovirus Indonesian Isolates

    Directory of Open Access Journals (Sweden)

    Murwantoko .

    2015-11-01

    Full Text Available generated by an Adobe application 11.5606 Iridovirus was known as agents that caused serious systemic disease in freshwater and marine fishes. The mortality up to 100% of orange-spotted grouper (Epinephelus coioides due to iridovirus infection has been reported in Indonesia. The gene encoding capsid protein of iridovirus is supposed to be conserved and has the potency for the development of control methods. The objectives of this study are to clone the gene encoding capsid protein iridovirus and to analyze their sequences. The   spleen tissues of orange-spotted grouper were collected and extracted their DNA. The DNA fragment of capsid protein of iridovirus genes were amplified by PCR using designed primers with the extraction DNA as templates. The amplified DNA fragments were cloned in pBSKSII and sequenced.  The genes encoding capsid protein of iridovirus from Jepara and Bali were successfully amplified and cloned. The Jepara clone (IJP03 contained complete open reading frame (ORF of the gene composed by 1362 bp nucleotides which encoded 453 amino acids. Those Jepara and Bali (IGD01 clones shared 99.8% similarity in nucleotide level and 99.4% at amino acid level. Based on those sequences, Indonesian iridovirus was belonged to genus Megalocystivirus and shared 99,6-99,9% similarity on nucleotide level with DGIV, ISKNV, MCIV, and ALIV Normal 0 36 false false false

  11. [A review of the genomic and gene cloning studies in trees].

    Science.gov (United States)

    Yin, Tong-Ming

    2010-07-01

    Supported by the Department of Energy (DOE) of U.S., the first tree genome, black cottonwood (Populus trichocarpa), has been completely sequenced and publicly release. This is the milestone that indicates the beginning of post-genome era for forest trees. Identification and cloning genes underlying important traits are one of the main tasks for the post-genome-era tree genomic studies. Recently, great achievements have been made in cloning genes coordinating important domestication traits in some crops, such as rice, tomato, maize and so on. Molecular breeding has been applied in the practical breeding programs for many crops. By contrast, molecular studies in trees are lagging behind. Trees possess some characteristics that make them as difficult organisms for studying on locating and cloning of genes. With the advances in techniques, given also the fast growth of tree genomic resources, great achievements are desirable in cloning unknown genes from trees, which will facilitate tree improvement programs by means of molecular breeding. In this paper, the author reviewed the progress in tree genomic and gene cloning studies, and prospected the future achievements in order to provide a useful reference for researchers working in this area.

  12. Construction of BAC Libraries from Flow-Sorted Chromosomes.

    Science.gov (United States)

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

    Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

  13. Algorithms and software tools for ordering clone libraries: application to the mapping of the genome of Schizosaccharomyces pombe.

    Science.gov (United States)

    Mott, R; Grigoriev, A; Maier, E; Hoheisel, J; Lehrach, H

    1993-04-25

    A complete set of software tools to aid the physical mapping of a genome has been developed and successfully applied to the genomic mapping of the fission yeast Schizosaccharomyces pombe. Two approaches were used for ordering single-copy hybridisation probes: one was based on the simulated annealing algorithm to order all probes, and another on inferring the minimum-spanning subset of the probes using a heuristic filtering procedure. Both algorithms produced almost identical maps, with minor differences in the order of repetitive probes and those having identical hybridisation patterns. A separate algorithm fitted the clones to the established probe order. Approaches for handling experimental noise and repetitive elements are discussed. In addition to these programs and the database management software, tools for visualizing and editing the data are described. The issues of combining the information from different libraries are addressed. Also, ways of handling multiple-copy probes and non-hybridisation data are discussed.

  14. Successful pod infections by Moniliophthora roreri result in differential Theobroma cacao gene expression depending on the clone's level of tolerance.

    Science.gov (United States)

    Ali, Shahin S; Melnick, Rachel L; Crozier, Jayne; Phillips-Mora, Wilberth; Strem, Mary D; Shao, Jonathan; Zhang, Dapeng; Sicher, Richard; Meinhardt, Lyndel; Bailey, Bryan A

    2014-09-01

    An understanding of the tolerance mechanisms of Theobroma cacao used against Moniliophthora roreri, the causal agent of frosty pod rot, is important for the generation of stable disease-tolerant clones. A comparative view was obtained of transcript populations of infected pods from two susceptible and two tolerant clones using RNA sequence (RNA-Seq) analysis. A total of 3009 transcripts showed differential expression among clones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes indicated shifts in 152 different metabolic pathways between the tolerant and susceptible clones. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) analyses of 36 genes verified the differential expression. Regression analysis validated a uniform progression in gene expression in association with infection levels and fungal loads in the susceptible clones. Expression patterns observed in the susceptible clones diverged in tolerant clones, with many genes showing higher expression at a low level of infection and fungal load. Principal coordinate analyses of real-time qRT-PCR data separated the gene expression patterns between susceptible and tolerant clones for pods showing malformation. Although some genes were constitutively differentially expressed between clones, most results suggested that defence responses were induced at low fungal load in the tolerant clones. Several elicitor-responsive genes were highly expressed in tolerant clones, suggesting rapid recognition of the pathogen and induction of defence genes. Expression patterns suggested that the jasmonic acid-ethylene- and/or salicylic acid-mediated defence pathways were activated in the tolerant clones, being enhanced by reduced brassinosteroid (BR) biosynthesis and catabolic inactivation of both BR and abscisic acids. Finally, several genes associated with hypersensitive response-like cell death were also induced in tolerant clones. © 2014

  15. Direct screening of libraries of yeast clones for alpha-amylase activity on raw starch hydrolysis.

    Science.gov (United States)

    Wong, Dominic W S; Batt, Sarah B; Lee, Charles C; Robertson, George H

    2003-10-01

    High-throughput screening for high-activity barley alpha-amylase mutants expressed in Saccharomyces cerevisiae is hampered by the interference of reducing agents, particularly the glucose used in yeast growth media. The present investigation employed colorimetric and chemiluminescent detection systems that enable direct and rapid screening of activities on raw starch substrate. Active clones could be separated into two groups, based on high total activity or high specific activity.

  16. Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3

    Energy Technology Data Exchange (ETDEWEB)

    Haiming Chen; Lalioti, M.D.; Perrin, G.; Antonarakis, S.E. [Univ. of Geneva Medical School (Switzerland)] [and others

    1996-07-01

    In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-binding cassette transporter gene family and is homologous to Drosophila w (and to 2 genes from other species) and to a lesser extent to Drosophila brown (bw) and scarlet (st) genes that are all involved in the transport of eye pigment precursor molecules. A DNA polymorphism with 62% heterozygosity due to variation of a poly (T) region in the 3{prime} UTR of the hW has been identified and used for the incorporation of this gene to the genetic map of chromosome 21. The hW is located at 21q22.3 between DNA markers D21S212 and D21S49 in a P1 clone that also contains marker BCEI. The gene is expressed at various levels in many human tissues. The contributions of this gene to the Down syndrome phenotypes, to human eye color, and to the resulting phenotypes of null or missense mutations are presently unknown. 56 refs., 8 figs., 1 tab.

  17. Cloning of xylanase gene of Streptomyces flavogriseus in Escherichia coli and bacteriophage lambda-induced lysis for the release of cloned enzyme.

    Science.gov (United States)

    Srivastava, R; Ali, S S; Srivastava, B S

    1991-03-01

    The xylanase gene of Streptomyces flavogriseus was cloned in pUC8 plasmid and expressed in Escherichia coli lysogenic for lambda cI857. lambda-Induced lysis of E. coli at 42 degrees C allowed efficient release of cloned enzyme activity in extracellular environment. The xylanase gene was located in the 0.8-kb HindIII fragment and coded for 18,000 Mr xylanase.

  18. Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos

    Directory of Open Access Journals (Sweden)

    Wang Ya-Ping

    2008-03-01

    Full Text Available Abstract Background Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. Results A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp in length, with a 342 bp open reading frame (ORF encoding a putative protein of 113 amino acids (aa. Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. Conclusion Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.

  19. Cloning and sequence analysis of hyaluronoglucosaminidase (nagH gene of Clostridium chauvoei

    Directory of Open Access Journals (Sweden)

    Saroj K. Dangi

    2017-09-01

    Full Text Available Aim: Blackleg disease is caused by Clostridium chauvoei in ruminants. Although virulence factors such as C. chauvoei toxin A, sialidase, and flagellin are well characterized, hyaluronidases of C. chauvoei are not characterized. The present study was aimed at cloning and sequence analysis of hyaluronoglucosaminidase (nagH gene of C. chauvoei. Materials and Methods: C. chauvoei strain ATCC 10092 was grown in ATCC 2107 media and confirmed by polymerase chain reaction (PCR using the primers specific for 16-23S rDNA spacer region. nagH gene of C. chauvoei was amplified and cloned into pRham-SUMO vector and transformed into Escherichia cloni 10G cells. The construct was then transformed into E. cloni cells. Colony PCR was carried out to screen the colonies followed by sequencing of nagH gene in the construct. Results: PCR amplification yielded nagH gene of 1143 bp product, which was cloned in prokaryotic expression system. Colony PCR, as well as sequencing of nagH gene, confirmed the presence of insert. Sequence was then subjected to BLAST analysis of NCBI, which confirmed that the sequence was indeed of nagH gene of C. chauvoei. Phylogenetic analysis of the sequence showed that it is closely related to Clostridium perfringens and Clostridium paraputrificum. Conclusion: The gene for virulence factor nagH was cloned into a prokaryotic expression vector and confirmed by sequencing.

  20. Cloning and DNA sequence analysis of a Lactococcus bacteriophage lysin gene.

    Science.gov (United States)

    Shearman, C; Underwood, H; Jury, K; Gasson, M

    1989-08-01

    A gene for the lysin of Lactococcus lactis bacteriphage phi vML3 was cloned using an Escherichia coli/bacteriophage lambda host-vector system. The gene was detected by its expression of antimicrobial activity against L. lactis cells in a bioassay. The cloned fragment was analysed by sub-cloning on to E. coli plasmid vectors and by restriction endonuclease and deletion mapping. Its entire DNA sequence was determined and an open reading frame for the lysin structural gene was identified. The sequenced lysin gene would express a protein of 187 amino acids with a molecular weight of 21,090, which is in good agreement with that of a protein detected after in vitro transcription and translation of DNA encoding the gene. Expression of the lysin gene in E. coli and B. subtilis from an adjacent bacteriophage promoter was readily detected but in L. lactis expression of lysin was found to be lethal. The bacteriophage phi vML3 lysin had sequence homology with protein 15 of B. subtilis bacteriophage PZA. This protein is involved in DNA packaging during bacteriophage maturation rather than in host cell lysis. The cloning and analysis of the phi vML3 lysin gene is of importance in further understanding lactic streptococcal bacteriophages, for the development of positive selection vectors and for biotechnological applications of relevance to the dairy industry.

  1. Cloning of a chitinase gene from Ewingella americana, a pathogen of the cultivated mushroom, Agaricus bisporus

    Directory of Open Access Journals (Sweden)

    P.W. Inglis

    2000-09-01

    Full Text Available We have isolated a gene encoding a chitinase (EC 3.2.1.14 from Ewingella americana, a recently described pathogen of the mushroom Agaricus bisporus. This gene, designated chiA (EMBL/Genbank/DDBJ accession number X90562, was cloned by expression screening of a plasmid-based E. americana HindIII genomic library in Escherichia coli using remazol brilliant violet-stained carboxymethylated chitin incorporated into selective medium. The chiA gene has a 918-bp ORF, terminated by a TAA codon, with a calculated polypeptide size of 33.2 kDa, likely corresponding to a previously purified and characterised 33-kDa endochitinase from E. americana. The deduced amino acid sequence shares 33% identity with chitinase II from Aeromonas sp. No. 10S-24 and 7.8% identity with a chitinase from Saccharopolyspora erythraeus. Homology to other chitinase sequences was otherwise low. The peptide sequence deduced from chiA lacks a typical N-terminal signal sequence and also lacks the chitin binding and type III fibronectin homology units common to many bacterial chitinases. The possibility that this chitinase is not primarily adapted for the environmental mineralisation of pre-formed chitin, but rather for the breakdown of nascent chitin, is discussed in the context of mushroom disease.O gene que codifica uma quitinase (EC 3.2.1.14 foi isolado de Ewingella americana, recentemente descrita como patógeno do cogumelo Agaricus bisporus. Este gene, denominado chiA (EMBL/Genebank/DDBJ número de acesso X9061, foi clonado e selecionado a partir de livraria genômica construída por digestão do DNA de E. americana com HindIII e ligação em plasmídio de expressão em E. coli, utilizando meio seletivo contendo quitina carboximetilada, corada com "remazol brilliant violet'' para seleção de clones. O gene chiA apresenta uma ORF de 918 bp, código terminador TAA, tendo o tamanho do polipeptídeo sido calculado como 33,2 kDa, o qual corresponde ao tamanho de 33 kDa da endoquitinase

  2. Identification of yak lactate dehydrogenase B gene variants by gene cloning

    Institute of Scientific and Technical Information of China (English)

    ZHENG YuCai; ZHAO XingBo; ZHOU Jing; PIAO Ying; JIN SuYu; HE QingHua; HONG Jian; LINing; WU ChangXin

    2008-01-01

    Native polyacrylamide gel electrophoresis showed that two types of lactate dehydrogenase (LDH) existed in yaks. Based on the electrophoresis characteristics of LDH isoenzymes, yak LDH variants were speculated to be the gene mutation on H subunit encoded by B gene. According to the mobility in electrophoresis, the fast-band LDH type was named LDH-Hf and the slow-band LDH type LDH-Hs. In order to reveal the gene alteration In yak LDH variants, total RNA was extracted from heart tissues of yaks with different LDH variants, and cDNAs of the two variants were reverse transcripted. Two variants of B genes were cloned by RT-PCR. Sequence analysis revealed that four nucleotides differed between LDH-Bf and LDH-Bs, which resulted in two amino acids alteration. By Deepview software analysis of the conformation of yak LDH1 variants and H subunit, these four nucleotides altered two amino acids that generated new hydrogen bonds to change the hydrogen bonds network, and further caused subtle conformstionsl changes between the two LDH variants.

  3. Identification of yak lactate dehydrogenase B gene variants by gene cloning

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Native polyacrylamide gel electrophoresis showed that two types of lactate dehydrogenase (LDH) existed in yaks. Based on the electrophoresis characteristics of LDH isoenzymes, yak LDH variants were speculated to be the gene mutation on H subunit encoded by B gene. According to the mobility in electrophoresis, the fast-band LDH type was named LDH-Hf and the slow-band LDH type LDH-Hs. In order to reveal the gene alteration in yak LDH variants, total RNA was extracted from heart tissues of yaks with different LDH variants, and cDNAs of the two variants were reverse transcripted. Two variants of B genes were cloned by RT-PCR. Sequence analysis revealed that four nucleotides differed between LDH-Bf and LDH-Bs, which resulted in two amino acids alteration. By Deepview software analysis of the conformation of yak LDH1 variants and H subunit, these four nucleotides altered two amino acids that generated new hydrogen bonds to change the hydrogen bonds network, and further caused subtle conformational changes between the two LDH variants.

  4. Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli.

    Science.gov (United States)

    Knietsch, Anja; Waschkowitz, Tanja; Bowien, Susanne; Henne, Anke; Daniel, Rolf

    2003-03-01

    Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C(2) to C(4)) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any

  5. Effect of methamidophos on soil fungi community in microcosms by plate count, DGGE and clone library analysis

    Institute of Scientific and Technical Information of China (English)

    LI Xinyu; ZHANG Huiwen; WU Minna; ZHANG Yan; ZHANG Chenggang

    2008-01-01

    Methamidophos was widely used a pesticide in northern China. The potential influences of methamidophos on soil fungal community in black soil were assessed by plate count, 28S rDNA-PCR-DGGE, and clone library analysis. Three methamidophos levels (50, 150, and 250 mg/kg) were tested in soil microcosms. Results from plate count during a 60-d microcosm experiment showed that high concentrations of methamidophos (250 mg/kg) could significantly stimulate fungal populations. DCGE (denaturing gradient gel electrophoresis) fingerprinting patterns showed a significant difference between the responses of culturable and total fungi communities under the stress of methamidophos. Shannon diversity indices calculated from DGGE profiles indicated that culturable fungi in all microcosms with methamidophos treatment increased after 1 week of incubation. However, the diversity indices of total fungi decreased in the first week, as compared to the stimulation of culturable fungi. At the 8th week, however, all the microcosms treated by methamidophos were similar to the control microcosms in community structure as suggested by the Shannon diversity indices for both culturable and total fungi. In contrast, after 1 week the fungal structure of culturable and unculturable both were disturbed to different extent under the stresses of methamidophos by clustering analysis. Clone sequencing analysis indicated the stimulation of pathogenic and unculturable fungal populations by methamidophos treatment, suggetsing potential risks of plant disease outbreak.

  6. Assessment of the fungal diversity and succession of ligninolytic endophytes in Camellia japonica leaves using clone library analysis.

    Science.gov (United States)

    Hirose, Dai; Matsuoka, Shunsuke; Osono, Takashi

    2013-01-01

    Fungal assemblages in live, newly shed and partly decomposed leaves of Camellia japonica were investigated with a clone library analysis to assess the fungal diversity and succession in a subtropical forest in southern Japan. Partly decomposed leaves were divided into bleached and adjacent nonbleached portions to estimate the fungi functionally associated with lignin decomposition in the bleached portions, with an emphasis on Coccomyces sinensis (Rhytismataceae, Ascomycota). From 144 cloned 28S ribosomal DNA (rDNA) sequences, 48 operational taxonomic units (OTUs) were defined based on a sequence similarity threshold of 98%. Forty-one (85%) of the 48 OTUs belonged to the Ascomycota and seven OTUs (15%) to the Basidiomycota. Twenty-six OTUs (54%) were detected only once (singletons). The number of OTUs and the diversity indices of the fungal assemblages in the different leaves were in this order: live leaves > newly shed leaves > bleached portions > nonbleached portions of partly decomposed leaves. The fungal assemblages were similar in newly shed leaves and the bleached portions of partly decomposed leaves. Ligninolytic fungi of the genera Coccomyces, Lophodermium and Xylaria were frequently detected in the bleached portions. OTU3, identified as Coccomyces sinensis, was detected in live and newly shed leaves and the bleached portions of partly decomposed leaves, suggesting that this fungus latently infects live leaves, persists after leaf fall and takes part in lignin decomposition.

  7. Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers

    NARCIS (Netherlands)

    Brons, Jolanda; van Elsas, J.D.

    2008-01-01

    To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel

  8. Construction of a yeast artificial chromosome contig encompassing the human acidic fibroblast growth factor (FGF1) gene: Toward the cloning of the ANLL/MDS tumor-suppressor gene

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Ing-Ming; Gilmore, E.C.; Liu, Yang; Payson, R.A. (Ohio State Univ., Columbus, OH (United States))

    1994-02-01

    The region surrounding the human acidic fibroblast growth factor (FGF1) locus on chromosome 5q31 is of particular interest since it represents a critical region consistently lost in acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS) patients who have a demonstrable deletion of the distal portion of the long arm of chromosome 5. It is proposed that an ANLL/MDS leukemia suppressor gene resides on 5q31. The authors have previously shown that the gene is most likely localized between FGF1 and PDGFRB/CSF1R loci. The region has also been linked to at least four other genetic diseases, Treacher Collins syndrome, diastrophic dysplasia, limb-girdle muscular dystrophy, and an autosomal dominant deafness, by linkage analysis. Here, they describe yeast artificial chromosomes (YAC) spanning 450 kb around the FGF1 gene. Six YAC clones were isolated from a human YAC library and their restriction enzyme maps were determined. The overlap of the clones with each other and with FGF1 cosmid and phage clones was characterized. Three of the YAC clones were found to contain the entire FGF1 gene, which spans more than 100 kb. Proximal and distal ends of several of these YAC clones were isolated for further overlap cloning. The proximal ends of both Y2 and Y4 were localized to previously isolated FGF1 DNA by sequence analysis. The distal ends of these two clones also hybridized to a human-hamster hybrid containing chromosome 5 as the only human genetic material. These results suggest that these YAC clones represent colinear DNA around the FGF1 locus. None of the YAC clones were found to contain the CD 14 and GRL genes, the closest known proximal and distal markers (relative to the centromere) to the FGF1 gene, respectively. This contig is useful for the overlap cloning of the 5q31 region and for reverse genetic strategies for the isolation of disease genes in the region. 46 refs., 7 figs., 5 tabs.

  9. Identification of polybacterial communities in patients with postoperative, posttraumatic, and endogenous endophthalmitis through 16S rRNA gene libraries.

    Science.gov (United States)

    Jayasudha, Rajagopalaboopathi; Narendran, Venkatapathy; Manikandan, Palanisamy; Prabagaran, Solai Ramatchandirane

    2014-05-01

    Endophthalmitis is a potential vision-threatening complication following surgical procedures (postoperative endophthalmitis [POE]), trauma (posttraumatic endophthalmitis [PTE]), and bacteremic seeding of the eye from a distant infection site (endogenous endophthalmitis [EE]). Several studies have revealed the polybacterial characteristics of endophthalmitis, which make the administration of antibiotics to treat the disease challenging. However, until now, the polybacterial communities of POE, PTE, and EE have not been precisely studied. Hence, the present study was designed to identify the bacterial community of endophthalmitis through 16S rRNA gene libraries. Of the 40 intraocular samples tested, 30 libraries were constructed with bacterial nested-PCR-positive samples. The obtained recombinant clones were screened through amplified rRNA gene restriction analysis (ARDRA) to identify unique clones. The multiple types of ARDRA patterns (P=0.345) and diverse bacterial sequences (P=0.277) within the libraries revealed the polybacterial nature of infection in POE, PTE, and EE. Moreover, to the best of our knowledge, this is the first report on polybacterial infection in EE. Gram-positive bacteria, including Bacillus spp. (n=19), Streptococcus spp. (n=18), Staphylococcus spp. (n=6), Exiguobacterium spp. (n=3), Gemella spp. (n=2), Enterococcus spp. (n=2), a Lysinibacillus sp. (n=1), a Clostridium sp. (n=1), and a Nocardia sp. (n=1), and Gram-negative bacteria, including Serratia spp. (n=18), Pseudomonas spp. (n=10), Enterobacter spp. (n=8), Acinetobacter spp. (n=3), Pantoea spp. (n=3), a Haemophilus sp. (n=1), and a Massilia sp. (n=1), were identified. Interestingly, among them, 10 bacterial species were not previously reported to be associated with endophthalmitis or other ocular infections. Besides, the presence of 4 unidentifiable clones suggests the possibility of novel organisms that might cause eye infections. Therefore, it is recommended that, in addition to the

  10. Cloning and localization of vip3A gene of Bacillus thuringiensis.

    Science.gov (United States)

    Wu, Zeng Ling; Guo, Wen Yi; Qiu, Jun Zhi; Huang, Tian Pei; Li, Xun Bo; Guan, Xiong

    2004-09-01

    An insecticidal protein gene, vip3A, was cloned from Bacillus thuringiensis strain WB50. The nucleotide sequence of 2,460 bp (GenBank acc. No. AY295778) showed 99% homology with the known vip3A genes. Using specific primers for vip3A gene, PCR was performed to demonstrate that the gene was not located on the bacterial chromosome and this was confirmed by Southern blotting using an internal fragment (486 bp) from vip3A gene as a probe. The gene was carried on a plasmid of 31.8 kb.

  11. Cloning and alignment of WaaF gene of Campylobacter jejuni Lulei

    Directory of Open Access Journals (Sweden)

    XING Cong-cong

    2012-04-01

    Full Text Available Objective To clone the WaaF gene of Campylobacter jejuni, and analyse its relationship with WaaF genetic evolution. Methods Amplified WaaF gene of Campylobacter jejuni Lulei by PCR, and constructed pGEM-T-WaaF cloning plasmid. Downloaded five WaaF associated with Guillain-Barré syndrome (GBS and one WaaF not associated with GBS, and then constructed phylogenetic tree. Results pGEM-T-WaaF cloning plasmid was constructed successfully. WaaF presented cluster phenomenon in Campylobacter jejuni associated with GBS. Conclusion WaaF gene of Campylobacter jejuni Lulei is the fragment of 807 bp, and has the nearest relationship with the genetic evolution of Lichang.

  12. Cloning and expression of swine myostatin gene and its application in animal immunization trial

    Institute of Scientific and Technical Information of China (English)

    MA; Xianyong; CAO; Yongchang; SHU; Dingming; BI; Yingzuo

    2005-01-01

    We have amplified swine myostatin (MSTN) gene by reverse transcription polymerase chain reaction (RT-PCR) and cloned it into pGEM-T Easy vector. The cloned swine MSTN gene consists of 1128 nucleotides, which has been submitted to GenBank (acquired registered code- AY448008). The cloned swine MSTN gene was successfully expressed in E. coli without the first 25 amino acids. Crude extraction of the expressed recombinant MSTN protein was used to immunize mice to investigate the effects on their bodyweights. We show here that the body weights of the immunized mice were higher than that of the controls, even though the difference was not significant. Surprisingly, the progenies of the immunized mice also were heavier than the controls. Especially at day 3, the average body weight of the immunized mice was 10.5% higher than that of the controls , which is significant (p < 0.05).

  13. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov

    2008-01-01

    Background: The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion...... of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results: Here, we present a USER Friendly cloning based...... technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium...

  14. Specific genetic modifications of domestic animals by gene targeting and animal cloning

    Directory of Open Access Journals (Sweden)

    Zhou Jiangfeng

    2003-11-01

    Full Text Available Abstract The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined.

  15. Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; You-Yong Lu

    2002-01-01

    AIM: To develop and optimize cDNA representationaldifference analysis (cDNA RDA) method and to identify andclone garlic up-regulated genes in human gastric cancer(HGC) cells.METHODS: We performed cDNA RDA method by usingabundant double-stranded cDNA messages provided by twoself-constructed cDNA libraries (Allitridi-trested and paternalHGC cell line BGC823 cells cDNA libraries respectively).BamH Ⅰ and Xho I restriction sites harbored in the libraryvector were used to select representations. Northern andSlot blots analyses were employed to identify the obtaineddifference products.RESJLTS: Fragments released from the cDNA library vectorafter restriction endonuclease digestion acted as goodmarker indicating the appropriate digestion degree for libraryDNA. Two novel expressed sequence tags (ESTs) and arecombinant gene were obtained. Slot blots result showed a8-fold increase of gila-derived nexin/protease nexin 1 (GDN/PN1 ) gene expression level and 4-fold increase of hepatitis Bvirus x-interacting protein (XIP) mRNA level in BGC823 cellsafter Allitridi treatment for 72 h.CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAsinduced by Allitridi provide valuable molecular evidence forelucidating the garlic' s efficacies against neurodegenerativeand inflammatory diseases. Isolation of a recombinant geneand two novel ESTs further show cDNA RDA based on cDNAlibraries to be a powerful method with high specificity andreproducibility in cloning differentially expressed genes.

  16. Using Transcriptomics to Identify Differential Gene Expression in Response to Salinity among Australian Phragmites australis Clones.

    Science.gov (United States)

    Holmes, Gareth D; Hall, Nathan E; Gendall, Anthony R; Boon, Paul I; James, Elizabeth A

    2016-01-01

    Common Reed (Phragmites australis) is a frequent component of inland and coastal wetlands in temperate zones worldwide. Ongoing environmental changes have resulted in the decline of this species in many areas and invasive expansion in others. In the Gippsland Lakes coastal waterway system in south-eastern Australia, increasing salinity is thought to have contributed to the loss of fringing P. australis reed beds leading to increased shoreline erosion. A major goal of restoration in this waterway is to address the effect of salinity by planting a genetically diverse range of salt-tolerant P. australis plants. This has prompted an interest in examining the variation in salinity tolerance among clones and the underlying basis of this variation. Transcriptomics is an approach for identifying variation in genes and their expression levels associated with the exposure of plants to environmental stressors. In this paper we present initial results of the first comparative culm transcriptome analysis of P. australis clones. After sampling plants from sites of varied surface water salinity across the Gippsland Lakes, replicates from three clones from highly saline sites (>18 g L(-1) TDS) and three from low salinity sites (<6 g L(-1)) were grown in containers irrigated with either fresh (<0.1 g L(-1)) or saline water (16 g L(-1)). An RNA-Seq protocol was used to generate sequence data from culm tissues from the 12 samples allowing an analysis of differential gene expression. Among the key findings, we identified several genes uniquely up- or down-regulated in clones from highly saline sites when irrigated with saline water relative to clones from low salinity sites. These included the higher relative expression levels of genes associated with photosynthesis and lignan biosynthesis indicative of a greater ability of these clones to maintain growth under saline conditions. Combined with growth data from a parallel study, our data suggests local adaptation of certain clones to

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

    Science.gov (United States)

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

    2008-02-01

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

  18. Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    易继林; 田耕

    2003-01-01

    To clone the murine α-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine α-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. A fter transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

  19. Molecular cloning and gene expression analysis of cystatin C-like proteins in spinyhead croaker Collichthys lucidus.

    Science.gov (United States)

    Song, W; Jiang, K J; Zhang, F Y; Zhao, M; Ma, L B

    2016-03-24

    Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. In this study, a cDNA library was constructed from Collichthys lucidus using the SMART technique. A complete cDNA sequence with high identity to the conserved sequence of the cystatin C gene was cloned from the library using EST analysis and rapid amplification of cDNA ends (RACE), then subjected to further investigation. The full-length cDNA of cystatin C from C. lucidus (Clcys) was 699 bp long, including a 5'-terminal untranslated region (5'-UTR) of 52 bp, a 3'-UTR of 290 bp, and an open-reading frame of 357 bp. The gene encoded a polypeptide of 118 amino acids, constituting a predicted molecular weight of 12.875 kDa and a theoretical isoelectric point of 8.81. The amino acid sequence of Clcys possessed typical features of type II cystatins and had the highest identity with cystatin C of Pseudosciaena crocea (89%); therefore, it clustered with the cystatin C group in the UPGMA phylogenetic tree. Quantitative real-time reverse transcription analysis revealed that the highest expression was found in the kidney, followed by the liver, heart, and testis, with the lowest expression in muscle. Interestingly, Clcys had relatively low identity with cystatin C genes from other fish and mammals, and its expression pattern did not possess features of a housekeeping gene. Based on these findings, we suspect that the classification of cystatins in fish is somewhat confusing, and the identification of more cystatin gene sequences is needed before a definite conclusion can be drawn.

  20. Construction of a library of cloned short tandem repeat (STR) alleles as universal templates for allelic ladder preparation.

    Science.gov (United States)

    Wang, Le; Zhao, Xing-Chun; Ye, Jian; Liu, Jin-Jie; Chen, Ting; Bai, Xue; Zhang, Jian; Ou, Yuan; Hu, Lan; Jiang, Bo-Wei; Wang, Feng

    2014-09-01

    Short tandem repeat (STR) genotyping methods are widely used for human identity testing applications, including forensic DNA analysis. Samples of DNA containing the length-variant STR alleles are typically separated and genotyped by comparison to an allelic ladder. Here, we describe a newly devised library of cloned STR alleles. The library covers alleles X and Y for the sex-determining locus Amelogenin and 259 other alleles for 22 autosomal STR loci (TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01, vWA, D13S317, D16S539, D18S51, D21S11, D2S1338, D6S1043, D12S391, Penta E, D19S433, D11S4463, D17S974, D3S4529 and D12ATA63). New primers were designed for all these loci to construct recombinant plasmids so that the library retains core repeat elements of STR as well as 5'- and 3'-flanking sequences of ∼500 base pairs. Since amplicons of commercial STR genotyping kits and systems developed in laboratories are usually distributed from 50 to STR alleles. The sequencing results showed all repeat structures we obtained for TPOX, CSF1PO, D7S820, TH01, D16S539, D18S51 and Penta E were the same as reported. However, we identified 102 unreported repeat structures from the other 15 STR loci, supplementing our current knowledge of repeat structures and leading to further understanding of these widely used loci.

  1. Molecular cloning, gene structure and expression profile of two mouse peroxisomal 3-ketoacyl-CoA thiolase genes

    Directory of Open Access Journals (Sweden)

    Latruffe Norbert

    2004-03-01

    Full Text Available Abstract Background In rats, two peroxisomal 3-ketoacyl-CoA thiolase genes (A and B have been cloned, whereas only one thiolase gene is found in humans. The aim of this study was thus to clone the different mouse thiolase genes in order to study both their tissue expression and their associated enzymatic activity. Results In this study, we cloned and characterized two mouse peroxisomal 3-ketoacyl-CoA thiolase genes (termed thiolase A and B. Both thiolase A and B genes contain 12 exons and 11 introns. Using RNA extracted from mouse liver, we cloned the two corresponding cDNAs. Thiolase A and B cDNAs possess an open reading frame of 1272 nucleotides encoding a protein of 424 amino acids. In the coding sequence, the two thiolase genes exhibited ≈97% nucleotide sequence identity and ≈96% identity at the amino acid level. The tissue-specific expression of the two peroxisomal 3-ketoacyl-CoA thiolase genes was studied in mice. Thiolase A mRNA was mainly expressed in liver and intestine, while thiolase B mRNA essentially exhibited hepatic expression and weaker levels in kidney, intestine and white adipose tissue. Thiolase A and B expressions in the other tissues such as brain or muscle were very low though these tissues were chiefly involved in peroxisomal disorders. At the enzymatic level, thiolase activity was detected in liver, kidney, intestine and white adipose tissue but no significant difference was observed between these four tissues. Moreover, thiolase A and B genes were differently induced in liver of mice treated with fenofibrate. Conclusion Two mouse thiolase genes and cDNAs were cloned. Their corresponding transcripts are mostly expressed in the liver of mice and are differently induced by fenofibrate.

  2. Cloning of Dense Granular (GRA 7 Gene of Toxoplasma gondii into pTZ57RT Vectors for Sub-Cloning in Prokaryotic and Eukaryotic Plasmids

    Directory of Open Access Journals (Sweden)

    Zahra Arab-Mazar

    2014-11-01

    Full Text Available Background: Serological assay based on dense granular (GRA proteins of Toxoplasma gondii (T. gondii is actually the most popular laboratory diagnostic tool to detection of toxoplasmosis. We aimed to construct a recombinant GRA7-pTZ57RT plasmid vectors that it is suitable for sub-cloning and GRA7 protein production.Materials and Methods: Souris mice were used for maintaining of T. gondii tachyzoites by serial intraperitoneal passage. The tachyzoites’ DNA was extracted, and the GRA7 gene was amplified by PCR. The purified DNA was inserted into pTZ57RT cloning vectors, and then transformed into TOP10 competent cells. Finally, cloning and transformation were confirmed by restriction enzymatic digestion and gene sequencing.Results: Agarose gel electrophoresis analysis on PCR products of genomic DNA, revealed 726 bp bands that were equal to the GRA7 gene. Both white (recombinant and blue (non-recombinant colonies appeared on ampicillin-LB agar. Results of enzymatic digestion and gene sequencing confirmed successful cloning and transformation procedures.Conclusion: The GRA7 gene of T. gondii was cloned into pTZ57RT plasmid, which is suggested to be further used as DNA vaccine or sub-cloned for production of recombinant GRA7 protein.

  3. Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

    Institute of Scientific and Technical Information of China (English)

    LI Bi-feng; MAO Hua-ming; YANG Shu-Li; ZHU Ya-xin; GU Zhao-bing; CHEN Yuan; LENG Jing; GOU Xiao; FENG Li; LI Qing; XI Dong-mei

    2016-01-01

    Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as wel as reeds and other plant species, and grow to higher mature live weights than Yunnan Yelow cattle maintained in similar harsh environments. The aim of this study was to identify speciifc celulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38400 clones with an average insert size of 35.5 kb. TheUmcel-1 gene was isolated from this library. Investigation of the celulase activity of 24 random clones led to the identiifcation of theUmcel-1 gene, which exhibited the most potent celulase activity. Sequencing theUmcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putativegene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the celulase (GenBank accession no. YP_004310852.1) fromClostridium lentocelum DSM 5427, with 44% identity and 62% similarity. TheUmcel-1 gene was heterologously expressed inEscherichia coli BL21, and recombinant Umcel-1 was puriifed. The activity of puriifed recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl celulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the ifrst evidence for a celulase produced by bacteria in gayal rumen.

  4. Identification and functional analysis of a new glyphosate resistance gene from a fungus cDNA library.

    Science.gov (United States)

    Tao, Bo; Shao, Bai-Hui; Qiao, Yu-Xin; Wang, Xiao-Qin; Chang, Shu-Jun; Qiu, Li-Juan

    2017-08-01

    Glyphosate is a widely used broad spectrum herbicide; however, this limits its use once crops are planted. If glyphosate-resistant crops are grown, glyphosate can be used for weed control in crops. While several glyphosate resistance genes are used in commercial glyphosate tolerant crops, there is interest in identifying additional genes for glyphosate tolerance. This research constructed a high-quality cDNA library form the glyphosate-resistant fungus Aspergillus oryzae RIB40 to identify genes that may confer resistance to glyphosate. Using a medium containing glyphosate (120mM), we screened several clones from the library. Based on a nucleotide sequence analysis, we identified a gene of unknown function (GenBank accession number: XM_001826835.2) that encoded a hypothetical 344-amino acid protein. The gene was named MFS40. Its ORF was amplified to construct an expression vector, pGEX-4T-1-MFS40, to express the protein in Escherichia coli BL21. The gene conferred glyphosate tolerance to E. coli ER2799 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Cloning, chromosomal localization, SNP detection and association analysis of the porcine IRS-1 gene.

    Science.gov (United States)

    Niu, P-X; Huang, Z; Li, C-C; Fan, B; Li, K; Liu, B; Yu, M; Zhao, S-H

    2009-11-01

    Insulin receptor substrate-1(IRS-1) gene is one member of the Insulin receptor substrate (IRS) gene family, which plays an important role in mediating the growth of skeletal muscle and the molecular metabolism of type 2 diabetes. Here, we cloned a 3,573 bp fragment of the partial CDS sequence of porcine IRS-1 gene by in silicon cloning strategy and RT-PCR method. The porcine IRS-1 gene was assigned to SSC15q25 by using IMpRH. Sequencing of PCR products from Duroc and Tibetan pig breeds identified one SNP in exon 1 of porcine IRS-1 gene (C3257A polymorphisms). Association analysis of genotypes with the growth traits, anatomy traits, meat quality traits and physiological biochemical indexes traits showed that different genotypes at locus 3,257 of IRS-1 have significant differences in carcass straight length in pigs (P = 0.0102 \\ 0.05).

  6. Cloning of TRP Gene of Bombyx mori%家蚕TPR基因的克隆

    Institute of Scientific and Technical Information of China (English)

    牛伟涛

    2011-01-01

    [ Objective ] The aim was to clone Bornbyx rnori TRP gene. [ Method ] The total RNA of Bombyx mori was extracted during its pupal pedod with Trizol method, and then the TRP gene was cloned by RT-PCR. [ Result] The full-length cDNA of TRP gene was successfully obtained, and the ORF fragment (858 bp) of TRP gene was cloned by PCR. [ Conclusion] TRP gene of Bombyx mori was cloned for the first time,which could provide solid basis for the study on its function.%[目的]克隆家蚕TPR基因,为研究该基因功能提供依据.[方法]利用Trizol法提取家蚕蛹期总RNA,再用RT-PCR的方法对家蚕TPR基因进行克隆.[结果]成功得到家蚕TPR基因全cDNA序列,并用PCR扩增得到了TPR基因的ORF,全长858 bp.[结论]首次对家蚕TPR家族基因进行了克隆,为今后对该基因功能研究奠定了坚实的基础.

  7. Cloning of TRP Gene of Bombyx mori%家蚕TPR基因的克隆

    Institute of Scientific and Technical Information of China (English)

    牛伟涛

    2011-01-01

    [ Objective ] The aim was to clone Bombyx mori TRP gene. [ Method ] The total RNA of Bombyx mori was extracted during its pupal period with Trizol method, and then the TRP gene was cloned by RT-PCR. [ Result] The full-length cDNA of TRP gene was successfully obtained, and the ORF fragment (858 bp) of TRP gene was cloned by PCR. [Conclusion] TRP gene of Bombyx mori was cloned for the first time, which could provide solid basis for the study on its function.%[目的]克隆家蚕TPR基因,为研究该基因功能提供依据.[方法]利用Trizol法提取家蚕蛹期总RNA,再用RT-PCR方法对家蚕TPR基因进行克隆.[结果]成功得到家蚕TPR基因全cDNA序列,并用PCR扩增得到了TPR基因的ORF,全长858 bp.[结论]首次对家蚕TPR家族基因进行了克隆,为今后对该基因功能研究奠定了坚实的基础.

  8. Exression and Cloning of Apoptosis-related Gene and Its Association with Hepatocellular Carcinoma in Qidong

    Institute of Scientific and Technical Information of China (English)

    LUDongdong; ZHANGXiran; 等

    2002-01-01

    Objective:To explore the molecular basis of hepatocarcinogenesis by cloning and expressing a novel liver cancer apoptosis -related gene.Methods:With homologous screening and RT-PCR,we had cloned an apoptosis-related gene APG from liver cancer cells,compared its expression in hepatocellular carcinoma(HCC) tissue and paracarcinoma tissue,and analyzed its sequence from these tissues.The association of APG gene expression with HCC was investigated.Results:A new gene APG was cloned with a full-legth cDNA of 563 bp.Sequencing analysis showed heterogeneity of APG gene from hepatocarcinoma tissue and from paracarcinoma tissue.Among 50 cases of liver cancer,APG gene expressions were down-regulated in 42 cases(84%) ,while up-regulated in 8 cases(16%,P0.05).Conclusion APG is an appoptosis-relate gene and down-regualted in HCC.Its expression is associated with many clinical and pathologic features of HCC,suggesting that APG gene is probably involved in the tumorigenesis of HCC.

  9. Cloning and Expression of the Lactococcus lactis purDEK Genes, Required for Growth in Milk

    DEFF Research Database (Denmark)

    Nilsson, Dan; Kilstrup, Mogens

    1998-01-01

    An operon containing the genes purD and purE and part of the purK gene was cloned from the facultative anaerobic gram positive bacterium Lactococcus lactis by complementation of the purD mutation in Escherichia coli SO609. The genes encode enzymes in the de novo pathway of purine nucleotides....... The expression of the genes was regulated approximately 35-fold at the transcription level by the availability of purines in the growth medium. Deletion analysis of the nucleotide region upstream of purD indicated that a region of 145 bp is enough to give regulated expression of the reporter lacLM genes, which...

  10. Development of a Gene Cloning System in Methanogens.

    Science.gov (United States)

    1987-03-27

    resistance genes, and genes coding for enzymes that produce colored products will be tested as markers for plasmid transformation. A functional plasmid... Halobacterium volcanii 99 0 0 0 Escherichia coli 216 0 0 0 None 348 0 0 0 abbreviations: FU, 5-fluorouracil; MP, 6-mercaptopurine. Colonies were

  11. Toward functional genomics in bacteria: analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus.

    Science.gov (United States)

    Rondon, M R; Raffel, S J; Goodman, R M; Handelsman, J

    1999-05-25

    As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.

  12. Cloning and Expression Pattern of a Gene Encoding a Putative Plastidic ATP/ADP Transporter from Helianthus tuberosus L.

    Institute of Scientific and Technical Information of China (English)

    Kun MENG; Tuan-Jie CHANG; Xiang LIU; Song-Biao CHEN; Yong-Qin WANG; Ai-Jun SUN; Hong-Lin XU; Xiao-Li WEI; Zhen ZHU

    2005-01-01

    Herein, we report the cloning and molecular characterization of a full cDNA encoding a putative plastidic ATP/ADP transporter, designated HtAATP, for Helianthus tuberosus L. The ATP/ADP translocator protein was isolated from the tuber-cDNA library of H. tuberosus for the first time. The predicted HtAATP protein was judged as a plastidic ATP/ADP translocator protein from its high homology at the amino acid sequence level to the two Arabidopsis thaliana plastidic ATP/ADP translocator proteins AATP1 and AATP2 (84.8% and 79.9% identity, respectively). Amino acid sequence analysis of the primary structure of HtAATP revealed that it belonged to the plastidic ATP/ADP transporter family. Hydropathy prediction indicated that HtAATP gene product is a highly hydrophobic membrane protein that contains 10 transmembrane domains to form a spanning topology. Southern blotting analysis showed that the HtAATP gene is a single-copy gene in the H. tuberosus genome. Tissue distribution analysis showed that the HtAATP gene is prominently expressed in sink tissues. A stable expression pattern in tubers at different developmental stages implies an active involvement of HtAATP during carbohydrate formation.

  13. Cloning, expression, and purification of a new antibacterial substance gene from larvae of Musca domestica (Diptera: Muscidae).

    Science.gov (United States)

    Pei, Zhihua; Bian, Lu; Zhang, Hui; Gao, Yunhang; Ma, Hongxia

    2014-01-01

    Musca domestica L. (Diptera: Muscidae), the housefly, exhibits unique immune defenses and can produce antibacterial substances upon stimulation with bacteria. On the basis of the cDNA library constructed using the suppression subtractive hybridization method, a 1188-bp antibacterial substance gene, which we named AS566, was amplified by rapid amplification of cDNA ends from M. domestica larva stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In this study, the full-length AS566 gene was cloned and inserted into a His-tagged Escherichia coli (Enterobacteriaceae: Escherichia) prokaryotic expression system to enable production of the recombinant protein. The recombinant AS566 protein was purified in denatured form from inclusion bodies and renatured to obtain functionally active AS566 protein. The bacteriostatic activity of the recombinant purified AS566 protein was assessed using the Oxford plate assay system and the results indicated that AS566 had antibacterial activity against six bacteria, including an E. coli clinical isolate, S. pullorum, Streptococcus bovis (Streptococcaceae: Streptococcus), Streptococcus suis, and Staphylococcus aureus (Staphylococcaceae: Staphylococcus) in vitro. The antibacterial activity of AS566 toward Gram- bacteria was two times greater than that against Gram+ bacteria. The sequencing results and BLAST analysis showed that the antibacterial substance gene AS566 was not homologous to any other antibacterial substance genes in GenBank. The antibacterial mechanisms of the newly discovered AS566 protein warrant further study.

  14. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and aminoacid homology with the yeast DNA repair gene RAD10.

    NARCIS (Netherlands)

    M. van Duin (Mark); J. de Wit (Jan); H. Odijk (Hanny); A. Westerveld (Andries); A. Yasui (Akira); M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1986-01-01

    textabstractThe human excision repair gene ERCC-7 was cloned after DNA mediated gene transfer to the CHO mutant 43-38, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-7 cDNA and partial characterization of the gene. ERCC.1 has a size

  15. Heterologous expression of Avermectins biosynthetic gene cluster by construction of a Bacterial Artificial Chromosome library of the producers

    Directory of Open Access Journals (Sweden)

    Qian Deng

    2017-03-01

    Full Text Available Avermectins, a group of polyketide natural products, are widely used as anthelmintics in agriculture. Metabolic engineering and combinatorial biosynthesis were extensively employed to improve Avermectins production and create novel Avermectin derivatives, including Ivermectin and Doramectin. It is labor intensive and time cost to genetically manipulate Avermectins producer Streptomyces avermitilis in vivo. Cloning and heterologous expression of Avermectins biosynthetic gene cluster will make it possible to tailor the cluster in vitro. We constructed a Bacterial Artificial Chromosome (BAC library of S. avermitilis ATCC 31267 with inserted DNA fragments ranged from 100 to 130 Kb. Five recombinant BAC clones which carried the Avermectins biosynthetic gene cluster ave (81 Kb in size were screened out from the library. Then, ave was hetero-expressed in S. lividans. Three Avermectin components, A2a, B1a and A1a were detected from the cell extracts of recombinant strains. It will facilitate the development of Avermectin derivatives by polyketide synthase domain swapping and provide functional element for Avermectins synthetic biology study.

  16. Cloning of novel rice blast resistance genes from two rapidly evolving NBS-LRR gene families in rice.

    Science.gov (United States)

    Guo, Changjiang; Sun, Xiaoguang; Chen, Xiao; Yang, Sihai; Li, Jing; Wang, Long; Zhang, Xiaohui

    2016-01-01

    Most rice blast resistance genes (R-genes) encode proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. Our previous study has shown that more rice blast R-genes can be cloned in rapidly evolving NBS-LRR gene families. In the present study, two rapidly evolving R-gene families in rice were selected for cloning a subset of genes from their paralogs in three resistant rice lines. A total of eight functional blast R-genes were identified among nine NBS-LRR genes, and some of these showed resistance to three or more blast strains. Evolutionary analysis indicated that high nucleotide diversity of coding regions served as important parameters in the determination of gene resistance. We also observed that amino-acid variants (nonsynonymous mutations, insertions, or deletions) in essential motifs of the NBS domain contribute to the blast resistance capacity of NBS-LRR genes. These results suggested that the NBS regions might also play an important role in resistance specificity determination. On the other hand, different splicing patterns of introns were commonly observed in R-genes. The results of the present study contribute to improving the effectiveness of R-gene identification by using evolutionary analysis method and acquisition of novel blast resistance genes.

  17. Using transcriptomics to identify differential gene expression in response to salinity among Australian Phragmites australis clones

    Directory of Open Access Journals (Sweden)

    Gareth Donald Holmes

    2016-04-01

    Full Text Available Common Reed (Phragmites australis is a frequent component of inland, and coastal, wetlands in temperate zones worldwide. Ongoing environmental changes have resulted in the decline of this species in many areas and invasive expansion in others. In the Gippsland Lakes coastal waterway system in south-eastern Australia, increasing salinity is thought to have contributed to the loss of fringing P. australis reed beds leading to increased shoreline erosion. A major goal of restoration in this waterway is to address the effect of salinity by planting a genetically-diverse range of salt-tolerant P. australis lineages. This has prompted an interest in examining the variation in salinity tolerance among lineages and the underlying basis of this variation. Transcriptomics is an approach for identifying variation in genes and their expression levels associated with the exposure of plants to environmental stressors. In this paper we present initial results of the first comparative culm transcriptome analysis of P. australis clones. After sampling plants from sites of varied surface water salinity across the Gippsland Lakes, replicates from three clones from highly saline sites (>18 g L-1 TDS and three from low salinity sites (<6 g L-1 were grown in containers irrigated with either fresh (<0.1 g L-1 or saline water (16 g L-1. An RNA-Seq protocol was used to generate sequence data from culm tissues from the 12 samples allowing an analysis of differential gene expression. Among the key findings, we identified several genes uniquely up- or down-regulated in clones from highly saline sites when irrigated with saline water relative to clones from low salinity sites. These included the relative higher expression levels of genes associated with photosynthesis and lignan biosynthesis indicative of a greater ability of these clones to maintain growth under saline conditions. Combined with growth data from a parallel study, our data suggests local adaptation of

  18. Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique

    Institute of Scientific and Technical Information of China (English)

    Gui-Qin Bai; Yan Liu; Jun Cheng; Shu-Lin Zhang; Ya-Fei Yue; Yan-Ping Huang; Li-Ying Zhang

    2005-01-01

    AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection.METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BarmH-I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ.After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used.The mRNA of HepG2 cells transfected with pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector was isolated,and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR)method, and cDNA was synthesized. After digestion with restriction enzyme RcaI, cDNA fragments were obtained.Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification.RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete S was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86

  19. Computational analysis of the human HSPH/HSPA/DNAJ family and cloning of a human HSPH/HSPA/DNAJ expression library

    NARCIS (Netherlands)

    Hageman, Jurre; Kampinga, Harm H.

    In this manuscript, we describe the generation of a gene library for the expression of HSP110/HSPH, HSP70/HSPA and HSP40/DNAJ members. First, the heat shock protein (HSP) genes were collected from the gene databases and the gene families were analyzed for expression patterns, heat inducibility,

  20. Cloning of phenazine carboxylic acid genes of Fusarium fujikuroi ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-08

    Mar 8, 2010 ... can described the structure and function of the biosynthetic gene clusters ... Guetsky R, Shtienberg D, Elad Y, Fischer E, Dinoor A (2002). Improving ..... Current Trends and Future Prospects, Haworth Press, New York,. London ...

  1. Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

  2. Cloning and characterization of zebra fish SPATA4 gene and analysis of its gonad specific expression.

    Science.gov (United States)

    Liu, Shangfeng; Liu, Bowen; He, Shan; Zhao, Ying; Wang, Zhao

    2005-06-01

    The spermatogenesis associated 4 gene (SPATA4, previously named TSARG2) was first cloned in human tissues and was reported to be a candidate spermatocyte apoptosis-related gene that is expressed specifically in testis. Analysis of SPATA4 expression and regulation in zebra fish may provide insight into the understanding of the complicated process of gonadogenesis. In this study, we cloned and characterized the SPATA4 gene from zebra fish (Danio rerio), which is homologous to human and mouse SPATA4. Zebra fish SPATA4 consists of six exons separated by five introns, as all SPATA4 genes in vertebrates. A promoter region was predicted using homologous blast and cloned for further study, and possible transcription factors were analyzed in this region. The putative protein encoded by this gene was analyzed using bioinformatics methods. Multi-tissue RT-PCR results demonstrated that the zebra fish SPATA4 gene is expressed specifically in testis and slightly in ovary. Analysis of the SPATA4 sequence and its spatial expression pattern indicate that this gene is highly conserved and may play an important role in the process of zebra fish gonadogenesis.

  3. Cloning and Analysis of Genes SAMS From Glycine soja

    Institute of Scientific and Technical Information of China (English)

    FAN Jinping; BAI Xi; LI Yong; JI Wei; WANG Xi; ZHU Yanming

    2008-01-01

    S-adenosylmethionine (SAM) plays important role in trans-methyl reactions. Under the condition of drought (30%PEG),salinity (200 mmol·L-1 NaCI) and low temperature (4℃),total RNA was extracted from the leaf and the first strand of eDNA was synthesized with reverse transcription.S-adenosylmethionine synthetase gene (SAMS gene) was amplified by PeR with the first strand eDNA as template and a pair of primers which was based on constructed ESTs sequence.Full-length SAMS gene sequence was obtained by BLAST comparison. According to the analysis, completed sequence of SAMS gene was integrality.The sequence of the SAMS gene was 1185 bp in length with an opening reading frame (ORF) encoding 394 amino acids.The cDNA sequence showed a significant homology to the SAM genes from Phaseolus lunatus (89%),Medicago sativa (85%).A prokaryotie expression vectors based on pET-32b had been constructed and prokaryotie expression was analyzed in order to lay a strong foundation for resist adversity function analysis through situation of genie expression analysis.

  4. The S-layer gene of Lactobacillus helveticus CNRZ 892 : cloning, sequence and heterologous expression

    NARCIS (Netherlands)

    Callegari, M.L.; Riboli, B.; Sanders, J.W; Cocconcelli, P.S.; Kok, J.; Venema, G; Morelli, L.

    1998-01-01

    Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) composed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein (slpH) has been cloned in Escherichia coli and sequenced. slpH consists of 440 codons and is preceded by a ribosome-binding site (RB

  5. The chicken CCAAT/Enhancer Binding Protein alpha gene. Cloning, characterisation and tissue distribution

    NARCIS (Netherlands)

    Calkhoven, CF; Gringhuis, SI; Ab, G

    1997-01-01

    We present the cloning and sequencing of the gene encoding the chicken CCAAT/Enhancer Binding Protein alpha (cC/EBP alpha). The coding region and 1.5 kb of 5' flanking DNA form a CpG island. Comparison of the chicken C/EBP alpha sequence to the homologous proteins of other species reveals several ev

  6. Cloning and bioinformatic analysis of lovastatin biosynthesis regulatory gene lovE

    Institute of Scientific and Technical Information of China (English)

    HUANG Xin; LI Hao-ming

    2009-01-01

    Background Lovastatin is an effective drug for treatment of hyperlipidemia.This study aimed to clone Iovastatin biosynthesis regulatory gene lovE and analyze the structure and function of its encoding protein.Methods According to the Iovastatin synthase gene sequence from genebank,primers were designed to amplify and clone the Iovastatin biosynthesis regulatory gene lovE from Aspergillus terms genomic DNA.Bioinformatic analysis of lovE and its encoding animo acid sequence was performed through intemet resources and software like DNAMAN.Results Target fragment lovE,almost 1500 bp in length,was amplified from Aspergillus terms genomic DNA and the secondary and three-dimensional structures of LovE protein were predicted.Conclusion In the Iovastatin biosynthesis process lovE is a regulatory gene and LovE protein is a GAL4-1ike transcriptional factor.

  7. Potato virus X isolation and purification and coat protein gene cloning

    Directory of Open Access Journals (Sweden)

    L. Duplat

    2011-12-01

    Full Text Available In this work we describe the construction of potato virus X coat protein (PVX-CP gene clones from a Colombian isolate. Virus was isolated from field potato plants cultured at páramo de San Jorge. PVX particles were purified from Nicotiana tabacum leaves infected with a local lesion taken from Datura stramonium plants. The genomic RNA present in the virus particles consisted of a single species of about 6 Kb. cDNA synthesis representing genomic RNA was followed by Digoxigenin incorporation after priming with oligonucleotide 4DT/KPN. PCR amplification of CP gene sequence was made by using oligonucleotides OX6 and L/Kpn. An expected fragment of 870 bp, besides some 600-123 bp fragments, was obtained after PCR. The blunt-ended fragments were clonned into the PCR cloning pMOSblue plasmid. The insert size of the selected recombinant cDNA clones was determined by restriction analysis of the plasmidDNAwith Sma I and Hind III enzymes. Positive clones were also screened by direct colony PCR screening. The selected recombinant cDNA clones were stored in glycerol at .70 °C.

  8. Over-expression of GSH1 gene and disruption of PEP4 gene in self-cloning industrial brewer's yeast.

    Science.gov (United States)

    Wang, Zhao-Yue; He, Xiu-Ping; Zhang, Bo-Run

    2007-11-01

    Foam stability is often influenced by proteinase A, and flavor stability is often affected by oxidation during beer storage. In this study, PEP4, the gene coding for proteinase A, was disrupted in industrial brewing yeast. In the meantime, one copy of GSH1 gene increased in the same strain. GSH1 is responsible for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for synthesis of glutathione which is one kind of important antioxidant and beneficial to beer flavor stability. In order to improve the brewer's yeast, plasmid pYPEP, pPC and pPCG1 were firstly constructed, which were recombined plasmids with PEP4 gene, PEP4's disruption and PEP4's disruption+GSH1 gene respectively. These plasmids were verified to be correct by restriction enzymes' assay. By digesting pPCG1 with AatII and PstI, the DNA fragment for homologous recombination was obtained carrying PEP4 sequence in the flank and GSH1 gene internal to the fragment. Since self-cloning technique was applied in the study and the modified genes were from industrial brewing yeast itself, the improved strains, self-cloning strains, were safe to public. The genetic stability of the improved strains was 100%. The results of PCR analysis of genome DNA showed that coding sequence of PEP4 gene had been deleted and GSH1 gene had been inserted into the locus of PEP4 gene in self-cloning strains. The fermentation ability of self-cloning strain, SZ-1, was similar to that of the host. Proteinase A could not be detected in beer brewed with SZ-1, and GSH content in the beer increased 35% compared to that of the host, Z-1.

  9. Cloning and characterization of the human USP22 gene promoter.

    Directory of Open Access Journals (Sweden)

    Jianjun Xiong

    Full Text Available Ubiquitin-specific processing enzyme 22 (USP22 plays a direct role in regulating cell cycle, and its overexpression has been reported to be involved in tumor progression. However, little is known about the regulation of USP22 transcription. In this study, we cloned and characterized the human USP22 promoter. Using 5' RACE (rapid amplification of cDNA ends analysis, the transcriptional initiation site was identified. Promoter deletion analysis showed that the sequence between -210 and -7 contains the basal promoter for USP22 in human fibroblast and tumor cells. Surprisingly, mutations in a putative Sp1 binding site immediately upstream of the USP22 transcriptional start site (-13 to -7 resulted in a significant induction of promoter activity. Further study revealed that Sp1 binds to this site in human normal fibroblast cells, and treatment with the Sp1 inhibitor mithramycin A led to a marked increase in USP22 transcript levels. Forced expression of exogenous Sp1 repressed the USP22 promoter activity in HeLa cells. In contrast, knockdown of Sp1 enhanced USP22 promoter activity and mRNA levels. These data suggest that Sp1 is a crucial regulator of USP22 transcription.

  10. Cloning and characterization of the beta-amylase gene from Bacillus polymyxa.

    OpenAIRE

    Friedberg, F; Rhodes, C

    1986-01-01

    The gene for beta-amylase was isolated from Bacillus polymyxa by molecular cloning in B. subtilis. B. subtilis cells containing this gene express and secrete an amylase which resembles the B. polymyxa beta-amylase and barley beta-amylase in terms of the products it generates during carbohydrate hydrolysis. Starch hydrolysis with this beta-amylase produces maltose, not glucose, whereas maltotriose and cycloheptaose are resistant to the action of this beta-amylase. The enzyme has a molecular we...

  11. Cloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli.

    Science.gov (United States)

    Tokuyama, S; Hatano, K

    1995-03-01

    The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage lambda-4 from the DNA library of Amycolatopsis sp. TS-1-60, a rare actinomycete, using antiserum against the enzyme. The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector. The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da. The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp. TS-1-60. The guanosine plus cytosine content of the AAR gene was about 70%. Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118. The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp. TS-1-60. When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp. TS-1-60. In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found. However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Cloning and nucleotide sequence of the Enterobacter aerogenes signal peptidase II (lsp) gene.

    OpenAIRE

    Isaki, L; Kawakami, M; Beers, R; Hom, R; Wu, H.C.

    1990-01-01

    In Escherichia coli, prolipoprotein signal peptidase is encoded by the lsp gene, which is organized into an operon consisting of ileS, lsp, and three open reading frames, designated genes x, orf-149, and orf-316. The Enterobacter aerogenes lsp gene was cloned and expressed in E. coli. The nucleotide sequence of the Enterobacter aerogenes lsp gene and a part of its flanking sequences were determined. A high degree of homology was found between the E. coli ileS-lsp operon and the corresponding ...

  13. Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos.

    Science.gov (United States)

    Park, Mi-Ryung; Im, Gi-Sun; Kim, Sung Woo; Hwang, Seongsoo; Park, Jae-Hong; Kim, Hyun; Do, Yoon Jung; Park, Soo Bon; Yang, Bo-Suck; Song, Young Min; Cho, Jae-Hyeon; Ko, Yeoung-Gyu

    2013-06-01

    The abnormal development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is considered to be associated with consequent changes in gene expression following errors in epigenetic reprogramming. In this study, we carried out SCNT using donor fibroblast cells derived from 3-way hybrids (Landrace×Duroc×Yorkshire). A total of 655 SCNT embryos were transferred, and 6.97±2.3 cloned fetuses were successfully recovered from three surrogates at gestational day 30. An analysis of the 6.97±2.3 cloned embryos revealed that most had severe extraembryonic defects. The extraembryonic tissue from the SCNT embryos was abnormally small compared with that of the control. To investigate the differentially expressed genes between the SCNT and control extraembryonic tissues, we compared the gene expression profiles of the extraembryonic tissues from gestational day 30 cloned pig embryos with those from the control using an annealing control primer-based GeneFishing polymerase chain reaction. As a result, we found that a total of 50 genes were differentially expressed by utilizing 120 ACPs, 38 genes of which were known. Among them, 26 genes were up-regulated, whereas 12 genes were down-regulated. Real-time RT-PCR showed that apoptosis-related genes were expressed significantly higher in SCNT extraembryonic tissue than in the control, whereas metabolism-related genes were expressed at significantly lower levels in the SCNT extraembryonic tissue. These observations strongly indicate that early gestational death of SCNT embryo is caused, at least in part, by the disruption of developing extraembryonic tissues as a result of aberrant gene expression, which results in abnormal apoptosis and metabolism.

  14. Cloning and characterization of the densoviruses susceptible gene ...

    African Journals Online (AJOL)

    USER

    2010-06-21

    Jun 21, 2010 ... parvoviruses that are highly pathogenic for invertebrates and are commonly isolated from arthropod hosts (Bergoin and Tijssen, 2000). Bombyx mori ... Nid-1 gene control non-infection to BmDNV-1 (Eguchi et al., 1986) and a ...

  15. Cloning and functional characterization of a class III chitinase gene ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-17

    Dec 17, 2008 ... encoded by VvChiF III showed a high identity to that of a class III ... gene corresponds to the Glyco-hydro-18 super family that consisting of a signal peptide with the ..... broad-spectrum plant defence mechanism has been well.

  16. Cloning and sequencing of the ferredoxin gene of blue-green alga Anabaena siamensis

    Science.gov (United States)

    Li, Shou-Dong; Song, Li-Rong; Liu, Yong-Ding; Zhao, Jin-Dong

    1998-03-01

    The structure gene for ferredoxin, petFI, from Anabaena siamensis has been amplified by polymerase chain reaction(PCR) and cloned into cloning vector pGEM-3zf(+). The nucleotide sequence of petFI has been determined with silver staining sequencing method. There is 96.8% homology between coding region of petFI from A. siamensis and that of petFI from A. sp. 7120. Amino acid sequences of seven strains of blue-green algae are compared.

  17. Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library

    Directory of Open Access Journals (Sweden)

    Eddy Edward M

    2007-07-01

    Full Text Available Abstract Background The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes. Results We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein. Conclusion We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.

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

    Science.gov (United States)

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

    1991-08-01

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

  19. Construction and evaluation of normalized cDNA libraries enriched with full-length sequences for rapid discovery of new genes from Sisal (Agave sisalana Perr.) different developmental stages.

    Science.gov (United States)

    Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

    2012-10-12

    To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing.

  20. Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages

    Science.gov (United States)

    Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

    2012-01-01

    To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing. PMID:23202944

  1. Cloning, sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene.

    Science.gov (United States)

    De Zoysa, P A; Connerton, I F; Watson, D C; Johnston, J R

    1991-08-01

    The cloned NADP-specific glutamate dehydrogenase (GDH) genes of Aspergillus nidulans (gdhA) and Neurospora crassa (am) have been shown to hybridize under reduced stringency conditions to genomic sequences of the yeast Schwanniomyces occidentalis. Using 5' and 3' gene-specific probes, a unique 5.1 kb BclI restriction fragment that encompasses the entire Schwanniomyces sequence has been identified. A recombinant clone bearing the unique BclI fragment has been isolated from a pool of enriched clones in the yeast/E. coli shuttle vector pWH5 by colony hybridization. The identity of the plasmid clone was confirmed by functional complementation of the Saccharomyces cerevisiae gdh-1 mutation. The nucleotide sequence of the Schw. occidentalis GDH gene, which consists of 1380 nucleotides in a continuous reading frame of 459 amino acids, has been determined. The predicted amino acid sequence shows considerable homology with GDH proteins from other fungi and significant homology with all other available GDH sequences.

  2. Cloning and functional characterization of the SUR2/SYR2 gene encoding sphinganine hydroxylase in Pichia ciferrii.

    Science.gov (United States)

    Bae, Jung-Hoon; Sohn, Jung-Hoon; Park, Chang-Seo; Rhee, Joon-Shick; Choi, Eui-Sung

    2004-04-15

    Saccharomyces cerevisiae sphinganine C4-hydroxylase encoded by the SUR2 gene catalyses the conversion of sphinganine to phytosphingosine. We isolated the SUR2 gene from Pichia ciferrii using nucleotide sequence homology to S. cerevisiae SUR2 to study hydroxylation of sphinganine in the sphingoid base overproducing yeast P. ciferrii. A positive clone was confirmed by nucleotide sequencing. A syringomycin-E resistance phenotype of a S. cerevisiae sur2-null mutant was complemented by expression of the cloned P. ciferrii SUR2 gene. Restoration of phytosphingosine production in the complemented strain was also confirmed, indicating that the cloned gene is a functional homologue of S. cerevisiae SUR2. .

  3. Comparison of inherently essential genes of Porphyromonas gingivalis identified in two transposon-sequencing libraries.

    Science.gov (United States)

    Hutcherson, J A; Gogeneni, H; Yoder-Himes, D; Hendrickson, E L; Hackett, M; Whiteley, M; Lamont, R J; Scott, D A

    2016-08-01

    Porphyromonas gingivalis is a Gram-negative anaerobe and keystone periodontal pathogen. A mariner transposon insertion mutant library has recently been used to define 463 genes as putatively essential for the in vitro growth of P. gingivalis ATCC 33277 in planktonic culture (Library 1). We have independently generated a transposon insertion mutant library (Library 2) for the same P. gingivalis strain and herein compare genes that are putatively essential for in vitro growth in complex media, as defined by both libraries. In all, 281 genes (61%) identified by Library 1 were common to Library 2. Many of these common genes are involved in fundamentally important metabolic pathways, notably pyrimidine cycling as well as lipopolysaccharide, peptidoglycan, pantothenate and coenzyme A biosynthesis, and nicotinate and nicotinamide metabolism. Also in common are genes encoding heat-shock protein homologues, sigma factors, enzymes with proteolytic activity, and the majority of sec-related protein export genes. In addition to facilitating a better understanding of critical physiological processes, transposon-sequencing technology has the potential to identify novel strategies for the control of P. gingivalis infections. Those genes defined as essential by two independently generated TnSeq mutant libraries are likely to represent particularly attractive therapeutic targets.

  4. Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library.

    Science.gov (United States)

    Le Paslier, M C; Pierce, R J; Merlin, F; Hirai, H; Wu, W; Williams, D L; Johnston, D; LoVerde, P T; Le Paslier, D

    2000-04-15

    A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.

  5. Cloning and sequencing of a Moraxella bovis pilin gene.

    Science.gov (United States)

    Marrs, C F; Schoolnik, G; Koomey, J M; Hardy, J; Rothbard, J; Falkow, S

    1985-07-01

    Moraxella bovis pili have been shown to play a major role in both infectivity and protective immunity of bovine infectious keratoconjunctivitis. Sonicated M. bovis DNA from the piliated strain EPP63 was inserted into the vector lambda gt11 with EcoRI linkers. Recombinant phage were screened with an oligonucleotide probe based on the amino-terminal portion of the DNA sequence of a Neisseria gonorrhoeae pilin gene. Two candidate phages produced a protein that comigrated with EPP63 beta pilin in sodium dodecyl sulfate-polyacrylamide gels and bound anti-pilus antisera. The 1.9-kilobase insert from one of these, lambda gt11M182, was subcloned in both orientations into pBR322, forming the plasmids pMxB7 and pMxB9, both of which produced beta pilin, as did pMxB12, a HindIII deletion derivative of pMxB7. In HB101(pMxB12), the M. bovis pilin protein was shown to be primarily localized in the inner membrane. The entire 939-base-pair insert of pMxB12 was sequenced, revealing a ribosome binding site just upstream of the coding region and an AT-rich region further upstream containing some potential RNA polymerase recognition sites. The translation of the sequence predicts a six-amino-acid leader sequence preceding the phenylalanine that begins the mature protein. Codon usage analysis of the M. bovis beta pilin gene revealed greater use of the CUA codon for leucine than usual for a well-expressed Escherichia coli gene. Comparisons of the M. bovis EPP63 beta pilin protein sequence with other pilin gene sequences are presented.

  6. Cloning and sequencing of a Moraxella bovis pilin gene.

    OpenAIRE

    1985-01-01

    Moraxella bovis pili have been shown to play a major role in both infectivity and protective immunity of bovine infectious keratoconjunctivitis. Sonicated M. bovis DNA from the piliated strain EPP63 was inserted into the vector lambda gt11 with EcoRI linkers. Recombinant phage were screened with an oligonucleotide probe based on the amino-terminal portion of the DNA sequence of a Neisseria gonorrhoeae pilin gene. Two candidate phages produced a protein that comigrated with EPP63 beta pilin in...

  7. 基于克隆文库筛选的池设计%Pooling designs based on clone library screenings

    Institute of Scientific and Technical Information of China (English)

    管宇

    2007-01-01

    池设计(pooling design)是基于克隆文库筛选(clone library screening)的一种实验设计,要求从大量的克隆体中筛选出其中含有某组特定核苷酸串的克隆体,它属于组合群试.一个池设计方案用一个二元关联矩阵(称为分离矩阵)表示,行对应于试验,列对应于克隆集合的体;考虑到生物实验存在误差,分离矩阵还需要有查错和纠错能力.利用子集或子空间之间的包含关系可以构建出分离矩阵,它的每行和每列中元素1的个数分别都一样,但试验总次数即行数明显大于信息下界.

  8. Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction.

    Science.gov (United States)

    Lazinski, David W; Camilli, Andrew

    2013-01-01

    The amplification of DNA fragments, cloned between user-defined 5' and 3' end sequences, is a prerequisite step in the use of many current applications including massively parallel sequencing (MPS). Here we describe an improved method, called homopolymer tail-mediated ligation PCR (HTML-PCR), that requires very little starting template, minimal hands-on effort, is cost-effective, and is suited for use in high-throughput and robotic methodologies. HTML-PCR starts with the addition of homopolymer tails of controlled lengths to the 3' termini of a double-stranded genomic template. The homopolymer tails enable the annealing-assisted ligation of a hybrid oligonucleotide to the template's recessed 5' ends. The hybrid oligonucleotide has a user-defined sequence at its 5' end. This primer, together with a second primer composed of a longer region complementary to the homopolymer tail and fused to a second 5' user-defined sequence, are used in a PCR reaction to generate the final product. The user-defined sequences can be varied to enable compatibility with a wide variety of downstream applications. We demonstrate our new method by constructing MPS libraries starting from nanogram and sub-nanogram quantities of Vibrio cholerae and Streptococcus pneumoniae genomic DNA.

  9. Cloning and embryonic expression of zebrafish PLAG genes.

    Science.gov (United States)

    Pendeville, Hélène; Peers, Bernard; Kas, Koen; Voz, Marianne L

    2006-03-01

    PLAG transcription factors play important roles in oncogenesis. To date three members of this subfamily of zinc finger proteins have been identified in humans and mice: PLAG1, PLAGL1 and PLAGL2. In this study, we identified zebrafish orthologs of PLAG1 and PLAGL2 and a novel member of this family, PLAGX. We examined the temporal expression of these three genes by quantitative real time RT-PCR and found that all three genes are maternally provided, expressed at low level during early somitogenesis and, during late somitogenesis and beyond, PLAG expression increases to reach a plateau level around 5 dpf. Whole mount in situ experiments revealed that PLAG1, PLAGL2 and PLAGX display a similar pattern of expression characterized by a low ubiquitous expression overcame by high expression in some restricted compartments such as the ventricular zone of the brain, the pectoral fin buds, the developing pharyngeal arches and the axial vasculature. We show that this pattern resembles the one observed for the proliferative marker PCNA, suggesting that the PLAG genes are expressed more strongly in zones of active proliferation. This hypothesis was proven for the ventricular zone shown to be a highly proliferative zone using the anti-phosphohistone H3 antibody that detects cells in mitosis.

  10. Molecular cloning and in vitro expression of a silent phenoxazinone synthase gene from Streptomyces lividans.

    Science.gov (United States)

    Madu, A C; Jones, G H

    1989-12-14

    Phenoxazinone synthase (PHS) catalyzes a step in actinomycin D biosynthesis in Streptomyces antibioticus. Two sequences from Streptomyces lividans that hybridize to the phs gene of S. antibioticus have been cloned in Escherichia coli K-12 using the plasmid pBR322. Although there was some similarity in the restriction maps of the two cloned fragments, neither insert appeared to be a direct subset of the other nor of the S. antibioticus phs gene. In vitro expression studies, in a streptomycete coupled transcription-translation system, showed that a 3.98-kb SphI fragment encoded a PHS-related protein. These observations provide additional support for the existence of silent genes for antibiotic production in streptomycetes.

  11. Molecular Cloning and Characterization of the Actin-depolymerizing Factor Gene in Gossypium barbadense

    Institute of Scientific and Technical Information of China (English)

    MA Zhi-ying; CHI Ji-na; WANG Xing fen; ZHOU Hong-mei; ZHANG Gui-yin

    2008-01-01

    @@ Sea Island cotton (Gossypium barbadense L.) has been highly valued in Verticillium wilt resistance and many fiber qualities including fiber length,strength,and fineness.To identify whether it had some special genes in fiber development in comparison with the upland cotton (G.hirsutum L.),an actin-depolymerizing factor (ADF) gene was cloned and characterized in this research.A 420 bp open reading frame of the cloned gene,termed GbADF1,encoded a protein of 139 amino acids,which included39.57% nonpolar amino acids,17.27% acidic amino acids,15.83% basic amino acids,and 31.92% hydrophobic amino aids.

  12. Research progress on isolation and cloning of functional genes in tea plants

    Institute of Scientific and Technical Information of China (English)

    MA Chunlei; CHEN Liang

    2007-01-01

    Tea,which has many sanitarian functions,is one of the most popular non-alcoholic soft and healthy beverages in the world.In many countries,as well as in China,tea (Camellia sinensis) is an important cash crop.It has great value as a source of secondary metabolic products.Molecular biology of tea plants has been one of the most active and kinetic research fields of tea science for the last decade.Isolation and cloning of important functional genes of tea plants have a critical significance on elucidating the molecular mechanism of high quality,yield and resistance,as well as genetic manipulating via biotechnological approaches for tea plants.In this paper,we introduced the research progress on the isolation and cloning of functional genes in tea plants.In addition,the brief prospect on the research of functional genes of tea plants in the near future is also given out.

  13. Identification and molecular cloning of glutamate decarboxylase gene from Lactobacillus casei

    Directory of Open Access Journals (Sweden)

    Yasaman Tavakoli

    2015-09-01

    Full Text Available Gamma-amino butyric acid (GABA possesses several physiological functions such as neurotransmission, induction of hypotension, diuretic and tranquilizer effects. Production of GABA-enriched products by lactic acid bacteria has been a focus of different researches in recent years because of their safety and health-promoting specifities. In this study, glutamate decarboxylase (gad gene of a local strains Lactobacillus casei was identified and cloned. In order to clone the gad gene from this strain, the PCR was carried out using primers designed based on conserved regions. The PCR product was purified and ligated into PGEM-T vector. Comparison of obtained sequences shows that this fragment codes the pyridoxal 5′-phosphate binding region. This strain could possibly be used for the industrial GABA production and also for development of functional fermented foods. Gad gene manipulation can also either decrease or increase the activity of enzyme in bacteria.

  14. Apoptosis Gene Hunting Using Retroviral Expression Cloning: Identification of Vacuolar ATPase Subunit E

    Directory of Open Access Journals (Sweden)

    Claire L. Anderson

    2003-01-01

    Full Text Available Over the past 10-15 years there has been an explosion of interest in apoptosis. The delayed realisation that cell death is an essential part of life for any multicellular organism has meant that, despite the recent and rapid developments of the last decade, the precise biochemical pathways involved in apoptosis remain incomplete and potentially novel genes may, as yet, remain undiscovered. The hunt is therefore on to bridge the remaining gaps in our knowledge. Our contribution to this research effort utilises a functional cloning approach to isolate important regulatory genes involved in apoptosis. This mini-review focuses on the use and advantages of a retroviral expression cloning strategy and describes the isolation and identification of one such potential apoptosis regulatory gene, namely that encoding vacuolar ATPase subunit E.

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

    Science.gov (United States)

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

    2008-11-01

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

  16. Molecular cloning and SNP association analysis of chicken PMCH gene.

    Science.gov (United States)

    Sun, Guirong; Li, Ming; Li, Hong; Tian, Yadong; Chen, Qixin; Bai, Yichun; Kang, Xiangtao

    2013-08-01

    The pre-melanin-concentrating hormone (PMCH) gene is an important gene functionally concerning the regulations of body fat content, feeding behavior and energy balance. In this study, the full-length cDNA of chicken PMCH gene was amplified by SMART RACE method. The single nucleotide polymorphisms (SNPs) in the PMCH gene were screened by comparative sequence analysis. The obtained non-synonymous coding SNPs (ncSNPs) were designed for genotyping firstly. Its effects on growth, carcass characteristics and meat quality traits were investigated employing the F2 resource population of Gushi chicken crossed with Anak broiler by AluI CRS-PCR-RFLP. Our results indicated that the cDNA of chicken PMCH shared 67.25 and 66.47% homology with that of human and bovine PMCH, respectively. The deduced amino acid sequence of chicken PMCH (163 amino acids) were 52.07 and 50.89% identical to those of human and bovine PMCH, respectively. The PMCH protein sequence is predicted to have several functional domains, including pro-MCH, CSP, IL7, XPGI and some low complexity sequence. It has 8 phosphorylation sites and no signal peptide sequence. gga-miR-18a, gga-miR-18b, gga-miR-499 microRNA targeting site was predicted in the 3' untranslated region of chicken PMCH mRNA. In addition, a total of seven SNPs including an ncSNP and a synonymous coding SNP, were identified in the PMCH gene. The ncSNP c.81 A>T was found to be in moderate polymorphic state (polymorphic index=0.365), and the frequencies for genotype AA, AB and BB were 0.3648, 0.4682 and 0.1670, respectively. Significant associations between the locus and shear force of breast and leg were observed. This polymorphic site may serve as a useful target for the marker assisted selection of the growth and meat quality traits in chicken.

  17. Cloning and sequencing of cagA gene fragment of Helicobacter pylori with coccoid form

    Institute of Scientific and Technical Information of China (English)

    Ke-Xia Wang; Xue-Feng Wang

    2004-01-01

    AIM: To clone and sequence the cagA gene fragment of Helicobacter pylori ( H pylori) with coccoid form.METHODS: H pylori strain NCTC11637 were transformed to coccoid form by exposure to antibiotics in subinhibitory concentrations. The coccoid H pyloriwas collected. cagA gene of the coccoid H pylori strain was amplified by PCR.After purified, the target fragment was cloned into plasmid pMD-18T. The recombinant plasmid pMD-18T-cagA was transformed into E. coli JM109. Positive clones were screened and identified by PCR and digestion with restriction endonucleases. The sequence of inserted fragment was then analysed.RESULTS: cagA gene of 3 444 bp was obtained from the coccoid H pylori genome DNA. The recombinant plasmid pMD-18T-cagA was constructed, then it was digested by BamH Ⅰ+Sac Ⅰ, and the product of digestion was identical with the predicted one. Sequence analysis showed that the homology of coccoid and the reported original sequence H pylori was 99.7%.CONCLUSION: The recombinant plasmid containing cagA gene from coccoid H pylori has been constructed successfully.The coccoid H pylori contain completed cagA gene, which may be related to pathogenicity of them.

  18. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Davanloo, P.; Rosenberg, A.H.; Dunn, J.J.; Studier, F.W.

    1984-04-01

    The complete coding sequence of the gene for bacteriophage T7 RNA polymerase (T7 gene 1) has been cloned in the plasmid pBR322. Large amounts of active enzyme can be accumulated in Escherichia coli when the cloned gene is transcribed from the lac UV5 promoter. A protease activity that apparently can nick the protein without causing it to fall apart can be a problem during purification, but a procedure is described that gives good yields of essentially homogeneous, highly active enzyme suitable for biochemical and physical studies. T7 RNA polymerase has a stringent specificity for its own promoters and will selectively transcribe DNA that has been linked to such a promoter. This specificity makes the enzyme useful both for producing specific RNAs in vitro and for directing the expression of selected genes inside the cell. Having the cloned gene also makes possible a detailed mutational analysis of the functioning of T7 RNA polymerase. 25 references, 3 figures.

  19. 细胞内PCR法克隆抗体可变区基因%Antibody variable genes cloning by in-cell PCR

    Institute of Scientific and Technical Information of China (English)

    袁清安; 韩素文; 俞炜源; 孔德清; 贺秉坤

    2001-01-01

    报道一种克隆免疫脾细胞中抗体可变区基因的新方法。抗原免疫小鼠的脾细胞经分散、固定、渗透处理,直接在细胞内进行反转录,再采用半巢式PCR扩增,获得的序列经测序证明是抗体可变区编码序列。这种不经mRNA纯化、从细胞内直接获取目的基因的方法既可用于抗体库的建立,亦可用于新基因的快速克隆。%A novel method in cloning antibody variable genes from immunized spleen cells was described in the report. After the antigen sensitized spleen was dispersed, the cells were treated by fixation and permeabilization. Reverse transcription and polymerase chain reaction (RT-PCR) was conducted inside the cells and semi-nested PCR was used to amplify the variable genes that proved to be. The method, without mRNA purification and direct cloning inside the cells, may provide a new way on construction of antibody library or fast cloning of novel genes.

  20. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

    Directory of Open Access Journals (Sweden)

    Emanuele Sasso

    2015-01-01

    Full Text Available Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications.

  1. Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch.

    Science.gov (United States)

    Zhijian, Cao; Yun, Xie; Chao, Dai; Shunyi, Zhu; Shijin, Yin; Yingliang, Wu; Wenxin, Li

    2006-06-01

    Many studies have been carried on peptides and genes encoding scorpion toxins from the venom of Mesobuthus martensii Karsch (synonym: Buthus martensii Karsch, BmK), such as Na+, K+ and Cl- channel modulators. In this study, a novel calcium channel toxin-like gene BmCa1 was isolated and characterized from the venom of Mesobuthus martensii Karsch. First, a partial cDNA sequence of the Ca2+ channel toxin-like gene was identified by random sequencing method from a venomous gland cDNA library of Mesobuthus martensii Karsch. The full-length sequence of BmCa1 was then obtained by 5'RACE technique. The peptide deduced from BmCa1 precursor nucleotide sequence contains a 27-residue signal peptide and a 37-residue mature peptide. Although BmCa1 and other scorpion toxins are different at the gene and protein primary structure levels, BmCa1 has the same precursor nucleotide organization and cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins. Genomic DNA sequence of BmCa1 was also cloned by PCR. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns of 72 bp and 1076 bp in length, respectively. BmCa1 is the first calcium channel toxin-like gene cloned from the venom of Mesobuthus martensii Karsch and potentially represents a novel class of calcium channel toxins in scorpion venoms.

  2. Molecular Cloning and Functional Characterization of a Salt Tolerance-Associated Gene IbNFU1 from Sweetpotato

    Institute of Scientific and Technical Information of China (English)

    WANG Lian-jun; HE Shao-zhen; ZHAI Hong; LIU De-gao; WANG Yan-nan; LIU Qing-chang

    2013-01-01

    Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. Based on the EST sequence selected from salt-stressed suppression subtractive hybridization (SSH) cDNA library constructed with a salt-tolerant mutant LM79, a NFU gene, termed IbNFU1, was cloned from sweetpotato (Ipomoea batatas (L.) Lam.) via rapid amplification of cDNA ends (RACE). The cDNA sequence of 1 117 bp contained an 846 bp open reading frame encoding a 281 amino acids polypeptide with a molecular weight of 30.5 kDa and an isoelectric point (pI) of 5.12. IbNFU1 gene contained a conserved Cys-X-X-Cys motif in C-terminal of the iron-sulfur cluster domain. The deduced amino acid sequence had 66.08 to 71.99%sequence identity to NFU genes reported in Arabidopsis thaliana, Eucalyptus grandis and Vitis vinifera. Real-time quantitative PCR analysis revealed that the expression level of IbNFU1 gene was significantly higher in the roots of the mutant LM79 compared to the wild-type Lizixiang. Transgenic tobacco (cv. Wisconsin 38) plants expressing IbNFU1 gene exhibited significantly higher salt tolerance compared to the untransformed control plants. It is proposed that IbNFU1 gene has an important function for salt tolerance of plants.

  3. Molecular cloning and characterization of the nontypeable Haemophilus influenzae 2019 rfaE gene required for lipopolysaccharide biosynthesis.

    Science.gov (United States)

    Lee, N G; Sunshine, M G; Apicella, M A

    1995-03-01

    The lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae (NTHi) is an important factor in pathogenesis and virulence. In an attempt to elucidate the genes involved in LOS biosynthesis, we have cloned the rfaE gene from NTHi 2019 by complementing a Salmonella typhimurium rfaE mutant strain with an NTHi 2019 plasmid library. The rfaE mutant synthesizes lipopolysaccharide (LPS) lacking heptose, and the rfaE gene is postulated to be involved in ADP-heptose synthesis. Retransformation with the plasmid containing 4 kb of NTHi DNA isolated from a reconstituted mutant into rfaE mutants gave wild-type LPS phenotypes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed the conversion of the rfaE mutant LPS to a wild-type LPS phenotype. Sequence analysis of a 2.4-kb BglII fragment revealed two open reading frames. One open reading frame encodes the RfaE protein with a molecular weight of 37.6 kDa, which was confirmed by in vitro transcription and translation, and the other encodes a polypeptide highly homologous to the Escherichia coli HtrB protein. These two genes are transcribed from the same promoter region into opposite directions. Primer extension analysis of the rfaE gene revealed a single transcription start site at 37 bp upstream of the predicted translation start site. The upstream promoter region contained a sequence (TA AAAT) homologous to the -10 region of the bacterial sigma 70-dependent promoters at an appropriate distance (7 bp), but not sequence resembling the consensus sequence of the -35 region was found. These studies demonstrate the ability to use complementation of defined LPS defects in members of the family Enterobacteriaceae to identify LOS synthesis genes in NTHi.

  4. Cloning and Expression of Helicobacter pylori HpaA Gene

    Directory of Open Access Journals (Sweden)

    Moein Farshchian

    2009-01-01

    Full Text Available Objective: Helicobacter pylori is associated with chronic gastritis, peptic ulcers, gastric adenocarcinomaand gastric mucosa-associated lymphoid tissue (MALT lymphoma. Antibiotictherapies do not protect from potential re-infection and have a risk for development of drugresistance. Therefore, prophylactic vaccine mediated protection against H. pylori is an attractiveclinical interest. H. pylori adhesin A (HpaA is a conserved surface lipoprotein and playsimportant roles in the pathogenesis of infection. In this study the recombinant protein (rHpaAwas over-expressed in E.coli.Materials and Methods: The hpaA gene was amplified by PCR. Prokaryote expression vectorpET28a-hpaA was constructed, and used to transform E.coli BL21DE3. The expressionof recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot wereused to determine immunoreactivity of rHpaA by a rabbit polyclonal antibodies against wholecell of H. pylori.Results: The hpaA gene nucleotide sequence in the recombinant plasmid vector of pET-28-a-hpaA was consistent with that of H.pylori hpaA as published in the GenBank. SDS-PAGEdemonstrated that the constructed prokaryotic expression efficiently produced rHpaA at the1.5 mmol/L of IPTG. HpaA fusion protein was able to react with the rabbit polyclonal antibodyagainst whole cells of H. pylori.Conclusion: A prokaryotic expression system pET-28a-hpaA-BL21 with high efficiency of H.pylori hpaA gene was successfully established and the HpaA fusion protein showed satisfactoryimmunoreactivity. These results indicate that production of a specific recombinant proteinis an alternative and potentially more expeditious strategy for development of H. pylori vaccine.

  5. Cloning and characterization of a nitrite reductase gene related to ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-01

    Mar 1, 2010 ... Total RNA was extracted from nonembryogenic calli, embryogenic. Han et al. 1305 calli and ... 1 µL of cDNA, 0.5 µL of 10 mM dNTP, 2 µL of 10×PCR buffer, 1 µL of 10 µM each primer, 0.2 µL Taq DNA polymerase and 14.3 µL of. ddH2O. ... Sequence characterization and gene structure of. GhNiR. A 2,257 ...

  6. fimH gene cloning, of Escherichia coli uropathogen and examination of its subsequence diversity

    Directory of Open Access Journals (Sweden)

    Samaneh Ostad Mohammadi

    2013-09-01

    Full Text Available Background: Escherichia coli uropathogen is the most prevalent pathogen separated from urinary tract that often is originated from intestinal flora of the own person. Urinary tract infection is one of the most prevalent infectious diseases in Human. Whereas binding stage has an important role in bacteria colonization and then the infection is created, one of the most important strategies for inhibiting the infection is inhibiting the bacterial binding. As fimH protein is acting as adhesion it could be an appropriate candidate for producingvaccine. Material and Methods: First, genomic DNA of Escherichia coli bacteria extracted from strain 35218 ATCC. Upon designing primer for fimH gene, the PCR reaction has been applied with Taq DNA Polymerase and then pfu DNA polymerase enzymes. pBluescript (SK- plasmid has been applied for cloning the product of PCR. Using ClustalW and MEGA4 software, the subsequence was alignmented with the gene subsequence existing in gene bank and its gene diversity was examined. Results: After sequencing the cloned fimH gene using ClustalW and MEGA4 software, the result of this subsequence were alignmented with the subsequence of Escherichia coli containing fimH gene existing in gene bank and based on this alignment, N terminal on the protein surface and DNA are protected. Conclusion: N terminal domain of fimH gene is a conserved sequence among clinical isolates and it could be used for designing a vaccine against urinary tract infection.

  7. Molecular cloning and nucleotide sequence of chicken avidin-related genes 1-5.

    Science.gov (United States)

    Keinänen, R A; Wallén, M J; Kristo, P A; Laukkanen, M O; Toimela, T A; Helenius, M A; Kulomaa, M S

    1994-03-01

    Using avidin cDNA as a hybridisation probe, we detected a gene family whose putative products are related to the chicken egg-white avidin. Two overlapping genomic clones were found to contain five genes (avidin-related genes 1-5, avr1-avr5), which have been cloned, characterized and sequenced. All of the genes have a four-exon structure with an overall identity with the avidin cDNA of 88-92%. The genes appear to have no pseudogenic features and, in fact, two of these genes have been shown to be transcribed. The putative proteins share a sequence identity of 68-78% with avidin. The amino acid residues responsible for the biotin-binding activity of avidin and the bacterial biotin-binding protein, streptavidin, are highly conserved. Since avidin is induced in both a progesterone-specific manner and in connection with inflammation, these genes offer a valuable tool to study complex gene regulation in vivo.

  8. Molecular cloning and gene expression analysis of the leptin receptor in the Chinese mitten crab Eriocheir sinensis.

    Directory of Open Access Journals (Sweden)

    Hui Jiang

    Full Text Available BACKGROUND: Leptin is an adipocyte-derived hormone with multiple functions that regulates energy homeostasis and reproductive functions. Increased knowledge of leptin receptor function will enhance our understanding of the physiological roles of leptin in animals. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, a full-length leptin receptor (lepr cDNA, consisting of 1,353 nucleotides, was cloned from Chinese mitten crab (Eriocheir sinensis using rapid amplification of cDNA ends (RACE following the identification of a single expressed sequence tag (EST clone in a cDNA library. The lepr cDNA consisted of a 22-nucleotide 5'-untranslated region (5' UTR, a 402-nucleotide open reading frame (ORF and a 929-nucleotide 3' UTR. Multiple sequence alignments revealed that Chinese mitten crab lepr shared a conserved vacuolar protein sorting 55 (Vps55 domain with other species. Chinese mitten crab lepr expression was determined in various tissues and at three different reproductive stages using quantitative real-time RT-PCR. Lepr expression was highest in the intestine, thoracic ganglia, gonad, and accessory gonad, moderate in hepatopancreas and cranial ganglia, and low in muscle, gill, heart, haemocytes, and stomach. Furthermore, lepr expression was significantly higher in the intestine, gonad and thoracic ganglia in immature crabs relative to precocious and mature crabs. In contrast, lepr expression was significantly lower in the hepatopancreas of immature crabs relative to mature crabs. CONCLUSIONS/SIGNIFICANCE: We are the first to identify the lepr gene and to determine its gene expression patterns in various tissues and at three different reproductive stages in Chinese mitten crab. Taken together, our results suggest that lepr may be involved in the nutritional regulation of metabolism and reproduction in Chinese mitten crabs.

  9. [Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].

    Science.gov (United States)

    Lu, Jie; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen

    2003-02-01

    Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.

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

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhiwen; SUN Xiuqin; ZHANG Jinxing; ZAN Jindong

    2008-01-01

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

  11. Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12.

    Science.gov (United States)

    Sung, Y C; Parsell, D; Anderson, P M; Fuchs, J A

    1987-06-01

    The gene in Escherichia coli for cyanase, designated cynS, was localized to a BglII restriction site approximately 1.7 kilobases from the lacA end of the lac operon. The gene was cloned into the pUC13 vector. Maxicell analysis of plasmid-encoded proteins confirmed that the BglII site is in the region encoding the structural gene for cyanase. Cyanase-deficient strains had increased sensitivity to cyanate and were not able to use cyanate as a nitrogen source.

  12. Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12.

    OpenAIRE

    Sung, Y C; Parsell, D; Anderson, P. M.; Fuchs, J A

    1987-01-01

    The gene in Escherichia coli for cyanase, designated cynS, was localized to a BglII restriction site approximately 1.7 kilobases from the lacA end of the lac operon. The gene was cloned into the pUC13 vector. Maxicell analysis of plasmid-encoded proteins confirmed that the BglII site is in the region encoding the structural gene for cyanase. Cyanase-deficient strains had increased sensitivity to cyanate and were not able to use cyanate as a nitrogen source.

  13. Cloning and characterization of a tryptophanase gene from Enterobacter aerogenes SM-18

    OpenAIRE

    Kawasaki, K.; Yokota, A; Oita, S; Kobayashi, C.; Yoshikawa, S; KAWAMOTO, S.; Takao, S.; Tomita, F.

    1993-01-01

    A tryptophanase gene from Enterobacter aerogenes SM-18 was cloned and sequenced. The structural gene for tryptophanase, tnaA, consisted of 1389 bp encoding 462 amino acid residues, and its nucleotide sequence and deduced amino acid sequence showed significant homology to those of tnaA from Escherichia coli K12. A short open reading frame consisting of 31 amino acid residues was found upstream of tnaA, and it showed some similarity to the E. coli tnaC gene known to be a cis-acting regulatory e...

  14. Cloning and Sequence Analysis of Y-box Binding Protein Gene in Min Pig

    Institute of Scientific and Technical Information of China (English)

    Zhang Dong-jie; Liu Di; Wang Liang; He Xin-miao; Wang Wen-tao

    2014-01-01

    In order to study the gene sequence of Min pig Y-box binding protein (YB-1) gene, the complete coding sequence of Min pig YB-1 gene was cloned by RT-PCR, the sequence features were analyzed by some software and online website. The results showed that the complete CDS of Min pig Y-box was found to be 975 bp long, encoding 324 amino acids. It contained a conserved cold shock domain and several phosphorylation sites, but had no transmembrane domains, and was consistent with a protein found in the cytoplasm. Min pig YB-1 nucleotides shared high similarity (61.37%-97.66%) with other mammals.

  15. Cloning and analysing of 5‘ flanking region of Xenopus organizer gene noggin

    Institute of Scientific and Technical Information of China (English)

    TAOQINHUA; JINGYANG; 等

    1999-01-01

    Xenopus organizer specific gene noggin possesses nearly all the characterestic properties of the action of organizer to specify the embryonic body acis.To analyze how the maternal inherited factors control its expression pattern,we cloned the 5' regulatory region of noggin gene.The 1.5 kb upstream sequense could direct reporter gene to express in vivo and data from deletion analysis indicated that a 229 base pair fragmet is essential for activating noggin expression.We further demonstrated that the response elements within this regulatory region were indeed under the control of growth factor activin and Wnt signaling pathway components.

  16. Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

    Directory of Open Access Journals (Sweden)

    De Paepe Anne

    2004-02-01

    Full Text Available Abstract Background Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. Results As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. Conclusions The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

  17. Genomic and cDNA cloning of a novel mouse lipoxygenase gene

    NARCIS (Netherlands)

    Willems van Dijk, K.; Steketee, K.; Havekes, L.; Frants, R.; Hofker, M.

    1995-01-01

    A novel 12- and 15-lipoxygenase related gene was isolated from a mouse strain 129 genomic phage library in a screen with a human 15-lipoxygenase cDNA probe. The complete genomic sequence revealed 14 exons and 13 introns covering 7.3 kb of DNA. The splice junctions were verified from the cDNA

  18. Molecular cloning and nucleotide sequence of a transforming gene detected by transfection of chicken B-cell lymphoma DNA

    Science.gov (United States)

    Goubin, Gerard; Goldman, Debra S.; Luce, Judith; Neiman, Paul E.; Cooper, Geoffrey M.

    1983-03-01

    A transforming gene detected by transfection of chicken B-cell lymphoma DNA has been isolated by molecular cloning. It is homologous to a conserved family of sequences present in normal chicken and human DNAs but is not related to transforming genes of acutely transforming retroviruses. The nucleotide sequence of the cloned transforming gene suggests that it encodes a protein that is partially homologous to the amino terminus of transferrin and related proteins although only about one tenth the size of transferrin.

  19. Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family.

    Science.gov (United States)

    Tucker, M L; Durbin, M L; Clegg, M T; Lewis, L N

    1987-05-01

    A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.

  20. Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.

    Science.gov (United States)

    Laqueyrerie, A; Militzer, P; Romain, F; Eiglmeier, K; Cole, S; Marchal, G

    1995-01-01

    Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a

  1. Cloning and Sequencing of Leishmania major Thiol-Specific-Antioxidant Antigen (TSA Gene

    Directory of Open Access Journals (Sweden)

    F Tabatabaie

    2007-08-01

    Full Text Available Background: Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which, in the infected host are obli­gate intracellular parasite. TSA is the immuno-dominant antigen of Leishmania major which is considered as the most promising molecule for a recombinant or DNA vaccine against leishmaniasis. Methods: Genomic DNA of TSA protein was extracted and amplificated as a template. Then the PCR product was cloned into pTZ57R/T vector. Finally, the recombinant plasmid was extracted from transformed Escherichia coli (TG1 strain and sequenced. Results: MRHO/IR/75/ER (an Iranian strain of L. major and TSA gene (Accession number LmjF15.1080 were used. Se­quence analysis of cloned TSA gene into pTZ57R/T vector showed high homology of 90% with LmjF15.1080 (TSA gene and strain "LV39" (Accession no. AF069386 and strain "Friedlin" (Accession no.AF044679. Conclusion: We cloned TSA gene of L. major successfully. Recombinant plasmid was confirmed. It is ready to express recombinant protein for further studies.

  2. Cloning and characterization of the polyether salinomycin biosynthesis gene cluster of Streptomyces albus XM211.

    Science.gov (United States)

    Jiang, Chunyan; Wang, Hougen; Kang, Qianjin; Liu, Jing; Bai, Linquan

    2012-02-01

    Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.

  3. Cloning and Sequence Analysis of Light Variable Region Gene of Anti-human Retinoblastoma Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    Xiufeng Zhong; Yongping Li; Shuqi Huang; Bo Ning; Chunyan Zhang; Jianliang Zheng; Guanguang Feng

    2002-01-01

    Purpose: To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.Methods: Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody(McAb)against human retinoblastoma(RB), then transcripted reversely into cDNA with olig-dT primers.The variable region of the light chain (VL) gene fragments was amplified using polymeerase chain reaction(PCR) and further cloned into pGEM(R) -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method.Homologous analysis was done by NCBI BLAST.Results: The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions(FRs)and three complementarity-determining regions (CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.Conclusion: The light chain variable region gene of the McAb against human RB was amplified successfully , which belongs to the Vk9 gene family and utilizes Vk-Jk1 gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.

  4. Construction and analysis of a suppression subtractive hybridization library of regeneration-related genes in soybean.

    Science.gov (United States)

    Sun, J; Li, J; Liu, M; Zhang, B B; Li, D M; Wang, M; Zhang, C; Li, W B; Su, A Y; Wu, X X

    2015-01-30

    The development of a genetic transformation system is needed to address the problem of the low efficiency associated with soybean regeneration. To contribute to the enhancement of the soybean regenerative capacity, we explored the developmental mechanisms of soybean regeneration at the molecular level using a suppression subtractive hybridization cDNA library constructed from cotyledonary nodes of soybean cultivar DN50. A total of 918 positive clones were identified and screened, with most inserted fragments ranging from 100 to 750 bp. Of these, 411 differentially expressed functional expressed sequence tags were identified and annotated based on their similarity to orthologs and paralogs detected in GenBank using the nucleotide and translated nucleotide Basic Local Alignment Search Tools. Functional analysis revealed that the associated genes were involved in signal transduction, synthesis, and metabolism of macromolecules, glucose and protein synthesis and metabolism, light and leaf morphogenesis, regulation of apoptosis, cell defense, cell wall differentiation, and a variety of hormone and cytokinin-mediated signaling pathways. The information uncovered in our study should serve as a foundation for the establishment of an efficient and stable genetic transformation system for soybean regeneration.

  5. Molecular cloning and characterization of a cotton glucuronosyltranferase gene.

    Science.gov (United States)

    Wu, Yao-Ting; Liu, Jin-Yuan

    2005-05-01

    A glucuronosyltranferase gene has been isolated from cotton (Gossypium hirsutum) fiber cells using rapid amplification of the cDNA ends. The full-length cDNA, designated GhGlcAT1, is 1400 bp in length (AY346330) and contains an open reading frame of 1107 bp encoding a protein of 368 amino acids. Alignment of the GhGlcAT1 predicted amino acid sequence was shown to have high sequence similarity with animal glucuronosyltranferases. A phylogenic tree generated by the PHYLIP program package showed that GhGlcAT1 is clustered into the plant glucuronosyltranferase proteins and is distinct from those of other species. Homology modeling of the GhGlcAT1 structure using Homo sapiens native glucuronosyltranferase (1 kws and 1 fgg) structure as a template strongly suggests that the main-chain conformation and the folding patterns were similar to structural features characteristic of animal glucuronosyltranferases. Northern blot analysis showed that the transcripts of GhGlcAT1 were abundant in fiber cells, moderate in stem, but not detected in ovule, flower, seed, root and leaf. Transcripts were most abundant at 15dpa fiber. The transcription occurred at both the primary wall elongation stage and former stage of secondary cell thickening, suggesting that GhGLcAT1 may be involved in non-cellulose polysacchrides biosynthesis of the cotton cell wall.

  6. Phage Library Screening for the Rapid Identification and In Vivo Testing of Candidate Genes for a DNA Vaccine against Mycoplasma mycoides subsp. mycoides Small Colony Biotype

    Science.gov (United States)

    March, John B.; Jepson, Catherine D.; Clark, Jason R.; Totsika, Makrina; Calcutt, Michael J.

    2006-01-01

    A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia

  7. Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls.

    Directory of Open Access Journals (Sweden)

    Fei Gao

    Full Text Available Animal breeding via Somatic Cell Nuclear Transfer (SCNT has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF. Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver, using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.

  8. Cloning and bioinformatical analysis of the N-terminus of the sonic hedgehog gene.

    Science.gov (United States)

    Zhang, Yi; Zhao, Shu; Dong, Weiren; He, Suifen; Wang, Haihong; Zhang, Lihua; Tang, Yinjuan; Guo, Jiasong; Guo, Suiqun

    2013-01-25

    The sonic hedgehog protein not only plays a key role in early embryonic development, but also has essential effects on the adult nervous system, including neural stem cell proliferation, differentiation, migration and neuronal axon guidance. The N-terminal fragment of sonic hedgehog is the key functional element in this process. Therefore, this study aimed to clone and analyze the N-terminal fragment of the sonic hedgehog gene. Total RNA was extracted from the notochord of a Sprague-Dawley rat at embryonic day 9 and the N-terminal fragment of sonic hedgehog was amplified by nested reverse transcription-PCR. The N-terminal fragment of the sonic hedgehog gene was successfully cloned. The secondary and tertiary structures of the N-terminal fragment of the sonic hedgehog protein were predicted using Jpred and Phyre online.

  9. Cloning and bioinformatical analysis of the N-terminus of the sonic hedgehog gene

    Institute of Scientific and Technical Information of China (English)

    Yi Zhang; Shu Zhao; Weiren Dong; Suifen He; Haihong Wang; Lihua Zhang; Yinjuan Tang; Jiasong Guo; Suiqun Guo

    2013-01-01

    The sonic hedgehog protein not only plays a key role in early embryonic development, but also has essential effects on the adult nervous system, including neural stem cell proliferation, differentiation, migration and neuronal axon guidance. The N-terminal fragment of sonic hedgehog is the key functional element in this process. Therefore, this study aimed to clone and analyze the N-terminal fragment of the sonic hedgehog gene. Total RNA was extracted from the notochord of a Sprague-Dawley rat at embryonic day 9 and the N-terminal fragment of sonic hedgehog was amplified by nested reverse transcription-PCR. The N-terminal fragment of the sonic hedgehog gene was successfully cloned. The secondary and tertiary structures of the N-terminal fragment of the sonic hedgehog protein were predicted using Jpred and Phyre online.

  10. Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli.

    Science.gov (United States)

    Bautsch, W; Emde, M; Kretzschmar, T; Köhl, J; Suckau, D; Bitter-Suermann, D

    1992-06-01

    A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.

  11. Cloning and bioinformatics analysis of cDNA encoding cattle Smad4 gene

    Institute of Scientific and Technical Information of China (English)

    Xiaohui ZHANG; Shangzhong XU; Xue GAO; Hongyan REN; Jinbao CHEN

    2008-01-01

    The cDNA of cattle Smad4 gene was cloned by RT-PCR, 3' RACE and 5' RACE and got a 3503-bp full-long cDNA sequence. The cloned cattle Smad4 cDNA sequence had been send to GenBank and got an accession number: DQ494856. Cattle Smad4 gene consists of 12 exons and codes 553 amino acids. Cattle Smad4 cDNA shares 99%, 96%, 95%, 91% and 91% similarity in nucleic acid sequences, and 99%, 98%, 98%, 99% and 98% sim-ilarity in amino acid sequences with sheep, pig, human, rat and mouse, respectively. Smad4 cDNA was found in the testes, pancreas, liver, small intestine, ovary, lymph, car-diac muscle, skeleton muscle and thymus gland, which indicated that Smad4 was broadly expressed in cattle.

  12. Microarray analysis identifies a common set of cellular genes modulated by different HCV replicon clones

    Directory of Open Access Journals (Sweden)

    Gerosolimo Germano

    2008-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV RNA synthesis and protein expression affect cell homeostasis by modulation of gene expression. The impact of HCV replication on global cell transcription has not been fully evaluated. Thus, we analysed the expression profiles of different clones of human hepatoma-derived Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system. Results First, we compared the expression profile of HCV replicon clone 21-5 with both the Huh-7 parental cells and the 21-5 cured (21-5c cells. In these latter, the HCV RNA has been eliminated by IFN-α treatment. To confirm data, we also analyzed microarray results from both the 21-5 and two other HCV replicon clones, 22-6 and 21-7, compared to the Huh-7 cells. The study was carried out by using the Applied Biosystems (AB Human Genome Survey Microarray v1.0 which provides 31,700 probes that correspond to 27,868 human genes. Microarray analysis revealed a specific transcriptional program induced by HCV in replicon cells respect to both IFN-α-cured and Huh-7 cells. From the original datasets of differentially expressed genes, we selected by Venn diagrams a final list of 38 genes modulated by HCV in all clones. Most of the 38 genes have never been described before and showed high fold-change associated with significant p-value, strongly supporting data reliability. Classification of the 38 genes by Panther System identified functional categories that were significantly enriched in this gene set, such as histones and ribosomal proteins as well as extracellular matrix and intracellular protein traffic. The dataset also included new genes involved in lipid metabolism, extracellular matrix and cytoskeletal network, which may be critical for HCV replication and pathogenesis. Conclusion Our data provide a comprehensive analysis of alterations in gene expression induced by HCV replication and reveal modulation of new genes potentially useful

  13. Construction of Geobacillus thermoglucosidasius cDNA library and analysis of genes expressed in response to heat stress.

    Science.gov (United States)

    Tripathy, S; Maiti, N K

    2014-03-01

    Thermophiles exhibit various kinds of molecular mechanisms to survive in extreme environment, but their behavioral responses to long duration stress is poorly understood until date. In the present study, we have prospected for the genes differentially expressed in response to long duration heat stress in thermophilic bacteria. A cDNA library was constructed from Geobacillus thermoglucosidasius grown with a temperature upshift of 10 °C from optimum growth temperature of 45 °C for 16 h. A total of 451 clones from the library were sequenced with accurate base calling that generated 257 high quality sequences with an average read length of 350 bp. We queried our collection of single pass sequences against the NCBI non-redundant database using the BLASTX algorithm and obtained sequences that showed significant similarity (>60%) with heat shock proteins, metabolic proteins and hypothetical proteins. The expressed sequence tags (ESTs) expressed in response to heat stress were annotated that further commuted a strong interaction network among one another. The ESTs based on the best hits were validated by RT-PCR. Di- and tri-nucleotide repeat motifs were also found to be associated with 17 genes involved in heat shock response, metabolism, transport and transcriptional regulation. The present results provide the novel identification of the putative genes responsible for imparting tolerance to bacteria under heat stress and unveil their role for survival of life in environmental extremes.

  14. Rapid discrimination of Acinetobacter baumannii international clone II lineage by pyrosequencing SNP analyses of bla(OXA-51-like) genes.

    Science.gov (United States)

    Matsui, Mari; Suzuki, Satowa; Suzuki, Masato; Arakawa, Yoshichika; Shibayama, Keigo

    2013-08-01

    We found that Acinetobacter baumannii international clone II generally possesses unique GTA sequence at nucleotide positions 106-108 in the bla(OXA-51-like) genes. We exploited this to develop an easy and rapid method for discrimination of international clone II from other A. baumannii by employing pyrosequencing analyses of single nucleotide polymorphisms.

  15. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S;

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...

  16. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were us...

  17. Cloning and expression of Pectobacterium carotovorum endo-polygalacturonase gene in Pichia pastoris for production of oligogalacturonates

    Science.gov (United States)

    A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZaA vector and constitutively expressed in Pichia pastoris. The recombinant endo-PGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included ...

  18. Cloning of a Gene Cluster from Cellvibrio mixtus which Codes for Cellulase, Chitinase, Amylase, and Pectinase

    OpenAIRE

    1986-01-01

    The soil isolate Cellvibrio mixtus UQM2294 degraded a variety of polysaccharides including microcrystalline cellulose. Among 6,000 cosmid clones carrying C. mixtus DNA, constructed in Escherichia coli with pHC79, 50 expressed the ability to degrade one or more of the following substrates: carboxymethyl cellulose, chitin, pectin (polygalacturonic acid), cellobiose, and starch. These degradative genes are encoded in a single 94.1-kilobase segment of the C. mixtus genome; a preliminary order of ...

  19. A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

    OpenAIRE

    Wei, Lei; Yin, Li; Hu,Xiaole; Xu, Ying; Chen,Fang

    2014-01-01

    Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene ...

  20. Cloning and sequencing of the gene encoding thermophilic beta-amylase of Clostridium thermosulfurogenes.

    OpenAIRE

    Kitamoto, N; Yamagata, H; Kato, T.; Tsukagoshi, N; Udaka, S

    1988-01-01

    A gene coding for thermophilic beta-amylase of Clostridium thermosulfurogenes was cloned into Bacillus subtilis, and its nucleotide sequence was determined. The nucleotide sequence suggested that the thermophilic beta-amylase is translated from monocistronic mRNA as a secretory precursor with a signal peptide of 32 amino acid residues. The deduced amino acid sequence of the mature beta-amylase contained 519 residues with a molecular weight of 57,167. The amino acid sequence of the C. thermosu...

  1. Cloning of a Putative Pectate Lyase Gene Expressed in the Subventral Esophageal Glands of Heterodera glycines.

    Science.gov (United States)

    De Boer, J M; Davis, E L; Hussey, R S; Popeijus, H; Smant, G; Baum, T J

    2002-03-01

    We report the cloning of a Heterodera glycines cDNA that has 72% identity at the amino acid level to a pectate lyase from Globodera rostochiensis. In situ hybridizations showed that the corresponding gene (Hg-pel-1) is expressed in the subventral esophageal gland cells of second-stage juveniles. The deduced amino acid sequence of the H. glycines cDNA shows homology to class III pectate lyases of bacterial and fungal origin.

  2. [cDNA cloning and sequence analysis of pluripotency genes in tree shrews (Tupaia belangeri)].

    Science.gov (United States)

    Wang, Cai-Yun; Ma, Yun-Han; He, Da-Jian; Yang, Shi-Hua

    2013-04-01

    In this paper, partial sequences of the tree shrew (Tupaia belangeri) Klf4, Sox2, and c-Myc genes were cloned and sequenced, which were 382, 612, and 485 bp in length and encoded 127, 204, and 161 amino acids, respectively. Whereas, their cDNA sequence identities with those of human were 89%, 98%, and 89%, respectively. Their phylogenetic tree results indicated different topologies and suggested individual evolutional pathways. These results can facilitate further functional studies.

  3. Design of a ribozyme targeting human telomerase reverse transcriptase and cloning of it's gene

    Institute of Scientific and Technical Information of China (English)

    Zhi-Ming Hap; Jin-Yan Luo; Jin Cheng; Quan-Yin Wang; Guang-Xiao Yang

    2003-01-01

    AIM: To design a hammerhead ribozyme targeting humantelomerase reverse transcriptase (hTERT) and clone it's genefor future use in the study of tumor gene therapy.METHODS: Using the software RNAstructure, the secondarystructure of hTERT mRNA was predicted and the cleavagesite of ribozyme was selected. A hammerhead ribozymetargeting this site was designed and bimolecular fold betweenthe ribozyme and hTERT was predicted. The DNA encodingthe ribozyme was synthesized and cloned into pGEMEX-1and the sequence of the ribozyme gene was confirmed byDNA sequencing.RESULTS: Triplet GUC at 1742 of hTERT mRNA was chosenas the cleavage site of the ribozyme. The designed ribozymewas comprised of 22nt catalytic core and 17nt flankingsequence. Computer-aided prediction suggested that theribozyme and hTERT mRNA could cofold into a properconformation. Endonuclease restriction and DNA sequencingconfirmed the correct insertion of the ribozyme gene intothe vector pGEMEX-1.CONCLUSION: This fundamental work of successfuldesigning and cloning of an anti-hTERT hammerheadribozyme has paved the way for further study of inhibitingtumor cell growth by cleaving hTERT mRNA with ribozyme.

  4. [Cloning and bioinformatics analysis of SLA-DR genes in Hunan Shaziling pigs].

    Science.gov (United States)

    Tang, Yi-Ya; Xing, Xiao-Wei; Xue, Li-Qun; Huang, Sheng-Qiang; Wang, Wei

    2007-12-01

    In order to clone class II DRA and DRB genes of swine leukocyte antigen (SLA) in Hunan Shaziling pigs, to analyze their characteristics and polymorphism and to provide immunological basic parameters for xenotransplantation from pigs to humans. SLA-DRA and SLA-DRB genes in two Shaziling pigs with the absence of porcine endogenous retrovirus (PERV) env-c were amplified by RT-PCR, cloned into PUCm-T vectors, sequenced and analyzed through BLAST in NCBI and related software in ExPASY. The obtained SLA-DRA and SLA-DRB genes of Shaziling pigs were 1,177 and 909 nucleotides in length with their accession numbers in Genbank as EF143987 and EF143988. Bioinformatics analyses have shown that they both contain opening reading frame (ORF) and encode 252 and 266 amino acids respectively. Comparing the ORF and protein sequences of the Shaziling SLA-DRA and SLA-DRB genes with their counterpart sequences of human, the homologies of nucleotide sequences were 83% and 83%, and the homologies of amino acid sequences 83 % and 79% respectively. Further comparison with SLA sequences published in GenBank indicated that SLA-DRB gene found in Shaziling pigs has polymorphism while the homology of SLA-DRA gene is up to 100 % .

  5. Cloning and characterization of peter pan, a novel Drosophila gene required for larval growth.

    Science.gov (United States)

    Migeon, J C; Garfinkel, M S; Edgar, B A

    1999-06-01

    We identified a new Drosophila gene, peter pan (ppan), in a screen for larval growth-defective mutants. ppan mutant larvae do not grow and show minimal DNA replication but can survive until well after their heterozygotic siblings have pupariated. We cloned the ppan gene by P-element plasmid rescue. ppan belongs to a highly conserved gene family that includes Saccharomyces cerevisiae SSF1 and SSF2, as well as Schizosaccharomyces pombe, Arabidopsis, Caenorhabditis elegans, mouse, and human homologues. Deletion of both SSF1 and SSF2 in yeast is lethal, and depletion of the gene products causes cell division arrest. Mosaic analysis of ppan mutant clones in Drosophila imaginal disks and ovaries demonstrates that ppan is cell autonomous and required for normal mitotic growth but is not absolutely required for general biosynthesis or DNA replication. Overexpression of the wild-type gene causes cell death and disrupts the normal development of adult structures. The ppan gene family appears to have an essential and evolutionarily conserved role in cell growth.

  6. cDNA cloning and sequence analysis of NIb gene of soybean mosaic virus

    Institute of Scientific and Technical Information of China (English)

    刘俊君; 彭学贤; 莽克强

    1995-01-01

    cDNA of soybean mosaic virus (Beijing isolate, SMV-BJ) has been synthesized, using viralgenomic RNA as template and random hexanucleotides as primers. Based on the sequences of SMV-BJ coat protein (CP) gene as well as SMV- and WMV-II-related regions, oligonucleotides were made as primers for polymerase chain reaction (PCR). NIb gene of SMV-BJ was amplified by PCR, and cloned into pBluescript SK. The complete sequence was determined. The comparison of NIb genes between SMV-BJ and WMV-II . (USA) shows that similarities for nucleotide sequence reach 80.3%, and the deduced amino acid sequence. 91 3%. In consideration of the high identities in between the CP gene and the 3’-non-coding region between them, WMV-II might be considered as a watermelon strain of SMV Besides, some unexpected sequences were found in the 3’-region of 2 NIb gene clones. Following modification and splicing, a binary vector of NIb gene has been constructed for its expression in higher plant for the purpose of studying the possible repl

  7. Cloning and Homology Comparison of S Gene for Isolate TH-98 of Porcine Transmissible Gastroenteritis Virus

    Institute of Scientific and Technical Information of China (English)

    REN Xiao-feng; LI Yi-jing; LIU Bao-quan

    2003-01-01

    TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested onswine testicle (ST) monolayer ceil. Two pairs of primers were designed to amplify S gene by RT-PCR accord-ing to the published sequence of TGEV'S gene cDNA with Oligo version 4.1 and DNasis software. The productsof PCR were named Sa and Sb, of 2.3 kb and 2.1 kb respectively. Sa was inserted in EcoR I and Kpn I sitesafter Sb was cloned in Kpn I and Pst I multiple cloning sites of the same pUC18 plasmid. The recombinantpUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on thebasis of the genetic sites of S gene and pUC18 plasmid, which was identified as S gene of TGEV. RecombinantpUC-S was sequenced and analyzed in comparison with the other strains. Gene sequence comparison indicatedthat TH-98 shared 99, 97, 98, 97 and 94% identities with Purdue-115(US), Miller(US), TO14(Japan),FS772(British), 96-1933(British), respectively, their deduced amino acid homology was 99, 97, 97, 96 and93% correspondingly. In addition, the analysis report verified that pUC-S owned a complete open readingframe (ORF) including initiation codon, signal sequences, remaining sequences and termination codon aswell. Therefore, the results affirmed that S gene of TGEV TH-98 was extremely conservative.

  8. Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

    Science.gov (United States)

    Coskun, Cagil; Purali, Nuhan

    2016-06-01

    Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.

  9. [Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone].

    Science.gov (United States)

    Zeng, Qing-Ping; Feng, Li-Ling; Yang, Rui-Yi; Chen, Zhu-Hua

    2003-03-01

    To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.

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

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

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

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

    Science.gov (United States)

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

    2005-03-01

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

  12. [Cloning and sequencing the isopenicillin N synthetase(IPNS) gene from Streptomyces cattleya].

    Science.gov (United States)

    Wang, Y; Li, R

    1996-04-01

    Great homology existed between IPNS genes from surphur-containing beta-lactam antibiotics producers including procaryotes and eucaryotes. A DNA homologous band was confirmed in S. cattleya by Southern blot analysis using IPNS gene from S. lipmanii as a probe. A recombinant plasmid containing the cyclase gene involved in thienamycin biosynthesis and IPNS gene was obtained by complementary cloning with mutant from S. cattleya. DNA sequencing revealed that the IPNS gene of S. cattleya consists of 963 bp encoding a protein of 321 amino acids with ATG as start codon, TGA as stop codon. Pairwise comparison of the predicted amino acid sequences showed 56% and 64% similarity with IPNSs of S. clavuligerus and S. lipmanii, respectively.

  13. Cloning, sequencing and variability analysis of the gap gene from Mycoplasma hominis

    DEFF Research Database (Denmark)

    Mygind, Tina; Jacobsen, Iben Søgaard; Melkova, Renata

    2000-01-01

    The gap gene encodes the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The gene was cloned and sequenced from the Mycoplasma hominis type strain PG21(T). The intraspecies variability was investigated by inspection of restriction fragment length polymorphism (RFLP) patterns...... after polymerase chain reaction (PCR) amplification of the gap gene from 15 strains and furthermore by sequencing of part of the gene in eight strains. The M. hominis gap gene was found to vary more than the Escherichia coli counterpart, but the variation at nucleotide level gave rise to only a few...... to a 104-kDa band in addition to the expected 36-kDa band. The protein reacting at 104 kDa is a M. hominis protein with either an epitope similar to one on GAPDH, or it is an immunoglobulin binding protein...

  14. Suppression subtractive hybridization library construction and identification of epidermal bladder cell related genes in the common ice plant, Mesembryanthemum crystallinum L.

    Directory of Open Access Journals (Sweden)

    Siranet Roeurn

    2016-10-01

    Full Text Available Mesembryanthemum crystallinum L., a halophytic species, displays modified trichomes, epidermal bladder cells (EBC, on the surfaces of its aerial organs. EBCs serve to sequester excessive salt from underlying metabolically active tissues. To elucidate the molecular determinants governing EBC development in the common ice plant, we constructed a cDNA-based suppression subtractive hybridization library and identified genes differentially expressed between the wild-type and the EBC-less mutant. After hybridization, 38 clones were obtained. Among them, 24 clones had homology with plant genes of known functions, whose roles might not be directly related to EBC-morphology, while 14 clones were homologous to genes of unknown functions. After confirmation by northern blot analysis, 12 out of 14 clones of unknown functions were chosen for semi-quantitative RT-PCR analysis, and the results revealed that three clones designated as MW3, MW21, and MW31 preferentially expressed in the EBC-less mutant, whereas the other two designated as WM10 and WM28 preferentially expressed in the wild type. Among these genes, the expression of a putative jasmonate-induced gene, designated as WM28 was completely suppressed in the EBC-mutant. In addition, the deletion of C-box cis-acting element was found in the promoter region of WM28 in the EBC-less mutant. Overexpression of WM28 in Arabidopsis resulted in increased trichome number due to the upregulation of key trichome-related genes GLABRA1 (GL1, and GLABRA3 (GL3. These results demonstrate that WM28 can be an important factor responsible for EBC formation, and also suggest the similarity of developmental mechanism between trichome in Arabidopsis and EBC in common ice plant.

  15. Cloning large natural product gene clusters from the environment: Piecing environmental DNA gene clusters back together with TAR

    OpenAIRE

    Kim, Jeffrey H.; Feng, Zhiyang; Bauer, John D.; Kallifidas, Dimitris; Calle, Paula Y.; Brady, Sean F

    2010-01-01

    A single gram of soil can contain thousands of unique bacterial species, of which only a small fraction is regularly cultured in the laboratory. Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental sample...

  16. Cloning and molecular characterization of △12-fatty acid desaturase gene from Mortierella isabellina

    Institute of Scientific and Technical Information of China (English)

    Ming-Chun Li; Hang Li; Dong-Sheng Wei; Lai-Jun Xing

    2006-01-01

    AIM: To clone △12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS: Reverse transcriptional polymerase chain reaction (RT-PCR) was used to clone the open reading frame of △12-fatty acid desaturase gene (D12D) of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli(E.coli) strain BL21 using CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain INVSc1.RESULTS: Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E.coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of △12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △12-fatty acid desaturase,which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.CONCLUSION: Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.

  17. [Cloning, expression and identification of hpaA gene from a clinical isolate of Helicobacter pylori].

    Science.gov (United States)

    Mao, Ya-Fei; Yan, Jie; Li, Li-Wei

    2003-02-01

    To clone Helicobacter pylori adhesin (hpaA) gene,to construct the expression vector of the gene and to identify immunogenicity of the fusion protein. The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted hpaA gene was constructed. hpaA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein. In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned hpaA gene was from 94.25% approximate, equals 97.32%, while the homology of its putative amino acid sequence was as high as 95.38% approximate, equals 98.46%. The expression output of HpaA fusion protein in pET32a-hpaA-BL21DE3 system was approximately 40% of the total bacterial proteins. HpaA fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to preduce high titer antibody after the animal was immunized with the protein. An expression system with high efficiency of H.pylori hpaA gene has been established successfully. The expressed HpaA fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.

  18. cDNA Cloning and Sequence Analysis of Rice Sbel and Sbe3 Genes

    Institute of Scientific and Technical Information of China (English)

    CHENXiu-hua; LIUQiao-quan; WuHsin-kan; WANGZong-yang; GuMing-hong

    2004-01-01

    Two starch-branching enzyme (SBE) in rice, is known to be a key enzyme in amylopectin biosynthesis. The cDNA of two SBE(starch-branching enzyme) genes SheI and Shed encoding SBE Ⅰ and SBE Ⅲ (two major isoforms in rice) were cloned by an improved RT-PCR technique, from a template cDNA libray, derived from the total mRNAs extracted from the immature seeds of a japonica rice Wuyunjing 7. DNA sequence analysis showed that the size of the cloned SheI and Shed cDNAs were 2490 and 2481 bp long, respectively, including their entire coding sequences. Comparison analysis indicated that the nucleotide sequence of She3 was the same as that of shed (Genbank Accession No. D16201) as reported previously. There were only four base-pairs difference,which resulted in changes of two deduced amino acids between the cloned She1 cDNA and the reported she1 (Genbank Accession No. D11082). The cloned SheI and Shed cDNAs make it possible to improve rice starch quality through genetic engineering.

  19. Cloning and Sequence Analysis of Glycoprotein D Gene of Bovine Herpesvirus-1 Strain Luojing

    Institute of Scientific and Technical Information of China (English)

    LI Ji-chang; TONG Guang-zhi; QIU Hua-ji; ZHOU Yan-jun; XUE Qiang

    2003-01-01

    By means of PCR,the gene encoding gD of bovine herpesvirus-1 (BHV-1) strain Luojing was amplified,cloned and sequenced.The nucleotide sequence of this gD gene was 1 251 bp,encoding 417 amino acids.Comparied with the published P8-2 strain,the homology of the necleotide sequence is 99.92%,and that of the deduced amino acid sequence is 100%.The results indicated that gD of BHV-1 was highly conservative.

  20. Cloning and analysis of the four genes coding for Bpu10I restriction-modification enzymes.

    OpenAIRE

    Stankevicius, K; Lubys, A; Timinskas, A; Vaitkevicius, D; Janulaitis, A

    1998-01-01

    The Bpu 10I R-M system from Bacillus pumilus 10, which recognizes the asymmetric 5'-CCTNAGC sequence, has been cloned, sequenced and expressed in Escherichia coli . The system comprises four adjacent, similarly oriented genes encoding two m5C MTases and two subunits of Bpu 10I ENase (34.5 and 34 kDa). Both bpu10IR genes either in cis or trans are needed for the manifestation of R. Bpu 10I activity. Subunits of R. Bpu 10I, purified to apparent homogeneity, are both required for cleavage activi...

  1. Cloning and Sequencing of a Candida albicans Catalase Gene and Effects of Disruption of This Gene†

    OpenAIRE

    Wysong, Deborah R.; Christin, Laurent; Sugar, Alan M.; Robbins, Phillips W.; Diamond, Richard D.

    1998-01-01

    Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 102...

  2. Map-based cloning of the ALK gene, which controls the gelatinization temperature of rice.

    Science.gov (United States)

    Gao, Zhenyu; Zeng, Dali; Cui, Xia; Zhou, Yihua; Yan, Meixian; Huang, Danian; Li, Jiayang; Qian, Qian

    2003-12-01

    Gelatinization temperature (GT) is an important parameter for evaluating the cooking and eating quality of rice besides amylose content (AC). The inheritance of the genes affecting GT has been widely studied and is considered to be controlled by a major gene. Here, we report the map-based cloning of rice ALK that encodes the soluble starch synthase II (SSSII). Comparison between the DNA sequences from different rice varieties, together with the results obtained with digestion of the rice seeds in alkali solution, indicates that the base substitutions in coding sequence of ALK may cause the alteration in GT.

  3. Cloning of an epoxide hydrolase encoding gene from Rhodotorula mucilaginosa and functional expresion in Yarrowia lipolytica

    CSIR Research Space (South Africa)

    Labuschagne, M

    2007-01-01

    Full Text Available -joining method with the Kimura two-parameter distance measure. Confidence values were estimated from bootstrap analysis of 1000 replicates. The bar length corresponds to 10% amino acid dissimilarity amino acid) and the HGXP motif that contains the oxyanion... the isolation and cloning of an EH-encoding gene and its cDNA from Rhodotorula mucilaginosa and the functional expression of this gene in Y. lipolytica. Materials and methods Strains and culture conditions R. mucilaginosa (CBS 8596), Y. lipolytica strain...

  4. Cloning and Characterization of upp, a Gene Encoding Uracil Phosphoribosyltransferase from Lactococcus lactis

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1994-01-01

    Uracil phosphoribosyltransferase catalyzes the key reaction in the salvage of uracil in many microorganisms. The gene encoding uracil phosphoribosyltransferase (upp) was cloned from Lactococcus lactis subsp. cremoris MG1363 by complementation of an Escherichia coli mutant. The gene was sequenced...... construction of an internal deletion, a upp mutant was constructed by a double-crossover event. This implicated the utilization of a plasmid with a thermosensitive origin of replication and a new and easy way to screen for double crossover events in both gram-positive and gram-negative bacterial strains...

  5. Cloning, chromosome localization and features of a novel human gene, MATH2

    Indian Academy of Sciences (India)

    Lingchen Guo; Min Jiang; Yushu Ma; Haipeng Cheng; Xiaohua Ni; Yangsheng Jin; Yi Xie; Yumin Mao

    2002-04-01

    We report cloning and some features of a novel human gene, MATH2, which encodes a protein of 337 amino acid residues with a basic helix–loop–helix domain and exhibits 98% similarity to mouse Math2. Results of Northern blot analysis revealed two transcripts of the MATH2 gene of 1.7 kb and 2.4 kb in human brain. We localized MATH2 to chromosome 7 at 7p14–15 by matching with the Human Genome Sequence Database. Human MATH2 and mouse Math2 may have the same functions in the nervous system.

  6. Cloning and expression in Saccharomyces cerevisiae of chit2 gene from Beauveria bassiana

    Institute of Scientific and Technical Information of China (English)

    SONG Jin-zhu; YANG Xiao-xue; WANG Yun; YANG Qian

    2009-01-01

    To study recycled trashes from shrimps and crabs in the sea through chitinase secreted by microor-ganisms, the chitinase gene chit2 was cloned and sequenced from Beauveria bassiana by the polymerase chain reaction (PCR), and was ligated into the yeast expression vector pYES2. The expression vector plasmid was transformed into Saccharomyces cerevisiae H158. Gene expression took place upon induction with 2% galac-tose. The measurement of enzyme activity shows that the expression production can be expressed in active forms and secreted to the medium. The enzyme activity approaches the peak of 0. 63 U/mL when the culture time is 36 h.

  7. Cloning and analysizing the up-regulated expression of transthyretin-related gene(LR1) in rat liver regeneration following short interval successive partial hepatectomy

    Institute of Scientific and Technical Information of China (English)

    Cun-Shuan Xu; Yu-Chang Li; Jun-Tang Lin; Hui-Yong Zhang; Yun-Han Zhang

    2003-01-01

    AIM: Cloning and analysizing the up-regulated expressionof transthyretin-related gene following short intervalsuccessive partial hepatectomy (SISPH) to elucidate themechanism of differentiation, division, dedifferentiation andredifferentiation in rat liver regeneration (LR).METHODS: Lobus external sinister and lobus centralissinister, lobus centralis, lobus dexter, lobus candatus wereremoved one by one from rat liver at four different time points4, 36, 36 and 36 hr (total time: 4 hr, 40 hr, 76 hr, 112 hr)respectively. Suppression subtractive hybridization (SSH) wascarried out by using normal rat liver tissue as driver and thetissue following short interval successive partial hepatectomy(SISPH) as tester to construct a highly efficient forward-subtractive cDNA library. After screening, an interested ESTfragment was selected by SSH and primers were designedaccording to the sequence of the EST to clone the full-lengthcDNA fragment using RACE (rapid amplification of cDNAend). Homologous detection was performed between thefull-lenth cDNA and Genbank.RESULTS: Forward suppression subtractive hybridization(FSSH) library between 0 h and 112 h following SISPH wasconstructed and an up-regulated full-length cDNA (namedLR1), which was related with the transthyretin gene, wascloned by rapid amplification of cDNA end. It was suggestedthat the gene is involved in the cellular dedifferentiation inLR following SISPH.CONCLUSION: Some genes were up-regulated in 112 hfollowing SISPH in rat. LR1 is one of these up-regulatedexpression genes which may play an important role in rat LR.

  8. Cloning and characterisation of a glucoamylase gene (GlaM) from dimorphic zygomycete Mucor circinelloides

    DEFF Research Database (Denmark)

    Houghton-Larsen, J.; Pedersen, Per Amstrup

    2003-01-01

    This article reports a novel strategy for the cloning of glucoamylase genes using conserved sequences and semi-nested PCR and its application in cloning the GlaM glucoamylase gene and cDNA from the dimorphic zygomycete Mucor circinelloides. The deduced 609-amino-acid enzyme (including signal....... An alignment of the cloned gene and cDNA sequences showed that the gene contains three introns. The transcriptional start site and the site of polyadenylation were defined by primer extension and 3'RACE, respectively. The atypical Kozak sequence is identical to the one used in R. oryzae in positions -1 to -4....... Northern slot blots revealed that glucoamylase transcription is induced during growth on starch and repressed by glucose. In silico analysis of the 1.9-kb promoter sequence cloned by inverse PCR revealed the presence of several putative regulatory elements, most notably a 19-bp sequence containing six...

  9. Cloning and expression of a toxin gene from Pseudomonas fluorescens GcM5-1A.

    Science.gov (United States)

    Kong, Lingying; Guo, Daosen; Zhou, Shiyi; Yu, Xinlei; Hou, Guixue; Li, Ronggui; Zhao, Boguang

    2010-07-01

    Pseudomonas fluorescens GcM5-1A was isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, obtained from wilted Japanese black pine, Pinus thumbergii, in China. In this paper, a genomic library of the GcM5-1A strain was constructed and a toxin-producing clone was isolated by bioassay. Nucleotide sequence analysis revealed an open reading frame of 1,290 bp encoding a protein of 429 amino acids with N-terminal putative signal peptide of 36 amino acids, which shared a similarity of 83, 82 and 80% identity with hypothetical protein PFLU2919 from P. fluorescens SBW25, Dyp-type peroxidase family protein from P. fluorescens Pf-5 and Tat-translocated enzyme from P. fluorescens Pf0-1, respectively. The gene encoding a full-length protein or without the putative signal peptide was cloned and expressed as a soluble protein in E. coli. The recombinant protein was purified to electrophoretic homogeneity by affinity chromatography using a Ni2+ matrix column. Its relative molecular weight was estimated to be 48.5 kDa by SDS-PAGE for full-length protein, and 45.0 kDa for the recombinant protein without putative signal peptide. Bioassay results showed that the recombinant protein with or without the putative signal peptide was toxic to both suspension cells and P. thunbergii seedlings. HPLC analysis demonstrated that components in branch extracts of P. thunbergii were significantly changed after addition of the recombinant full-length protein and hydrogen peroxide, which indicated that it is probably a peroxidase. This study offers information that can be used to determine the mechanism of pine wilt disease caused by the PWN.

  10. Cloning and molecular characterisation of resuscitation promoting factor-like gene from Mycobacterium avium subspecies avium

    Directory of Open Access Journals (Sweden)

    R Kavitha

    2016-01-01

    Full Text Available Purpose: Resuscitation promoting factor (Rpf-like gene of Mycobacterium avium subspecies paratuberculosis has been known to stimulate the growth of mycobacteria and enhances the recovery of replicating cells from non-replicating phases. The objective of the study was to produce recombinant rpf-like protein of M. avium subspecies avium protein for purification and physico-chemical characterisation. Materials and Methods: The identified rpf gene of M. avium subspecies avium was cloned, subcloned, sequenced and expressed in Escherichia coli expression system for the production of the recombinant protein. The expressed recombinant Rpf protein was confirmed by Western blot and the extract was purified to yield a pure recombinant protein. Results: An rpf-like gene of 675 bp size in the M. avium subspecies avium was identified. This gene was expressed and the recombinant Rpf weighed 65 kDa as confirmed by Western blot. The M. avium recombinant Rpf protein was extracted under denatured conditions and purified yielding a recombinant protein with >90% purity. Conclusions: Identification, cloning, sequencing and expression of a rpf-like gene from M. avium suggest that RpfA is present in this species also, which might be involved in reactivation phenomenon in this high-risk pathogen.

  11. Molecular Cloning of TSARG6 Gene Related to Apoptosis in Human Spermatogenic Cells

    Institute of Scientific and Technical Information of China (English)

    Gang LIU; Guang-Xiu LU; Xiao-Wei XING

    2004-01-01

    Beginning from a mouse EST (GenBank accession No. BE644537) which was significantly up-regulated in cryptorchidism and represented a novel gene, we cloned a new gene (GenBank accessionNo. AY138810) which is related to apoptosis in human spermatogenic cells by means of GeneScan programand PCR technology. The gene whose full cDNA length is 1875 bp containing 8 exons and 7 introns islocated in human chromosome lq13.3. Its protein containing 316 amino acid residues is a new member ofHSP40 protein family because the sequence contains the highly conserved J domain which is present in allDna J-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. TSARG6protein displays a 45% identity in a 348-amino acid overlap with DJB5_HUMAN protein. The result ofRT-PCR and Northern blot analysis showed that TSARG6 is specifically expressed in adult testis and thetranscript is 1.8 kb. Based upon all these observations, it is considered that we cloned a new gene whichprobably inhibited human testis spermatogenesis apoptosis.

  12. Molecular cloning and expression analysis of Crustin-like gene from Chinese shrimp Fenneropenaeus chinensis

    Institute of Scientific and Technical Information of China (English)

    LIU Fengsong; LI Fuhua; XIANG Jianhai; DONG Bo; LIU Yichen; ZHANG Xiaojun; ZHANG Liusuo

    2008-01-01

    A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero-penaeus chinensis by 3'and 5' RACE.The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame,encoding 130 amino acids.The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids.The molecular mass of the deduced mature peptide is 12.3 ku.It is highly cationic with a theoretical isoelectric point of 8.5.The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus (Litopenaeus) setferus.Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes,gill,intestine,and RNA in situ hy-bridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues.Capillary elee-trephoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection.The level of Crustin mRNA began to restore at 72 h post-challenge.This indica-ted that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.

  13. Cloning and Expression Analysis of p26 Gene in Artemia sinica

    Institute of Scientific and Technical Information of China (English)

    Lijuan JIANG; Lin HOU; Xiangyang ZOU; Ruifeng ZHANG; Jiaqing WANG; Wenjing SUN; Xintao ZHAO; Jialu AN

    2007-01-01

    The protein p26 is a small heat shock protein that functions as a molecular chaperone to protect embryos by preventing irreversible protein damage during embryonic development. A 542 bp fragment of the p26 gene was cloned and sequenced. The fragment encoded 174 amino acid residues and the amino acid sequence contained the α-crystallin domain. Phylogenetic analysis showed that eight Artemia populations were divided into four major groups. Artemia sinica (YC) belonged to the East Asia bisexual group. Expression of the p26 gene at different developmental stages of A. sinica was quantified using real-time quantitative polymerase chain reaction followed by cloning and sequencing. The relationship between the quantity of p26 gene expression and embryonic development was analyzed. The results indicated that massive amounts of p26 were expressed during the development of A. sinica. At the developmental stage of 0 h, A. sinica expressed the highest level of p26. As development proceeded, expression levels of the p26 gene reduced significantly. There was a small quantity of p26 gene expression at the developmental stages of