WorldWideScience

Sample records for human cdna sequence

  1. 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.

  2. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.

  3. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

    DEFF Research Database (Denmark)

    Cowell, G M; Kønigshøfer, E; Danielsen, E M

    1988-01-01

    The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250...

  4. Nucleotide sequence of cloned cDNA for human pancreatic kallikrein.

    Science.gov (United States)

    Fukushima, D; Kitamura, N; Nakanishi, S

    1985-12-31

    Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.

  5. Sequence characterization of a human embryonic craniofacial cDNA library

    Energy Technology Data Exchange (ETDEWEB)

    Padanilam, B.J.; Barsel, S.; Solursh, M. [and others

    1994-09-01

    Broad-based sequencing approaches for the characterization of human cDNA libraries have proven successful in identifying large numbers of novel genes of specific tissue or developmental stages. To pursue our interests in human craniofacial development, stages. To pursue our interests in human craniofacial development, we have made use of both subtracted and unsubtracted cDNA libraries constructed from embryonic craniofacial tissue obtained from pooled samples at 42-54 days gestation. Single-pass sequencing was carried out using an ABI automated sequencer and T3 or T7 primers. Sequences were characterized using BLAST and GRAIL, and the identified homologous sequences grouped according to gene class and family. Four genes have been mapped using repeat sequence elements identified in the clones. Using primers developed from sequence data, other genes are being mapped using a panel of somatic cell hybrids. To date, a total of 786 sequences have been returned with 35% identifying no homologies, and 35% with strong homologies to previously identified genes. A number of genes previously identified to play a role in human embryonic development have been returned from the sequence comparisons providing evidence that the library is representative of this tissue and stage of development. Previous characterization of the library has also identified a number of novel embryonically expressed human homeobox genes. Genes felt to be of special relevance based on their homology to characterized genes known to play a role in development or that are members of novel classes but with high scores on GRAIL searches are being characterized using whole mount in situ hybridization with mouse embryos. Characterization of the library with respect to chromosomal mapping, gene types and make-up, and embryonic expression patterns will be presented.

  6. Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

    DEFF Research Database (Denmark)

    Ferreira, Elisa N; Pires, Lilian C; Parmigiani, Raphael B;

    2004-01-01

    The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification...... can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within...

  7. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. (The Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria (Australia))

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  8. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

    Energy Technology Data Exchange (ETDEWEB)

    Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L. (Univ. of Massachusetts Medical School, Worcester (USA))

    1988-06-01

    Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity.

  9. UMD‐Predictor: A High‐Throughput Sequencing Compliant System for Pathogenicity Prediction of any Human cDNA Substitution

    Science.gov (United States)

    Salgado, David; Desvignes, Jean‐Pierre; Rai, Ghadi; Blanchard, Arnaud; Miltgen, Morgane; Pinard, Amélie; Lévy, Nicolas; Collod‐Béroud, Gwenaëlle

    2016-01-01

    ABSTRACT Whole‐exome sequencing (WES) is increasingly applied to research and clinical diagnosis of human diseases. It typically results in large amounts of genetic variations. Depending on the mode of inheritance, only one or two correspond to pathogenic mutations responsible for the disease and present in affected individuals. Therefore, it is crucial to filter out nonpathogenic variants and limit downstream analysis to a handful of candidate mutations. We have developed a new computational combinatorial system UMD‐Predictor (http://umd‐predictor.eu) to efficiently annotate cDNA substitutions of all human transcripts for their potential pathogenicity. It combines biochemical properties, impact on splicing signals, localization in protein domains, variation frequency in the global population, and conservation through the BLOSUM62 global substitution matrix and a protein‐specific conservation among 100 species. We compared its accuracy with the seven most used and reliable prediction tools, using the largest reference variation datasets including more than 140,000 annotated variations. This system consistently demonstrated a better accuracy, specificity, Matthews correlation coefficient, diagnostic odds ratio, speed, and provided the shortest list of candidate mutations for WES. Webservices allow its implementation in any bioinformatics pipeline for next‐generation sequencing analysis. It could benefit to a wide range of users and applications varying from gene discovery to clinical diagnosis. PMID:26842889

  10. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data Description of data contents Phred's quality score. PHD format, one file to a single cDNA data, and co...ription Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive ...

  11. Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase.

    Science.gov (United States)

    Giallongo, A; Feo, S; Moore, R; Croce, C M; Showe, L C

    1986-01-01

    We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed. Images PMID:3529090

  12. Construction and packaging of pseudotype retrovirus containing human N—ras cDNA antisense sequence and its biological effects on human hepatoma cells

    Institute of Scientific and Technical Information of China (English)

    JIALIBIN; WANGXIANG; 等

    1990-01-01

    N-ras is one of the transforming genes in human hepatic cancer cells.It has been found that N-ras was overexpressed at the mRNA and protein level in hepatoma cells.In order to explore the biological roles of N-ras in human hepatic carcinogenesis and the potential application in control of cancer cell growth,a preudotype retrovirus containing antisense sequence of human N-ras was constructed and packaged.A recombinant retrovirus vector containing antisense or sense sequences of N-ras cDNA was constructed by pZIP-NeoSV(X)1.The pseudotype virus was packaged ang rescued by transfection and infection in PA317 and ψ 2 helper cells.It has been demonstrated that the pseudotype retrovirus containing antisense N-ras sequence did inhibit the growth of human PLC/PRF/5 hepatoma cells accompanied with inhibition of p21 expression,while the retrovirus containing sense sequence had none.The pseudotype virus had no effect on human diploid fibroblasts.

  13. Localization of the human fibromodulin gene (FMOD) to chromosome 1q32 and completion of the cDNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    Sztrolovics, R.; Grover, J.; Roughley, P.J. [McGill Univ., Montreal (Canada)] [and others

    1994-10-01

    This report describes the cloning of the 3{prime}-untranslated region of the human fibromodulin cDNA and its use to map the gene. For somatic cell hybrids, the generation of the PCR product was concordant with the presence of chromosome 1 and discordant with the presence of all other chromosomes, confirming that the fibromodulin gene is located within region q32 of chromosome 1. The physical mapping of genes is a critical step in the process of identifying which genes may be responsible for various inherited disorders. Specifically, the mapping of the fibromodulin gene now provides the information necessary to evaluate its potential role in genetic disorders of connective tissues. The analysis of previously reported diseases mapped to chromosome 1 reveals two genes located in the proximity of the fibromodulin locus. These are Usher syndrome type II, a recessive disorder characterized by hearing loss and retinitis pigmentosa, and Van der Woude syndrome, a dominant condition associated with abnormalities such as cleft lip and palate and hyperdontia. The genes for both of these disorders have been projected to be localized to 1q32 of a physical map that integrates available genetic linkage and physical data. However, it seems improbable that either of these disorders, exhibiting restricted tissue involvement, could be linked to the fibromodulin gene, given the wide tissue distribution of the encoded proteoglycan, although it remains possible that the relative importance of the quantity and function of the proteoglycan may avry between tissues. 11 refs., 1 fig.

  14. cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C{beta}4 (PLCB4)

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, R.A.; Ghalayini, A.J.; Anderson, R.E. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-09-01

    Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC{beta}4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC{beta}4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC{beta}4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by florescence in situ hybridization. 4 refs., 5 figs.

  15. Isolation of a human anti-haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX.

    Science.gov (United States)

    Jaye, M; de la Salle, H; Schamber, F; Balland, A; Kohli, V; Findeli, A; Tolstoshev, P; Lecocq, J P

    1983-04-25

    A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.Images

  16. cDNA cloning and sequencing of ostrich Growth hormone

    Directory of Open Access Journals (Sweden)

    Doosti Abbas

    2012-01-01

    Full Text Available In recent years, industrial breeding of ostrich (Struthio camelus has been widely developed in Iran. Growth hormone (GH is a peptide hormone that stimulates growth and cell reproduction in different animals. The aim of this study was to clone and sequence the ostrich growth hormone gene in E. coli, done for the first time in Iran. The cDNA that encodes ostrich growth hormone was isolated from total mRNA of the pituitary gland and amplified by RT-PCR using GH specific PCR primers. Then GH cDNA was cloned by T/A cloning technique and the construct was transformed into E. coli. Finally, GH cDNA sequence was submitted to the GenBank (Accession number: JN559394. The results of present study showed that GH cDNA was successfully cloned in E. coli. Sequencing confirmed that GH cDNA was cloned and that the length of ostrich GH cDNA was 672 bp; BLAST search showed that the sequence of growth hormone cDNA of the ostrich from Iran has 100% homology with other records existing in GenBank.

  17. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    Energy Technology Data Exchange (ETDEWEB)

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

  18. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    Science.gov (United States)

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  19. [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.

  20. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Jesionek-Kupnicka Dorota

    2009-08-01

    Full Text Available Abstract Background Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. Methods To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. Results We found P53 gene mutations in 16 cases (15 missense and 1 nonsense. Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. Conclusion In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis.

  1. Characterization of the human HOX 7 cDNA and identification of polymorphic markers.

    Science.gov (United States)

    Padanilam, B J; Stadler, H S; Mills, K A; McLeod, L B; Solursh, M; Lee, B; Ramirez, F; Buetow, K H; Murray, J C

    1992-09-01

    cDNA clones for a human HOX 7 gene obtained with homologous clones of Drosophila were used in human gene mapping studies. The human cDNA clone was isolated from a library constructed from human embryonic craniofacial material. The sequence of the cDNA demonstrates significant homology with mouse HOX 7. A search for RFLPs identified MboII and BstEII variants. A CA dinucleotide repeat with 5 alleles was also identified and allowed placement of HOX 7 into a defined linkage map. Evidence for linkage disequilibrium was found with markers tested. These results place the human HOX 7 gene in a defined position on 4p.

  2. CDNA encoding a polypeptide including a hevein sequence

    Science.gov (United States)

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  3. Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

    DEFF Research Database (Denmark)

    Lundwall, Åke B; Wetsel, Rick A; Kristensen, Torsten

    1985-01-01

    obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication......We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA...... clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence...

  4. Vector sequences - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us ...od - Number of data entries 7 entries - Joomla SEF URLs by Artio About This Database Database Description Download License Update His...tory of This Database Site Policy | Contact Us Vector sequences - Budding yeast cDNA sequencing project | LSDB Archive ...

  5. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

  6. Cloning and sequencing of Indian Water buffalo (Bubalus bubalis) interleukin-3 cDNA

    KAUST Repository

    Sugumar, Thennarasu

    2011-12-12

    Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.

  7. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 k......, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible...

  8. Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.

    Science.gov (United States)

    Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

    2016-08-24

    To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

  9. Complete cDNA sequence of the preproform of human pregnancy-associated plasma protein-A. Evidence for expression in the brain and induction by cAMP

    DEFF Research Database (Denmark)

    Haaning, Jesper; Oxvig, Claus; Overgaard, Michael Toft

    1996-01-01

    A cDNA that encodes the prepropeptide of pregnancy-associated plasma protein-A (preproPAPP-A), a putative metalloproteinase, has been cloned and sequenced. PAPP-A is synthesized in the placenta as a 1627-residue precursor preproprotein with a putative 22-residue signal peptide and a highly basic ...

  10. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  11. Analysis of cDNA sequence, protein structure and expression of parotid secretory protein in pig

    Institute of Scientific and Technical Information of China (English)

    YIN Haifang; FAN Baoliang; ZHAO Zhihui; LIU Zhaoliang; FEI Jing; LI Ning

    2003-01-01

    Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDNA end (RACE),based on the conserved signal peptide region among the known mammalian PSP. Theresult of homologous comparison shows that pig PSP and human PSP shares the high identity at the level of the primary, secondary and tertiary protein structure. A search for functionally significant protein motifs revealed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu- X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is important to its function. RT-PCR, Dot blot and Northern blot analysis demonstrated that PSP was strongly expressed in parotid glands, but not in other tissues.

  12. Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2009-01-01

    Full Text Available Abstract Background Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR and denervated (DL forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Results Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa. Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST contigs from the Ambystoma EST database more than doubled (3935 to 9411 the number of non-redundant human-A. mexicanum orthologous sequences. Conclusion Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

  13. THE TREE SHREW APOLIPOPROTEIN C-I cDNA: SEQUENCE AND ITS EXPRESSION

    Institute of Scientific and Technical Information of China (English)

    王克勤; 吕新跃; 吴钢; 薛红; 陈保生

    2001-01-01

    A rabbit anti-serum to tree shrew apolipoprotein C-I (apo C-l) was used to screen an expression cDNA li-braDy constructed by us from tree shrew (TS) liver tissue. Two apo C-I cDNA clones were obtained. The longerone consists of 380 nucleotides, including 21 bp and 95 bp at the 5' and 3' end of the non-translated region srespectively, and a 2 64-bp fragment in an open reading frame encoding 88 amino acids prepropeptide which con-ta-ins 26 amino acids of signal peptide and a mature protein (62 amino acids). Comparing the amino-acid se-quence deduced from this cDNA with those of the published mammalian apo C-Is reveals that it shared some struc-tural similarity with zat, mouse and dog apo C-l, but it had 5 more amino acids than that of human and baboon.The expression of apo C-I mRNA in 8 different tissues were also assayed with Northern blot. The results demonstrat-ed that liver had the highest expression, intestine had much less expression and no expression in other tissues,which is much different from human and other species. This study has laid down a good foundation for further study-ing on the function and the stucture of tree shrew apo C-I gene.``

  14. 人UCA1基因新剪接变异体全长cDNA序列的克隆%Cloning of the full-length cDNA sequence of a novel human UCA1 spliced variant

    Institute of Scientific and Technical Information of China (English)

    王宇; 陈葳; 李旭

    2012-01-01

    Objective To clone the full-length cDNA sequence of novel UCA1 spliced isoforms for understanding the exact mechanism of this type of alternative splicing. Methods The full-length cDNA was amplified from BLZ-211 cells by using the in silicon sequence elongation technique, 5'-RACE and 3'-RACE techniques. Products of RT-PCR were sequenced and further assembled. Results The new UCA1 spliced isoform sequence was 2 202 bp. Conclusion A combination of the in silicon sequence elongation, 5'-RACE and 3'-RACE techniques is an effective way to obtain the full-length cDNA, which will guide further research on the mechanism of this type of alternative splicing.%目的 克隆新的UCA1剪接变异体全长cDNA序列,为研究其可变剪接机制奠定基础.方法 用电子克隆技术和cDNA序列末端快速扩增技术(rapid amplification of cDNA ends,RACE)扩增细胞系BLZ-211 cDNA并进行产物测序和序列拼接.结果 新克隆的UCA1剪接变异体全长cDNA序列为2 202 bp.结论 综合采用电子克隆技术与RACE技术是获得全长cDNA序列的有效方法,为该基因的后续可变剪接机制的研究奠定了基础.

  15. Molecular cloning and sequence analysis of hamster CENP-A cDNA

    Science.gov (United States)

    Figueroa, Javier; Pendón, Carlos; Valdivia, Manuel M

    2002-01-01

    Background The centromere is a specialized locus that mediates chromosome movement during mitosis and meiosis. This chromosomal domain comprises a uniquely packaged form of heterochromatin that acts as a nucleus for the assembly of the kinetochore a trilaminar proteinaceous structure on the surface of each chromatid at the primary constriction. Kinetochores mediate interactions with the spindle fibers of the mitotic apparatus. Centromere protein A (CENP-A) is a histone H3-like protein specifically located to the inner plate of kinetochore at active centromeres. CENP-A works as a component of specialized nucleosomes at centromeres bound to arrays of repeat satellite DNA. Results We have cloned the hamster homologue of human and mouse CENP-A. The cDNA isolated was found to contain an open reading frame encoding a polypeptide consisting of 129 amino acid residues with a C-terminal histone fold domain highly homologous to those of CENP-A and H3 sequences previously released. However, significant sequence divergence was found at the N-terminal region of hamster CENP-A that is five and eleven residues shorter than those of mouse and human respectively. Further, a human serine 7 residue, a target site for Aurora B kinase phosphorylation involved in the mechanism of cytokinesis, was not found in the hamster protein. A human autoepitope at the N-terminal region of CENP-A described in autoinmune diseases is not conserved in the hamster protein. Conclusions We have cloned the hamster cDNA for the centromeric protein CENP-A. Significant differences on protein sequence were found at the N-terminal tail of hamster CENP-A in comparison with that of human and mouse. Our results show a high degree of evolutionary divergence of kinetochore CENP-A proteins in mammals. This is related to the high diverse nucleotide repeat sequences found at the centromere DNA among species and support a current centromere model for kinetochore function and structural plasticity. PMID:12019018

  16. Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan

    2001-09-01

    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

  17. Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan

    2013-03-01

    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

  18. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

  19. Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, E.K.; Horton, R.; Bloem, L.; Bach, R.; Williams, K.R.; Guha, A.; Kraus, J.; Lin, T.C.; Nemerson, Y.; Konigsberg, W.H.

    1987-08-01

    Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).

  20. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III (Univ. of Pennsylvania School of Medicine, Philadelphia (United States)); Billheimer, J.T. (E.I. DuPont de Nemours, Inc., Wilmington, DE (United States))

    1991-01-15

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP{sub 2}). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP{sub 2} amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP{sub 2}. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A){sup +} RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP{sub 2} gene in the human genome or that the SCP{sub 2} gene is very large. Coexpression of the SCP{sub 2} cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP{sub 2} plays a role in regulating steroidogenesis, among other possible functions.

  1. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality score...s Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality score...or-capping method, the sequence quality score generated by the Phred software, and links to SGD, dbEST and U...es. FASTA format. Quality Phred's quality score About This Database Database Desc...g yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive ...

  2. Characterization of Expressed Sequence Tags From a Gallus gallus Pineal Gland cDNA Library

    OpenAIRE

    2005-01-01

    The pineal gland is the circadian oscillator in the chicken, regulating diverse functions ranging from egg laying to feeding. Here, we describe the isolation and characterization of expressed sequence tags (ESTs) isolated from a chicken pineal gland cDNA library. A total of 192 unique sequences were analysed and submitted to GenBank; 6% of the ESTs matched neither GenBank cDNA sequences nor the newly assembled chicken genomic DNA sequence, three ESTs aligned with sequences designated to be on...

  3. Cloning and sequencing of dolphinfish (Coryphaena hippurus, Coryphaenidae) growth hormone-encoding cDNA.

    Science.gov (United States)

    Peduel, A D; Elizur, A; Knibb, W

    1994-01-01

    The cDNA encoding the preprotein growth hormone from the dolphinfish (Coryphaena hippurus) has been cloned and sequenced. The cDNA was derived by reverse transcription of RNA from the pituitary of a young fish using the method known as Rapid Amplification of cDNA Ends (RACE). An oligonucleotide primer corresponding to the 5' region of Pagrus major and the universal RACE primer enabled amplification using the Polymerase Chain Reaction (PCR). The dolphinfish and yellow-tail, Seriola quineqeradiata, are both members of the sub-order Percoidei (Perciforme) and their GH sequences show a high level of homology.

  4. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    Science.gov (United States)

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

  5. Construction of human and mouse brain cDNA libraries and isolation of full-length cDNAs

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    cDNA libraries from aborted human 3-month fetal brain,adult rat and mouse brain were constructed by using a yZAP express cDNA library construction kin.Low molecular weight fragments of the second strand cDNASA were removed by flowing through the Sepharose CL-4B column and the frractionated long,Middle,Short fragments and the combined fragments weire respectively inserted into clone vectors to construct the cDNA libraries of the brain of human 3-month fetus.The 5'ends of 1200 clones from each of human fetal brain cDNA libraries were sequenced.A total of 894 ESTs were obtained and some full-length clones were squenced.By andalyaing the se-quences,12 novel full-length cDNAs were obtained.

  6. Cloning, expression and mapping of the full-length cDNA of human CCTβ subunit

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Chaperonins assist the proper folding of target proteins without being a part of the substrates. The eukaryotic cytosolic chaperonin, CCT-Chaperonin Containing TCP-1 (tailless complex polypeptide-1), is mainly involved in the formation of cytoskeletal proteins and is essential for cell viability. Mammalian CCT is commonly a protein complex composed of 7-9 subunit species. We have isolated a novel full-length cDNA from human testis cDNA library. This cDNA of 1935 bp contains a 1605 bp open reading frame (ORF) encoding 535 amino acids (aa). The deduced protein of the cDNA is highly homologous to the CCTβ subunit of saccharomyces cerevisiae, schizosaccharomyces pombe, caenorhabditis elegans and mouse, etc. Especially high homology (97%) is found between the deduced protein and mouse CCTb. On the basis of such high homology, the protein encoded by the new gene was proposed to be a human CCTβ subunit. Northern hybridization showed that human CCTβ gene is expressed as a transcript of about 2.0 kb in various tissues. Overexpression was seen in testis with the expression level 3-24 times of those in other tissues. The CCTβ gene was mapped to human chromosome 12q14 by Radiation Hybrid Mapping. Through homologous search, the 5′-end of the cDNA sequence was found to share intermittent regional homology with the 3′-end of human genomic sequence (U91327). The genomic structure of the 5′-end of CCTβ was also described in detail through comparative analysis.

  7. Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States); Kaur, G.P. [Temple Univ. Medical School, Philadelphia, PA (United States)] [and others

    1997-05-01

    We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as {open_quotes}ST1C1{close_quotes} - although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated {open_quotes}SULT.{close_quotes} The new human cDNA was cloned from a fetal liver-spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5{prime}-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2. 14 refs., 2 figs.

  8. Budding yeast cDNA sequencing project: S03052-76_F01 [Budding yeast cDNA sequencing project

    Lifescience Database Archive (English)

    Full Text Available EST - Link to UCSC Genome Browser - Sequence >S03052-76_F01.phd NNNNNNNNNNNNNNNNNNNNNNNNNTNTAAAANNNNGANNNGANNNGTGGNTNTNTNTNT TNT...ANTTTNAANAAANAACNNNCCCTNNNNCNCNNNNNNNGAGNAAAAANNGGGTNTNNT NTTTTNNTNNTNTNTNNNNCNNN Qualit

  9. Download - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project Download First of all, please read the license of this database. Data ...names and data descriptions are about the downloadable data in this page. They might not correspond to the c...f the data. # Data name File Simple search and download 1 README README_e.html - 2 5'-end sequences of buddi...ng yeast full-length cDNA clones and quality scores yeast_seq_qual.zip (59.9MB) Simple search and download 3...Downlaod via FTP Joomla SEF URLs by Artio About This Database Database Description Download License Update H

  10. Cloning and sequencing of complete -crystallin cDNA from embryonic lens of Crocodylus palustris

    Indian Academy of Sciences (India)

    Raman Agrawal; Reena Chandrashekhar; Anurag Kumar Mishra; Jetty Ramadevi; Yogendra Sharma; Ramesh K Aggarwal

    2002-06-01

    -Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete -crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the -enolase gene from non-lenticular tissues. Quantitatively, the -crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile -crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5′- and 3′-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete -crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5′-and 3′-ends respectively. Further, it was found to be identical to its putative counterpart enzyme -enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of -crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.

  11. Molecular cloning and expression of a novel human cDNA containing CAG repeats.

    Science.gov (United States)

    Takeuchi, T; Chen, B K; Qiu, Y; Sonobe, H; Ohtsuki, Y

    1997-12-19

    A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.

  12. Molecular Cloning and Sequencing of Channel Catfish, Ictalurus punctatus, Cathepsin H and L cDNA

    Science.gov (United States)

    Cathepsin H and L, a lysosomal cysteine endopeptidase of the papain family, are ubiquitously expressed and involve in antigen processing. In this communication, the channel catfish cathepsin H and L transcripts were sequenced and analyzed. Total RNA from tissues was extracted and cDNA libraries we...

  13. Rapid Amplification of cDNA Ends for RNA Transcript Sequencing in Staphylococcus.

    Science.gov (United States)

    Miller, Eric

    2016-01-01

    Rapid amplification of cDNA ends (RACE) is a technique that was developed to swiftly and efficiently amplify full-length RNA molecules in which the terminal ends have not been characterized. Current usage of this procedure has been more focused on sequencing and characterizing RNA 5' and 3' untranslated regions. Herein is described an adapted RACE protocol to amplify bacterial RNA transcripts.

  14. Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz J; Boyd, Mette; Sandelin, Albin

    2013-01-01

    of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR...

  15. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

    DEFF Research Database (Denmark)

    Christensen, C B; Jørgensen, L; Jensen, A T

    2000-01-01

    Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L...

  16. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Science.gov (United States)

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  17. Cloning and Sequencing of Protein Kinase cDNA from Harbor Seal (Phoca vitulina Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2004-01-01

    Full Text Available Protein kinases (PKs play critical roles in signal transduction and activation of lymphocytes. The identification of PK genes provides a tool for understanding mechanisms of immunotoxic xenobiotics. As part of a larger study investigating persistent organic pollutants in the harbor seal and their possible immunomodulatory actions, we sequenced harbor seal cDNA fragments encoding PKs. The procedure, using degenerate primers based on conserved motifs of human protein tyrosine kinases (PTKs, successfully amplified nine phocid PK gene fragments with high homology to human and rodent orthologs. We identified eight PTKs and one dual (serine/threonine and tyrosine kinase. Among these were several PKs important in early signaling events through the B- and T-cell receptors (FYN, LYN, ITK and SYK and a MAP kinase involved in downstream signal transduction. V-FGR, RET and DDR2 were also expressed. Sequential activation of protein kinases ultimately induces gene transcription leading to the proliferation and differentiation of lymphocytes critical to adaptive immunity. PKs are potential targets of bioactive xenobiotics, including persistent organic pollutants of the marine environment; characterization of these molecules in the harbor seal provides a foundation for further research illuminating mechanisms of action of contaminants speculated to contribute to large-scale die-offs of marine mammals via immunosuppression.

  18. Revised sequence and expression of cyclin B cDNA from the starfish Asterina pectinifera.

    Science.gov (United States)

    Miyake, Y; Deshimaru, S; Toraya, T

    2001-05-01

    Cyclin B cDNA was cloned from the ovary of the starfish Asterina pectinifera and analyzed by RT-PCR and 3'- and 5'-RACE techniques. The cDNA consists of a 0.13-kb upstream untranslated region, a 1.22-kb coding region, and a 0.86-kb downstream untranslated region. The open reading frame encoded a polypeptide of 404 amino acid residues with a calculated molecular weight of 45,692. All the characteristic sequences, such as destruction and cyclin boxes, cyclin B motif, and cytoplasmic retention and nuclear export signals, were found in the newly cloned cyclin B cDNA. The deduced amino acid sequence of the cyclin B cDNA was highly homologous in the middle and carboxy terminal regions to that from mature eggs of the same organism, but quite different in the amino terminal region. Evidence was obtained which suggested that this cyclin B is expressed in immature and maturing oocytes and is the same as that cloned from mature eggs.

  19. Cloning and Sequence Analysis of cDNA Encoding MRJP3 of Apis cerana cerana

    Institute of Scientific and Technical Information of China (English)

    SU Song-kun; ZHNEG Huo-qing; CHEN Sheng-lu; ZHONG Bo-xiong; Stefan Albert

    2005-01-01

    By screening the worker (Apis cerana cerana) heads cDNA library using a fragment of the mrjp3 gene ofApis cerana as probe, 120 positive clones were obtained. The clone containing A. cerana cerana MRJP3 (AccMRJP3) cDNA was selected. Based on the sequencing of the inserts of the positive clone, a sequence of AccMRJP3 cDNA which is 1 887 bp long including a poly (A) tail was obtained. The AccMRJP3 cDNA encompassed an open-reading frame (ORF) with 1 779 bp encoding 593 amino acids. The un-translated regions (UTR) of the 5' end and 3' end are 46 bp and 160 bp in length,respectively. Similar to AmMRJP3 and AdMRJP3, the putative AccMRJP3 also has a repetitive region. The comparison of the repetitive region of AccMRJP3, AmMRJP3 and AdMRJP3 shows some differences between them.

  20. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

    DEFF Research Database (Denmark)

    Wewer, U M; Gerecke, D R; Durkin, M E

    1994-01-01

    Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... chain showed 86.1% sequence identity to the rat beta 2 chain, 50.0% to the human beta 1 chain, and 36.3% to the human beta 3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the beta 2 chain of laminin purified from human...

  1. Characterization of full-length sequenced cDNA inserts (FLIcs from Atlantic salmon (Salmo salar

    Directory of Open Access Journals (Sweden)

    Lunner Sigbjørn

    2009-10-01

    Full Text Available Abstract Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP, the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91% of the transcripts were annotated using Gene Ontology (GO terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS. The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS. This

  2. cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

    Institute of Scientific and Technical Information of China (English)

    王玉刚; 刘洪涛; 张颖妹; 马大龙

    1999-01-01

    By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell

  3. Molecular cloning and sequencing of a cDNA encoding partial putative molt-inhibiting hormone from Penaeus chinensis

    Science.gov (United States)

    Wang, Zai-Zhao; Xiang, Jian-Hai

    2002-09-01

    Total RNA was extracted from eyestalks of shrimp Penaeus chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymerase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  4. MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

    Total RNA was extracted from eyestalks of shrimp Penaeus chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymer ase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A s pecific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 ba se pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  5. MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

    Total RNA was extracted from eyestalks of shrimp Penaeue chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymerase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  6. Expressed sequence tags analysis of a liver tissue cDNA library from a highly inbred minipig line

    Institute of Scientific and Technical Information of China (English)

    CHEN You-nan; TAN Wei-dong; LU Yan-rong; QIN Sheng-fang; LI Sheng-fu; ZENG Yang-zhi; BU Hong; LI You-ping; CHENG Jing-qiu

    2007-01-01

    Background Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore,we investigated the liver expression profile of a highly inbred minipig line.Methods A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme.Results Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences.Conclusion These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.

  7. Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends.

    Science.gov (United States)

    Graham, A; Hopkins, B; Powell, S J; Danks, P; Briggs, I

    1991-05-31

    Functional cDNA clones for human NK-2 receptor were isolated from human lung RNA using a polymerase chain reaction (PCR) based method (RACE-PCR). In this method the cDNA was isolated as 5' end and 3'-end fragments; the entire cDNA was obtained by RNA-PCR. The sequence derived was 398 amino acids in length encoding an open-reading frame that was highly homologous to both the bovine and rat NK-2 receptor. The entire human cDNA sequence was cloned into a mammalian expression vector and mRNA was synthesised by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesised mRNA. The most potent of the three tachykinin peptides tested was neurokinin A. We have screened a human cosmid library and isolated a clone which contains the entire NK-2 receptor gene. The gene contains five exons and we have determined the complete sequence of the exons and the intron-exon junctions.

  8. Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).

    Science.gov (United States)

    Kasarda, D D; Okita, T W; Bernardin, J E; Baecker, P A; Nimmo, C C; Lew, E J; Dietler, M D; Greene, F C

    1984-01-01

    The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with amino acid sequences derived from both cDNA clones and is virtually identical to one of them. On the basis of sequence information, after loss of the signal sequence, the mature alpha-type gliadins may be divided into five different domains, two of which may have evolved from an ancestral gliadin gene, whereas the remaining three contain repeating sequences that may have developed independently. Images PMID:6589619

  9. 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.

  10. 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

  11. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    Energy Technology Data Exchange (ETDEWEB)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H. (Research Institutes, Postfach (West Germany))

    1988-06-01

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10{sup 6} independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I.

  12. Cloning and sequencing of murine T3 gamma cDNA from a subtractive cDNA library.

    Science.gov (United States)

    Haser, W G; Saito, H; Koyama, T; Tonegawa, S

    1987-10-01

    The coding sequences of the murine and human T3 gamma chains are of identical length (182 amino acids) and contain a remarkable conservation of residues. The most striking observation is the high degree of homology between the murine and human cytosolic domains (89%), suggesting that the effector function of the T3 complex may be extremely similar or identical within human and murine lymphocytes. Both murine and human T lymphocytes can express two T3 gamma mRNA transcripts, suggesting that a second polyadenylation signal is present downstream. A poly(A) tail is not found in the 3' untranslated region of the murine gamma presented here, indicating that the murine clones analyzed represent mRNA generated by reading through the overlapping poly(A) signals at position 850-860 and possibly terminating at a position that would produce the 1.0 kb transcript.

  13. Canine amino acid transport system Xc(-): cDNA sequence, distribution and cystine transport activity in lens epithelial cells.

    Science.gov (United States)

    Maruo, Takuya; Kanemaki, Nobuyuki; Onda, Ken; Sato, Reiichiro; Ichihara, Nobuteru; Ochiai, Hideharu

    2014-04-01

    The cystine transport activity of a lens epithelial cell line originated from a canine mature cataract was investigated. The distinct cystine transport activity was observed, which was inhibited to 28% by extracellular 1 mM glutamate. The cDNA sequences of canine cysteine/glutamate exchanger (xCT) and 4F2hc were determined. The predicted amino acid sequences were 527 and 533 amino acid polypeptides, respectively. The amino acid sequences of canine xCT and 4F2hc showed high similarities (>80%) to those of humans. The expression of xCT in lens epithelial cell line was confirmed by western blot analysis. RT-PCR analysis revealed high level expression only in the brain, and it was below the detectable level in other tissues.

  14. Rat serum amyloid P component. Analysis of cDNA sequence and gene expression.

    Science.gov (United States)

    Dowton, S B; McGrew, S D

    1990-09-01

    cDNA clones for rat serum amyloid P component (SAP) were isolated, and the derived amino acid sequence for pre-SAP was determined from the complete nucleotide sequence. Rat SAP is encoded by approximately 1 kb of mRNA, and the mature SAP protein is predicted to be 208 amino acids long. An increase in hepatic mRNA levels for rat SAP was found after administration of lipopolysaccharide, and SAP mRNA levels in livers of unstimulated male rats were lower than in hepatic RNA from female rats.

  15. Rice bicoid-related cDNA sequence and its expression during early embryogenesis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation.To clone and characterize the rice bicoid-related genes,one cDNA clone,Rb24 (EMBL accession number: AJ2771380),was isolated by screening of rice unmature seed cDNA library.Sequence analysis indicates that Rb24 contains a putative amino acid sequence,which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity,75% similarity) and involves a lys-9 in putative helix 3.Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner.The transcript was detected strongly in young panicles,but less in young leaves and roots.This results are further confirmed with paraffin section in situ hybridization.The signal is intensive in rice globular embryo and located at the apical tip of the embryo,then,along with the development of embryo,the signal is getting reduced and transfers into both sides of embryo.The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

  16. Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).

    OpenAIRE

    Kasarda, D.D.; Okita, T W; Bernardin, J. E.; Baecker, P A; Nimmo, C C; Lew, E J; Dietler, M D; Greene, F C

    1984-01-01

    The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with a...

  17. Molecular cloning of a cDNA encoding the human Sm-D autoantigen

    Energy Technology Data Exchange (ETDEWEB)

    Rokeach, L.A.; Haselby, J.A.; Hoch, S.O. (Agouron Institute, La Jolla, CA (USA))

    1988-07-01

    Antibodies to the Sm-D polypeptide antigen are closely associated with the rheumatic disease systemic lupus erythematosus. Sm-D exists in the cell as one of the core proteins of the small nuclear ribonucleoprotein complexes implicated in RNA processing. The authors have isolated a cDNA clone, D45-2, coding for the Sm-D human nuclear antigen by screening a human B-lymphocyte cDNA library with synthetic oligonucleotide probes. The 1633-base-pair clone contains an open reading frame (ORF) 357 nucleotides long, capable of encoding a 13,282-dalton polypeptide. The Sm-D coding region is initiated at an AUG codon downstream from a sequence with excellent match to the consensus for the eukaryotic ribosome-binding site. The Sm-D ORF is preceded by a 150-nucleotide-long untranslated leader and followed by a 1126-nucleotide-long untranslated region containing four putative poly(A) signals. The predicted amino acid sequence reveals a (Gly-Arg){sub 9} repeated motif at the C terminus, which may constitute one of the Sm-D immunoreactive determinants. Moreover, this C terminus shows interesting features: (i) a good homology to protamines as expected for a nucleic acid binding protein and (ii) a striking similarity to a region in the Epstein-Barr nuclear antigen.

  18. Human cDNA mapping using fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  19. Characterisation of full-length cDNA sequences provides insights into the Eimeria tenellatranscriptome

    Directory of Open Access Journals (Sweden)

    Amiruddin Nadzirah

    2012-01-01

    Full Text Available Abstract Background Eimeria tenella is an apicomplexan parasite that causes coccidiosis in the domestic fowl. Infection with this parasite is diagnosed frequently in intensively reared poultry and its control is usually accorded a high priority, especially in chickens raised for meat. Prophylactic chemotherapy has been the primary method used for the control of coccidiosis. However, drug efficacy can be compromised by drug-resistant parasites and the lack of new drugs highlights demands for alternative control strategies including vaccination. In the long term, sustainable control of coccidiosis will most likely be achieved through integrated drug and vaccination programmes. Characterisation of the E. tenella transcriptome may provide a better understanding of the biology of the parasite and aid in the development of a more effective control for coccidiosis. Results More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs. Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis. Conclusions This paper describes the generation and characterisation of full-length cDNA sequences from E

  20. Infectivity and complete nucleotide sequence of cucumber fruit mottle mosaic virus isolate Cm cDNA.

    Science.gov (United States)

    Rhee, Sun-Ju; Hong, Jin-Sung; Lee, Gung Pyo

    2014-07-01

    Three isolates of cucumber fruit mottle mosaic virus (CFMMV) were collected from melon, cucumber, and pumpkin plants in Korea. A full-length cDNA clone of CFMMV-Cm (melon isolate) was produced and evaluated for infectivity after T7 transcription in vitro (pT7CF-Cmflc). The complete CFMMV genome sequence of the infectious clone pT7CF-Cmflc was determined. The genome of CFMMV-Cm consisted of 6,571 nucleotides and shared high nucleotide sequence identity (98.8 %) with the Israel isolate of CFMMV. Based on the infectious clone pT7CF-Cmflc, a CaMV 35S-promoter driven cDNA clone (p35SCF-Cmflc) was subsequently constructed and sequenced. Mechanical inoculation with RNA transcripts of pT7CF-Cmflc and agro-inoculation with p35SCF-Cmflc resulted in systemic infection of cucumber and melon, producing symptoms similar to those produced by CFMMV-Cm. Progeny virus in infected plants was detected by RT-PCR, western blot assay, and transmission electron microscopy.

  1. Rabbit serum amyloid protein A: expression and primary structure deduced from cDNA sequences.

    Science.gov (United States)

    Rygg, M; Marhaug, G; Husby, G; Dowton, S B

    1991-12-01

    Serum amyloid A protein (SAA), the precursor of amyloid protein A (AA) in deposits of secondary amyloidosis, is an acute phase plasma apolipoprotein produced by hepatocytes. The primary structure of SAA demonstrates high interspecies homology. Several isoforms exist in individual species, probably with different amyloidogenic potential. The nucleotide sequences of two different rabbit serum amyloid A cDNA clones have been analysed, one (corresponding to SAA1) 569 base pairs (bp) long and the other (corresponding to SAA2) 513 bp long. Their deduced amino acid sequences differ at five amino acid positions, four of which are located in the NH2-terminal region of the protein. The deduced amino acid sequence of SAA2 corresponds to rabbit protein AA previously described except for one amino acid in position 22. Eighteen hours after turpentine stimulation, rabbit SAA mRNA is abundant in liver, while lower levels are present in spleen. None of the other extrahepatic organs studied showed any SAA mRNA expression. A third mRNA species (1.9 kb) hybridizing with a single-stranded RNA probe transcribed from the rabbit SAA cDNA, was identified. SAA1 and SAA2 mRNA were found in approximately equal amounts in turpentine-stimulated rabbit liver, but seem to be coordinately decreased after repeated inflammatory stimulation.

  2. Molecular Cloning of a Novel cDNA From Mus Muscular BALB/c Mice Encoding Glycosyl Hydrolase Family 1: A Homolog of HumanLactase-Phlorizin Hydrolase

    Institute of Scientific and Technical Information of China (English)

    WEI HE; ZHEN-YU JI; CHENG-YU HUANG

    2006-01-01

    Objective To study the mechanism of lactose intolerance (LI) by cloning the mouse lactase cDNA and recombining a vector. Methods Total murine RNA was isolated from the small intestine of a 4-week-old BALB/c mouse (♂).Gene-specific primers were designed and synthesized according to the cDNA sequences of lactase-phlorizin hydrolase (LPH) in human, rat, and rabbit. A coding sequence (CDS) fragment was obtained using RT-PCR, and inserted into a clone vector pNEB-193, then the cDNA was sequenced and analyzed using bioinformatics. Results The cDNA from the BALB/c mouse with 912 bp encoding 303 amino acid residues. Analysis of the deduced amino acid sequence using bioinformatics revealed that this cDNA shared extensive sequence homology with human LPH containing a conserved glycosy1 hydrolase family 1 motif important for regulating lactase intolerance. Conclusion BALB/c mouse LPH cDNA (GenBank accession No: AY751548) provides a necessary foundation for study of the biological function and regulatory mechanism of the lactose intolerance in mice.

  3. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class.

    OpenAIRE

    Gronwald, R G; Grant, F J; Haldeman, B A; Hart, C E; O'Hara, P J; Hagen, F S; Ross, R.; Bowen-Pope, D F; Murray, M. J.

    1988-01-01

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately ...

  4. Achieving high throughput sequencing of a cDNA library utilizing an alternative protocol for the bench top next-generation sequencing system.

    Science.gov (United States)

    Wan, Minxi; Faruq, Junaid; Rosenberg, Julian N; Xia, Jinlan; Oyler, George A; Betenbaugh, Michael J

    2013-02-15

    The development of next-generation sequencing (NGS) technologies has provided novel tools for genome analysis and expression profiling. A high throughput cDNA sequencing method using a bench top next-generation sequencing system, GS Junior, is now available. Here, we used an alternative protocol to the standard method for generating the cDNA library. This protocol can decrease the number of processing steps to manipulate RNA when constructing a cDNA library from an RNA sample, and does not require mRNA isolation from total RNA. Thus it can decrease the risk of RNA degradation and the cost for preparing a cDNA library. Also, the efficiency of sequencing data obtained with this approach is comparable to the standard method as verified by sequencing characteristics and expression levels of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

  5. Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: molecular sequence analysis of its cDNA and biochemical properties.

    Science.gov (United States)

    Vivas-Ruiz, Dan E; Sandoval, Gustavo A; Mendoza, Julio; Inga, Rosalina R; Gontijo, Silea; Richardson, Michael; Eble, Johannes A; Yarleque, Armando; Sanchez, Eladio F

    2013-07-01

    The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.

  6. Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis

    Directory of Open Access Journals (Sweden)

    S. S. Hasson

    2010-01-01

    Full Text Available Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

  7. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Alkhatib, H.M.; Chen, D.; Cherney, B.; Bhatia, K.; Notario, V.; Giri, C.; Stein, G.; Slattery, E.; Roeder, R.G.; Smulson, M.E.

    1987-03-01

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambdagt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambdagt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

  8. Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

    Directory of Open Access Journals (Sweden)

    Wayan T. Artama

    2015-10-01

    Full Text Available Rhoptry protein belongs to an excretory and secretory antigens (ESAs that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue. The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory

  9. Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

    Directory of Open Access Journals (Sweden)

    Murwantoko M

    2015-11-01

    Full Text Available Rhoptry protein belongs to an excretory and secretory antigens (ESAs that play an important role during active penetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targeted cell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasite successfully enter the cell target then Granule (GRA proteins are responsible for the formation of parasitophorus vacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently, this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone and sequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique. Total ribonucleic acid (RNA was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA was used as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor from Riboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinant plasmid was transformed into E. coli (XL1-Blue. The transformed E. coli XL-1 Blue were plated on LB agar containing X-Gal, IPTG and ampicillin. Recombinant clones (white colony were picked up and grown up in the LB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order to identify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolated using alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid was cut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward and M13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretory

  10. Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences.

    Science.gov (United States)

    Marhaug, G; Husby, G; Dowton, S B

    1990-06-15

    The nucleotide sequences of two mink serum amyloid A (SAA) cDNA clones have been analyzed, one (SAA1) 776 base pairs long and the other (SAA2) 552 base pairs long. Significant differences were discovered when derived amino acid sequences were compared with data for apoSAA isolated from high density lipoprotein. Previous studies of mink protein SAA and amyloid protein A (AA) suggest that only one SAA isotype is amyloidogenic. The cDNA clone for SAA2 defines the "amyloid prone" isotype while SAA1 is found only in serum. Mink SAA1 has alanine in position 10, isoleucine in positions 24, 67, and 71, lysine in position 27, and proline in position 105. Residue 10 in mink SAA2 is valine while arginine and asparagine are at positions 24 and 27, respectively, all characteristics of protein AA isolated from mink amyloid fibrils. Mink SAA2 also has valine in position 67, phenylalanine in position 71, and amino acid 105 is serine. It remains unknown why these six amino acid substitutions render SAA2 more amyloidogenic than SAA1. Eighteen hours after lipopolysaccharide stimulation, mink SAA mRNA is abundant in liver with relatively minor accumulations in brain and lung. Genes encoding both SAA isotypes are expressed in all three organs while no SAA mRNA was detectable in amyloid prone organs, including spleen and intestine, indicating that deposition of AA from locally synthesized SAA is unlikely. A third mRNA species (2.2 kilobases) was identified and hybridizes with cDNA probes for mink SAA1 and SAA2. In addition to a major primary translation product (molecular mass 14,400 Da) an additional product with molecular mass 28,000 Da was immunoprecipitable.

  11. Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus

    Directory of Open Access Journals (Sweden)

    Janeth Silva Pinheiro

    2008-01-01

    Full Text Available RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.

  12. Primary analysis of the expressed sequence tags in a pentastomid nymph cDNA library.

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    Full Text Available BACKGROUND: Pentastomiasis is a rare zoonotic disease caused by pentastomids. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, until now, the systematic classification of the pentastomids and the diagnosis of pentastomiasis are immature, and genetic information about pentastomid nylum is almost nonexistent. The objective of this study was to obtain information on pentastomid nymph genes and identify the gene homologues related to host-parasite interactions or stage-specific antigens. METHODOLOGY/PRINCIPAL FINDINGS: Total pentastomid nymph RNA was used to construct a cDNA library and 500 colonies were sequenced. Analysis shows one hundred and ninety-seven unigenes were identified. In which, 147 genes were annotated, and 75 unigenes (53.19% were mapped to 82 KEGG pathways, including 29 metabolism pathways, 29 genetic information processing pathways, 4 environmental information processing pathways, 7 cell motility pathways and 5 organismal systems pathways. Additionally, two host-parasite interaction-related gene homologues, a putative Kunitz inhibitor and a putative cysteine protease. CONCLUSION/SIGNIFICANCE: We first successfully constructed a cDNA library and gained a number of expressed sequence tags (EST from pentastomid nymphs, which will lay the foundation for the further study on pentastomids and pentastomiasis.

  13. Primary Analysis of the Expressed Sequence Tags in a Pentastomid Nymph cDNA Library

    Science.gov (United States)

    Yuan, Zhongying; Yin, Jianhai; Zang, Wei; Xu, Yuxin; Lu, Weiyuan; Wang, Yanjuan; Wang, Ying; Cao, Jianping

    2013-01-01

    Background Pentastomiasis is a rare zoonotic disease caused by pentastomids. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, until now, the systematic classification of the pentastomids and the diagnosis of pentastomiasis are immature, and genetic information about pentastomid nylum is almost nonexistent. The objective of this study was to obtain information on pentastomid nymph genes and identify the gene homologues related to host-parasite interactions or stage-specific antigens. Methodology/Principal Findings Total pentastomid nymph RNA was used to construct a cDNA library and 500 colonies were sequenced. Analysis shows one hundred and ninety-seven unigenes were identified. In which, 147 genes were annotated, and 75 unigenes (53.19%) were mapped to 82 KEGG pathways, including 29 metabolism pathways, 29 genetic information processing pathways, 4 environmental information processing pathways, 7 cell motility pathways and 5 organismal systems pathways. Additionally, two host-parasite interaction-related gene homologues, a putative Kunitz inhibitor and a putative cysteine protease. Conclusion/Significance We first successfully constructed a cDNA library and gained a number of expressed sequence tags (EST) from pentastomid nymphs, which will lay the foundation for the further study on pentastomids and pentastomiasis. PMID:23437150

  14. An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones

    Science.gov (United States)

    Butterfield, Yaron S. N.; Marra, Marco A.; Asano, Jennifer K.; Chan, Susanna Y.; Guin, Ranabir; Krzywinski, Martin I.; Lee, Soo Sen; MacDonald, Kim W. K.; Mathewson, Carrie A.; Olson, Teika E.; Pandoh, Pawan K.; Prabhu, Anna-Liisa; Schnerch, Angelique; Skalska, Ursula; Smailus, Duane E.; Stott, Jeff M.; Tsai, Miranda I.; Yang, George S.; Zuyderduyn, Scott D.; Schein, Jacqueline E.; Jones, Steven J. M.

    2002-01-01

    We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites. PMID:12034834

  15. Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; Lin Yang; Jian-Tao Cui; Wen-Mei Li; Rui-Fang Guo; You-Yong Lu

    2002-01-01

    AIM: To elucidate molecular mechanism of chemopreventiveefficacies of garlic against human gastric cancer (HGC):METHODS: HGC cell line BGC823 was treated with Allitridi (akind of garlic extract) and Allitridi-treated and parentalBGC823 cDNA librarles were constructed respectively byusing λZAP Ⅱ vector. cDNA Representatinal DifferenceAnalysis (cDNA RDA) was perfonmed using BamH Ⅰ cutting-site and abundant ~DNA messages provided by the Iibrarles.Northern blot analysls was applied to identifythe obtaineddifference prnducts.RESULTS: Two specific cDNA fragments were obtained andcharacterized to be derived from homo sapiens folatereceptorα (FRα) gene and calcyclin gene respectively.Northern blot results showed a 4-fold increase in FRα geneexpression level and 9-fold increase in calcyclin mRNA levelin BGC823 cells after Allilridi treatment for 72 h.CONCLUSION: The method of cDNA RDA based on cDNAlibraries combines the high specificity of cDNA RDA withabundant cDNA messages in cDNA library; this expands theapplication of cDNA library and increases the specificity ofcDNA RDA. Up-regulstion of FRα gene and calcyclin geneexpressions induced by Allitridi provide valuable molecularevidence for theefficacy of garlic in treating HGC as well asother diseases.

  16. ISOLATION OF HUMAN TRANSCRIPTS EXPRESSED IN 16HBE CELLS RELATED TO CHLOROPHYLLIN ANTITRANSFORMING ACTIVITY AGAINST ANTI-BPDE BY cDNA REPRESENTATIONAL DIFFERENCE ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    ZHU Li-jin; JIANG Yi-guo

    2005-01-01

    Objective: To analyze the differentially expressed cDNA sequences related to chlorophyllin (CHL) mediated inhibition of malignant transformation of human bronchia1 epithelial cell line (16HBE). Methods: 16HBE cells treated with chlorophyllin and anti-BPDE were conducted as tester, 16HBE cells treated only with anti-BPDE were conducted as driver, and cDNA representational difference analysis (cDNA RDA) was used to compare the differential gene expression between the two kinds of cells. The cDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA was sequenced and compared with database in GenBank by BLASTN. Results: Among the 5 cloned cDNA sequences, three were novel and were registered in dbEST database, two showed sequence homology to alpha-enolase and a newly found gene ribosomal protein S18/S6-like. Conclusion: These 5 cDNA sequences might play important roles in antitransforming effect of chlorophyllin.

  17. Interferon-gamma up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence-containing protein IGUP I-5111

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1993-01-01

    Treatment of proliferating and quiescent primary human keratinocytes with interferon-gamma (IFN-gamma) (100 U/ml, 23.5 h) followed by two-dimensional gel analysis revealed three proteins, IGUP I-3421 (M(r) = 48,200, pI = 6.06); IGUP I-3524 (M(r) = 56,900, pI = 5.92), a protein homologous to peptide......, which migrated with the AMA variant of keratinocyte protein IEF SSP 5111, is novel although it exhibits weak similarity to cytoskeletal proteins. IGUP I-5111 contains the RGD sequence found in many extracellular glycoprotein ligands of the integrin receptor family and it is found at least partially......-cultured, unfractionated psoriatic keratinocytes failed to reveal up-regulation of any of the three IFN-gamma-induced proteins suggesting that the effect of IFN-gamma in vivo may be modulated by the activity of other cytokine(s) or growth factor(s). Psoriatic keratinocytes were equally sensitive to IFN...

  18. 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

  19. Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing.

    Science.gov (United States)

    Shibata, Y; Carninci, P; Sato, K; Hayatsu, N; Shiraki, T; Ishii, Y; Arakawa, T; Hara, A; Ohsato, N; Izawa, M; Aizawa, K; Itoh, M; Shibata, K; Shinagawa, A; Kawai, J; Ota, Y; Kikuchi, S; Kishimoto, N; Muramatsu, M; Hayashizaki, Y

    2001-11-01

    We have developed a method to overcome sequencing problems caused by the presence of homopolymer stretches, such as polyA/T, in cDNA libraries. PolyA tails are shortened by cleaving before cDNA cloning with type IIS restriction enzymes, such as GsuI, placed next to the oligo-dT used to prime the polyA tails of mRNAs. We constructed four rice Cap-Trapper-selected, full-length normalized cDNA libraries, of which the average residual polyA tail was 4 bases or shorter in most of the clones analyzed Because of the removal of homopolymeric stretches, libraries prepared with this method can be used for direct sequencing and transcriptional sequencing without the slippage observed for libraries prepared with currently available methods, thus improving sequencing accuracy, operations, and throughput.

  20. Cloning, tissue expression pattern characterization and chromosome localization of human peptide methionine sulfoxide reductase cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Oxidation and reduction of some amino acids are one of the molecular mechanisms for regulating the function of proteins. The oxidation of methionine (Met) to methionine sulfoxide (Met(O)) results in decreasing or loss of the biological activity of related proteins. It was found that peptide methionine sulfoxide reductase (msrA) can reduce Met(O) to Met and therefore restored the biological function of the oxidized proteins. To reveal the methionine oxidation-reduction mechanism in human body, in this study, the cDNA sequence of bovine msrA was used as an information-probe to screen the human EST database. Based on a contig assembled from homologous ESTs, a 1 256-bp human MSRA cDNA was cloned from several human cDNA libraries. The cDNA contains an open reading frame (ORF) of 705 bp in length, which encodes 235 amino acid residues. Homology comparison revealed that human MSRA shares 88% and 61% identities with bovine and Escherichia coli msrA protein respectively. Expression pattern analysis revealed a single 1.6-kb transcript of human MSRA in most human tissues and with highest expression in kidney. By radiation hybrid panel mapping, the gene was localized to human chromosome 8p22-23 between markers D8S518 and D8S550. There are 2 human inherited diseases Keratolytic Winter Erythema and Microcephaly related genes in this region, it is inferred that human MSRA might be the candidate of the two diseases.

  1. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    OpenAIRE

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.

  2. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    2001-01-01

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus whi

  3. Molecular Cloning and Sequence Analysis of Full-Length cDNA Encoding Human Bone Morphogenetic Protein-7%人骨形态发生蛋白-7全长基因cDNA的克隆和序列分析

    Institute of Scientific and Technical Information of China (English)

    李新友; 刘淼; 李曙明; 姚煜; 王全颖; 杨广笑

    2003-01-01

    Objective: To clone the full-length human bone morphogenetic protein-7 ( BMP-7) gene and analyse its sequence, to aid in investigation of its function and structure. Methods: TotalRNA was isolated from Chinese fetal kidney by the acid guanidinium thiocyanate phenol-chloroformmethod. Two overlapping segments of human BMP-7 cDNA were obtained by reverse transcription(RT)-PCR. Fallowing application, the two segments were ligated to each other and subcloned intoPGEM-7 easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid. Sanger dideoxy chain-ter-mination method was used to sequence the cDNA. Results: There was 750 bp fragment obtained RT-PCR using # 2 primer from 5' end of BMP-7 gene (PCR by using # 2 and # 1) ,and 540 bp frag-merit from 3' end was generated by RT-PCR using # 4 primer (PCR using # 3 and # 4). Full-lengthcDNA encoding BMP-7 was obtained by religation of two segments. When compared with hBMP-7 se-quence in Gene bank (XM30619) ,our full-length BMP- 7 cDNA has a G instead of a T at nucleotide862. This change results in valine substituting for phenylalanine in the protein. Conclusion: This isthe first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney. BMP-7 cDNAplays an important role in healing injuries of the osteo-articular system. This makes BMP-7 is an at-tractive target for various clinical applications.%目的:克隆人骨形态发生蛋白-7(BMP-7)全长基因,并进行测序及序列分析,研究其结构和功能.方法:采用异硫氰酸胍一步法从人胎儿肾中提取总RNA,利用逆转录-聚合酶链反应(RT-PCR)的方法,分段扩增出hBMP-7全长基因cDNA,与PGEM-T easy连接成PGEM-T easy/hBMP-7重组质粒,采用Sanger双脱氧链终止法测定基因序列.结果:以4号特异引物引导的RT-PCR扩增出BMP-7基因3'端540 bp片段,以2号特异引物引导的RT-PCR扩增出BMP-7基因5'端750 bp片段,重组连接两片段得到BMP-7全长基因cDNA.与Genebank上发表的hBMP-7序列(XM30619)

  4. Random rapid amplification of cDNA ends (RRACE) allows for cloning of multiple novel human cDNA fragments containing (CAG)n repeats.

    Science.gov (United States)

    Carney, J P; McKnight, C; VanEpps, S; Kelley, M R

    1995-04-03

    We describe a new technique for isolating cDNA fragments in which (i) either a partial sequence of the cDNA is known or (ii) a repeat sequence is utilized. We have used this technique, termed random rapid amplification of cDNA ends (random RACE), to isolate a number of trinucleotide repeat (CAG)n-containing genes. Using the random RACE (RRACE) technique, we have isolated over a hundred (CAG)n-containing genes. The results of our initial analysis of ten clones indicate that three are identical to previously cloned (CAG)n-containing genes. Three of our clones matched with expressed sequence tags, one of which contained a CA repeat. The remaining four clones did not match with any sequence in GenBank. These results indicate that this approach provides a rapid and efficient method for isolating trinucleotide repeat-containing cDNA fragments. Finally, this technique may be used for purposes other than cloning repeat-containing cDNA fragments. If only a partial sequence of a gene is known, our system, described here, provides a rapid and efficient method for isolating a fragment of the gene of interest.

  5. Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project

    Directory of Open Access Journals (Sweden)

    Heinz Ruth A

    2003-09-01

    Full Text Available Abstract Background Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. Results Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4. The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60% did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences

  6. Full-Length Venom Protein cDNA Sequences from Venom-Derived mRNA: Exploring Compositional Variation and Adaptive Multigene Evolution.

    Science.gov (United States)

    Modahl, Cassandra M; Mackessy, Stephen P

    2016-06-01

    Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan) by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus), and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae) have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only venom, provides

  7. Full-Length Venom Protein cDNA Sequences from Venom-Derived mRNA: Exploring Compositional Variation and Adaptive Multigene Evolution.

    Directory of Open Access Journals (Sweden)

    Cassandra M Modahl

    2016-06-01

    Full Text Available Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus, and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only

  8. Full-Length Venom Protein cDNA Sequences from Venom-Derived mRNA: Exploring Compositional Variation and Adaptive Multigene Evolution

    Science.gov (United States)

    Modahl, Cassandra M.; Mackessy, Stephen P.

    2016-01-01

    Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan) by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus), and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae) have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only venom, provides

  9. 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.

  10. 人脑红蛋白(NGB)全长cDNA序列的克隆%Full-length cDNA Cloning of Human Neuroglobin by Using Electronic Sequence Elongation Technique and RACE Technique

    Institute of Scientific and Technical Information of China (English)

    王春丽; 张成岗

    2002-01-01

    人脑红蛋白(neuroglobin,NGB)是新发现的神经系统特异的携氧蛋白,然而其全长cDNA序列一直未见报道.采用电子序列延伸技术和cDNA序列末端快速扩增技术(rapid amplification of cDNA ends,RACE)研究发现,人NGB全长cDNA序列为1 09 bp,5′非编码区为375 bp,编码区(456 bp)可编码151个氨基酸,3′非编码区为1 078 bp,其中含27bp的poly(A)(GenBank接受号:AF422797).综合采用电子序列延伸技术与RACE技术是获得全长cDNA序列的有效方法,为后续的功能研究提供了重要基础.

  11. Uroporphyrinogen-III synthase: Molecular cloning, nucleotide sequence, expression of a mouse full-length cDNA, and its localization on mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Xu, W.; Desnick, R.J. [Mount Sinai School of Medicine, New York, NY (United States); Kozak, C.A. [National Institute of Health, Bethesda, MD (United States)

    1995-04-10

    Uroporphyrinogen-III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for the conversion of hydroxymethylbilane to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-S is the enzymatic defect in congenital erythropoietic porphyria (CEP), an autosomal recessive disorder. For the generation of a mouse model of CEP, the human URO-S cDNA was used to screen 2 X 10{sup 6} recombinants from a mouse adult liver cDNA library. Ten positive clones were isolated, and dideoxy sequencing of the entire 1.6-kb insert of clone pmUROS-1 revealed 5{prime} and 3{prime} untranslated sequences of 144 and 623 bp, respectively, and an open reading frame of 798 bp encoding a 265-amino-acid polypeptide with a predicted molecular mass of 28,501 Da. The mouse and human coding sequences had 80.5 and 77.8% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active monomeric enzyme in Escherichia coli. In addition, the analysis of two multilocus genetic crosses localized the mouse gene on chromosome 7, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 10. The isolation, expression, and chromosomal mapping of this full-length cDNA should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of CEP for characterization of the disease pathogenesis and evaluation of gene therapy. 38 refs., 1 tab.

  12. 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

  13. 16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Weller, R.; Ward, D.M. (Montana State Univ., Bozeman (United States)); Weller, J.W. (Univ. of Montana, Missoula (United States))

    1991-04-01

    Cloning and analysis of cDNAs synthesized from rRNAs is one approach to assess the species composition of natural microbial communities. In some earlier attempts to synthesize cDNA from 16S rRNA (16S rcDNA) from the Octopus Spring cyanobacterial mat, a dominance of short 16S rcDNAs was observed, which appear to have originated only from certain organisms. Priming of cDNA synthesis from small ribosomal subunit RNA with random deoxyhexanucleotides can retrieve longer sequences, more suitable for phylogenetic analysis. Here we report the retrieval of 16S rRNA sequences form three formerly uncultured community members. One sequence type, which was retrieved three times from a total of five sequences analyzed, can be placed in the cyanobacterial phylum. A second sequence type is related to 16S rRNAs from green nonsulfur bacteria. The third sequence type may represent a novel phylogenetic type.

  14. Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein.

    Science.gov (United States)

    Santhanam, R; Naz, R K

    2001-09-01

    A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-(lambda)gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility.

  15. Cloning and Expression Analysis of Downy Mildew Resistance-Related cDNA Sequences in Melon

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Melon downy mildew caused by Pseudoperonospora cubensis leads to significant losses in melon yields worldwide.Reverse-transcription Polymerase Chain Reaction (RT-PCR) was performed using cDNAs as templates from melonHuangdanzi induced with fungus Pseudoperonospora cubensis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. A polymorphic cDNA fragment which we named mp-19was cloned and sequenced. The Open Reading Frame (ORF) of this product comprised of 510 base pairs which encodes DNA or RNA-binding protein with 170 amino acids. The putative amino acid sequence of mp-19 appeared highly homologous with those of NBS-type resistant-genes isolated from other plants. Southern blot indicated that the melon genome contained more than 3 copies of mp-19. The obvious expression differences detected by semi-quantitative RTPCR could be observed between resistant-line Huangdanzi and susceptible-line Jiashi after Pseudoperonospora cubensis infection, which implied that mp-19 gene may be related to the resistance of downy mildew in melon.

  16. Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

    Science.gov (United States)

    Legname, G; Bellosta, P; Gromo, G; Modena, D; Keen, J N; Roberts, L M; Lord, J M

    1991-08-27

    Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.

  17. Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells.

    OpenAIRE

    1991-01-01

    The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-t...

  18. Identification and characterization of cDNA sequences encoding the HIS3 and LEU2 genes of the fungus Alternaria tenuissima

    Institute of Scientific and Technical Information of China (English)

    Ying Wan; Xuli Wang; Yun Huang; Dewen Qiu; Linghuo Jiang

    2008-01-01

    Alternaria tenuissima is a fungus widely present in the environment and could cause diseases in plants and humans.In this study,through a yeast genetic approach,cDNA sequences were isolated and characterized for the AtHIS3 and AtLEU2 genes.AtHIS3 cDNA encodes a protein of 238 amino acids,while AtLEU2 cDNA encodes a protein of 363 amino acids.Based on the phylogenetic analysis of amino acid sequences of AtHis3p and AtLeu2p,A.tenuissima is closely related to the plant pathogenic fungus Phaeosphaeria nodorum.This study provides two genetic markers for studies of functions of genes regulating development,morphology,and virulence of A.tenuissima.

  19. Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee.

    Science.gov (United States)

    Whitfield, Charles W; Band, Mark R; Bonaldo, Maria F; Kumar, Charu G; Liu, Lei; Pardinas, Jose R; Robertson, Hugh M; Soares, M Bento; Robinson, Gene E

    2002-04-01

    To accelerate the molecular analysis of behavior in the honey bee (Apis mellifera), we created expressed sequence tag (EST) and cDNA microarray resources for the bee brain. Over 20,000 cDNA clones were partially sequenced from a normalized (and subsequently subtracted) library generated from adult A. mellifera brains. These sequences were processed to identify 15,311 high-quality ESTs representing 8912 putative transcripts. Putative transcripts were functionally annotated (using the Gene Ontology classification system) based on matching gene sequences in Drosophila melanogaster. The brain ESTs represent a broad range of molecular functions and biological processes, with neurobiological classifications particularly well represented. Roughly half of Drosophila genes currently implicated in synaptic transmission and/or behavior are represented in the Apis EST set. Of Apis sequences with open reading frames of at least 450 bp, 24% are highly diverged with no matches to known protein sequences. Additionally, over 100 Apis transcript sequences conserved with other organisms appear to have been lost from the Drosophila genome. DNA microarrays were fabricated with over 7000 EST cDNA clones putatively representing different transcripts. Using probe derived from single bee brain mRNA, microarrays detected gene expression for 90% of Apis cDNAs two standard deviations greater than exogenous control cDNAs. [The sequence data described in this paper have been submitted to Genbank data library under accession nos. BI502708-BI517278. The sequences are also available at http://titan.biotec.uiuc.edu/bee/honeybee_project.htm.

  20. Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

    DEFF Research Database (Denmark)

    Lundwall, Åke B; Wetsel, Rick A; Kristensen, Torsten;

    1985-01-01

    DNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence......, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha......'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human...

  1. Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiao-hong; CHEN Zhi; YAO Hang-ping; CHEN Feng; ZHU Hai-hong; ZHOU Hong-juan

    2005-01-01

    Objective: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. Methods: The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript(SMART) technique and CDS Ⅲ/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction(LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to λTriplEx2 vector. Then λ phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. Results: The titers of unamplifed and amplified libraries were 1.94×106 pfu/ml and1.49×109 pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%.Conclusion: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

  2. cDNA sequence and protein bioinformatics analyses of MSTN in African catfish (Clarias gariepinus).

    Science.gov (United States)

    Kanjanaworakul, Poonmanee; Sawatdichaikul, Orathai; Poompuang, Supawadee

    2016-04-01

    Myostatin, also known as growth differentiation factor 8, has been identified as a potent negative regulator of skeletal muscle growth. The purpose of this study was to characterize and predict function of the myostatin gene of the African catfish (Cg-MSTN). Expression of Cg-MSTN was determined at three growth stages to establish the relationship between the levels of MSTN transcript and skeletal muscle growth. The partial cDNA sequence of Cg-MSTN was cloned by using published information from its congener walking catfish (Cm-MSTN). The Cg-MSTN was 1194 bp in length encoding a protein of 397 amino acids. The deduced MSTN sequence exhibited key functional sites similar to those of other members of the TGF-β superfamily, especially, the proteolytic processing site (RXXR motif) and nine conserved cysteines at the C-terminal. Expression of MSTN appeared to be correlated with muscle development and growth of African catfish. Protein bioinformatics revealed that the primary sequence of Cg-MSTN shared 98 % sequence identity with that of walking catfish Cm-MSTN with only two different residues, [Formula: see text]. and [Formula: see text]. The proposed model of Cg-MSTN revealed the key point mutation [Formula: see text] causing a 7.35 Å shorter distance between the N- and C-lobes and an approximately 11° narrow angle than those of Cm-MSTN. The substitution of a proline residue near the proteolytic processing site which altered the structure of myostatin may play a critical role in reducing proteolytic activity of this protein in African catfish.

  3. Assignment of casein kinase 2 alpha sequences to two different human chromosomes

    DEFF Research Database (Denmark)

    Boldyreff, B; Klett, C; Göttert, E

    1992-01-01

    Human casein kinase 2 alpha gene (CK-2-alpha) sequences have been localized within the human genome by in situ hybridization and somatic cell hybrid analysis using a CK-2 alpha cDNA as a probe. By in situ hybridization, the CK-2 alpha cDNA could be assigned to two different loci, one on 11p15.1-ter...

  4. Cloning and characterization of a cDNA encoding human differentiation antigen 5D4

    Institute of Scientific and Technical Information of China (English)

    马凤蓉; 朱立平; 汪燚; 赵方萄; 史耕先; 李波; 李国燕; 张淑珍; 王讯

    2000-01-01

    A 1 846 bp cDNA is isolated from a human tonsil cell λgt 11 cDNA library (ATCC No. 37546) with mAb 5D4 reactive strongly with human B cell line 3D5, but weakly with human B cell line Daudi and human T cell line Jurkat as a probe. RT-PCR also shows a strong reaction in 3D5 cell and a weak reaction in Daudi and Jurkat cell for 5D4 mRNA. There is an open reading frame from 88 to 1 209 bp in 5D4 cDNA encoding a 374 AA protein. Both the Northern blot analysis and the two consecutive stop codens before start coden demonstrate that the cDNA is a full-length cDNA. Secondary structure prediction suggests that there are a region from 295 to 334 AA in the protein with strong hydrophobicity and a transmembrane helix region with high score from 313 to 334 AA with an orientation from the inside to the outside of the cell.

  5. Update History of This Database - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us ...Budding yeast cDNA sequencing project Update History of This Database Date Update contents 2010/03/29 Buddin...tio About This Database Database Description Download License Update History of This Database Site Policy | Contact Us Update History

  6. Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

    Science.gov (United States)

    Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and seque...

  7. Molecular cloning of a cDNA encoding human calumenin, expression in Escherichia coli and analysis of its Ca2+-binding activity

    DEFF Research Database (Denmark)

    Vorum, H; Liu, X; Madsen, Peder;

    1998-01-01

    By microsequencing and cDNA cloning we have identified the transformation-sensitive protein No. IEF SSP 9302 as the human homologue of calumenin. The nucleotide sequence predicts a 315 amino acid protein with high identity to murine and rat calumenin. The deduced protein contains a 19 amino acid ...

  8. 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

  9. Cloning of human brevican cDNA and expression of its mRNA in human glioma

    Institute of Scientific and Technical Information of China (English)

    韩唏; 董艳; 由振东; 何成; 卢亦成

    2003-01-01

    Objective:To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma.Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human ahaplastic astrocytoma by RT-PCR,and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization.Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained.In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas(100%),whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined.Conclusion:The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

  10. Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

    Science.gov (United States)

    Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

    1997-01-01

    A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

  11. Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

    Science.gov (United States)

    Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

    1997-01-01

    A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

  12. Cloning and expression of a novel human HCUTA cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Copper is one of the most important trace elements to life. Human HCUTA is a novel cDNA encoding a 156aa protein, which may participate in human copper tolerance system. The HCUTA protein is highly similar to protein CUT A1 of E. coli. The whole opening reading frame of HCUTA cDNA was amplified by PCR and cloned into pET28a + express vector, and the HCUTA protein was effectively expressed in E. coli BL21 (DE3).

  13. cDNA, genomic sequence and overexpression of crystallin alpha-B Gene (CRYAB) of the Giant Panda

    Science.gov (United States)

    Hou, Yi-ling; Hou, Wan-ru; Ren, Zheng-long; Hao, Yan-zhe; Zhang, Tian

    2008-01-01

    αB-crystallin, a small heat-shock protein, has been shown to prevent the aggregation of other proteins under various stress conditions. Here we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%, 91.5%, 91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%, 97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinase II phosphorylation sites in the CRYAB protein of the Giant Panda. The cDNA of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein. The expression product obtained could be used for purification and study of its function further. PMID:19043608

  14. Studies of the hyperthermophile Thermotoga maritima by random sequencing of cDNA and genomic libraries. Identification and sequencing of the trpEG (D) operon.

    Science.gov (United States)

    Kim, C W; Markiewicz, P; Lee, J J; Schierle, C F; Miller, J H

    1993-06-20

    Random sequencing of cDNA and genomic libraries has been used to study the genome of the hyperthermophile Thermotoga maritima. To date, 175 unique clones have been analyzed by comparing short sequence tags with known proteins in the PIR and GenBank databases. We find that a significant proportion of sequences can be matched to previously identified protein from non-Thermotoga sources. A high match rate was obtained from an oligo(dT)-primed cDNA library, where one-third of all unique sequences analyzed (21/65) shared high amino acid sequence similarity with proteins in the PIR and GenBank databases. Also, approximately one-third of the unique sequences from a second cDNA library (28/89), constructed with random oligo primers, could be matched to sequences in PIR and GenBank. Identification of genes from the oligo(dT)-primed cDNA library indicates that some Thermotoga mRNAs are polyadenylated. Genes have also been identified from a 1 to 2 kb genomic DNA library. Here, (3/21) of genomic sequences analyzed could be matched to protein in PIR and GenBank. One of the genomic clones had high sequence similarity to the tryptophan synthesis gene anthranilate synthase component I (trpE). Using this sequence tag, the Thermotoga trp operon was isolated and sequenced. The Thermotoga maritima trp operon is arranged with trpE forming an overlapping transcript with a second protein consisting of a fusion of anthranilate synthase component II (trpG) and anthranilate phosphoribosyltransferse (trpD). With regard to the fusion, the operon organization is similar to Escherichia coli and Salmonella typhimurium, but lacks the classic attenuation system of enteric bacteria. Amino acid sequence comparison with 19 trpE, 18 trpG and 14 trpD genes from other organisms suggest that the Thermotoga trp genes resemble corresponding genes from other thermophiles more closely than expected.

  15. Regulation of human clotting factor IX cDNA expression in transgenic mice

    Institute of Scientific and Technical Information of China (English)

    胡以平; 邱信芳; 薛京伦; 刘祖洞

    1995-01-01

    To study the expression of human dotting factor IX cDNA in transgenic mice,Which is an es-sential work on gene therapy for hemophilia B,3 recombinant constructions containing different lengths ofhuman dotting factor IX cDNA have been introduced into the cultured cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These results show that the expression of the human clotting factor IX cDNA in the transgenic mice canbe determined by cis regulatory element(s).As compared With the results from other related works,it is sug-gested that the cis regulatory element(s)is resided in the 5’-end non-coding region.

  16. An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library

    Directory of Open Access Journals (Sweden)

    Wallis James G

    2007-07-01

    Full Text Available Abstract Background Castor seeds are a major source for ricinoleate, an important industrial raw material. Genomics studies of castor plant will provide critical information for understanding seed metabolism, for effectively engineering ricinoleate production in transgenic oilseeds, or for genetically improving castor plants by eliminating toxic and allergic proteins in seeds. Results Full-length cDNAs are useful resources in annotating genes and in providing functional analysis of genes and their products. We constructed a full-length cDNA library from developing castor endosperm, and obtained 4,720 ESTs from 5'-ends of the cDNA clones representing 1,908 unique sequences. The most abundant transcripts are genes encoding storage proteins, ricin, agglutinin and oleosins. Several other sequences are also very numerous, including two acidic triacylglycerol lipases, and the oleate hydroxylase (FAH12 gene that is responsible for ricinoleate biosynthesis. The role(s of the lipases in developing castor seeds are not clear, and co-expressing of a lipase and the FAH12 did not result in significant changes in hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Only one oleate desaturase (FAD2 gene was identified in our cDNA sequences. Sequence and functional analyses of the castor FAD2 were carried out since it had not been characterized previously. Overexpression of castor FAD2 in a FAH12-expressing Arabidopsis line resulted in decreased accumulation of hydroxy fatty acids in transgenic seeds. Conclusion Our results suggest that transcriptional regulation of FAD2 and FAH12 genes maybe one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm. The full-length cDNA library will be used to search for additional genes that affect ricinoleate accumulation in seed oils. Our EST sequences will also be useful to annotate the castor genome, which whole sequence is being generated by shotgun sequencing at

  17. Large-scale Identification of Expressed Sequence Tags (ESTs from Nicotianatabacum by Normalized cDNA Library Sequencing

    Directory of Open Access Journals (Sweden)

    Alvarez S Perez

    2014-12-01

    Full Text Available An expressed sequence tags (EST resource for tobacco plants (Nicotianatabacum was established using high-throughput sequencing of randomly selected clones from one cDNA library representing a range of plant organs (leaf, stem, root and root base. Over 5000 ESTs were generated from the 3’ ends of 8000 clones, analyzed by BLAST searches and categorized functionally. All annotated ESTs were classified into 18 functional categories, unique transcripts involved in energy were the largest group accounting for 831 (32.32% of the annotated ESTs. After excluding 2450 non-significant tentative unique transcripts (TUTs, 100 unique sequences (1.67% of total TUTs were identified from the N. tabacum database. In the array result two genes strongly related to the tobacco mosaic virus (TMV were obtained, one basic form of pathogenesis-related protein 1 precursor (TBT012G08 and ubiquitin (TBT087G01. Both of them were found in the variety Hongda, some other important genes were classified into two groups, one of these implicated in plant development like those genes related to a photosynthetic process (chlorophyll a-b binding protein, photosystem I, ferredoxin I and III, ATP synthase and a further group including genes related to plant stress response (ubiquitin, ubiquitin-like protein SMT3, glycine-rich RNA binding protein, histones and methallothionein. The interesting finding in this study is that two of these genes have never been reported before in N. tabacum (ubiquitin-like protein SMT3 and methallothionein. The array results were confirmed using quantitative PCR.

  18. Next-generation sequencing-based 5' rapid amplification of cDNA ends for alternative promoters.

    Science.gov (United States)

    Perera, Bambarendage P U; Kim, Joomyeong

    2016-02-01

    Mammalian genomes contain many unknown alternative first exons and promoters. Thus, we have modified the existing 5'RACE (5' rapid amplification of cDNA ends) approach into a next-generation sequencing (NGS)-based new protocol that can identify these alternative promoters. This protocol has incorporated two main ideas: (i) 5'RACE starting from the known second exons of genes and (ii) NGS-based sequencing of the subsequent cDNA products. This protocol also provides a bioinformatics strategy that processes the sequence reads from NGS runs. This protocol has successfully identified several alternative promoters for an imprinted gene, PEG3. Overall, this NGS-based 5'RACE protocol is a sensitive and reliable method for detecting low-abundant transcripts and promoters.

  19. Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo).

    Science.gov (United States)

    Słowińska, Mariola; Olczak, Mariusz; Wojtczak, Mariola; Glogowski, Jan; Jankowski, Jan; Watorek, Wiesław; Amarowicz, Ryszard; Ciereszko, Andrzej

    2008-06-01

    The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.

  20. Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen

    Energy Technology Data Exchange (ETDEWEB)

    Federsppiel, B.; Melhado, I.G.; Delaney, A.; Clark-Lewis, I. (Univ. of British Columbia, Vancouver (Canada)); Duncan, A.M.V. (Queens Univ., Kinston, Ontario (Canada)); Jirik, F.R. (Hospital for Sick Children, Toronto, Ontario (Canada))

    1993-06-01

    A family of proinflammatory cytokines sharing several structural features has been described and includes, for example, interleukin-8, monocyte chemoattractant protein-1, and melanocyte growth stimulatory activity. Recently, the receptors for interleukin-8 have been isolated and found to belong to the seven-transmembrane domain class of G protein-coupled receptors. As other members of this cytokine family likely interact with similar receptors, the polymerase chain reaction was employed to isolate related receptors from human peripheral blood adherent cells. Degenerate oligonucleotide primers based on the rabbit interleukin-8 receptor sequence were used. The corresponding full-length cDNA was isolated from a human spleen cDNA library. The predicted protein sequence of this clone, designated pBE1.3, was 93% identical to that of a cDNA isolated from bovine locus coeruleus, which apparently encodes a neuropeptide Y receptor, and also shows similarity with the interleukin-8 receptor and the human cytomegalovirus US28 sequences. The gene, designated D2S201E, was localized to human chromosome 2q21. By Northern blotting, transcripts hybridizing to this cDNA were present in a variety of tissues and cells, including those of hemopoietic origin. 32 refs., 5 figs.

  1. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    Tetranectin is a plasminogen-binding tetrameric protein originally isolated from plasma. Expression of tetranectin appears ubiquitous, although particularly high expression is noted in the stroma of malignant tumors and during mineralization. To dissect the molecular basis of tetranectin gene......% identity and 87% similarity at the amino acid level. Sequence comparisons between mouse and human tetranectin and some C-type lectins confirmed a complete conservation in the position of six cysteines as well as numerous other amino acid residues, indicating an essential structure for potential function......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed...

  2. Glutamate-gated chloride channel subunit cDNA sequencing of Cochliomyia hominivorax (Diptera: Calliphoridae): cDNA variants and polymorphisms.

    Science.gov (United States)

    Lopes, Alberto Moura Mendes; de Carvalho, Renato Assis; de Azeredo-Espin, Ana Maria Lima

    2014-09-01

    The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel) is one of the major myiasis-causing flies that injures livestock and leads to losses of ~US$ 2.7 billions/year in the Neotropics. Ivermectin (IVM), a macrocyclic lactone (ML), is the most used preventive insecticide for this parasite and targets the glutamate-gated chloride (GLUCLα) channels. Several authors have associated altered GluClα homologues to MLs resistance in invertebrates, although studies about resistance in NWS are limited to other genes. Here, we aimed to characterise the NWS GluClα (ChGluClα) cDNA and to search for alterations associated with IVM resistance in NWS larvae from a bioassay. The open reading frame of the ChGluClα comprised 1,359 bp and encoded a sequence of 452 amino acids. The ChGluClα cDNAs of the bioassay larvae showed different sequences that could be splice variants, which agree with the occurrence of alternative splicing in GluClα homologues. In addition, we found cDNAs with premature stop codons and the K242R SNP, which occurred more frequently in the surviving larvae and was located close to mutation (L256F) involved in ML resistance. Although these alterations were in low frequency, the ChGluClα sequencing will allow further studies to find alterations in the gene of resistant natural populations.

  3. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    Science.gov (United States)

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSESB.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt11Department of Reproductiv...

  4. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    Science.gov (United States)

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSESB.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt11Department of Reproductiv...

  5. Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

    DEFF Research Database (Denmark)

    de Souza, S J; Camargo, A A; Briones, M R;

    2000-01-01

    by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48......Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central...... coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes...

  6. Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus.

    Science.gov (United States)

    Olsen, B S; Johansen, I E

    2001-01-01

    The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.

  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. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  9. Generation and Analysis of Expressed Sequence Tags (ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

    Institute of Scientific and Technical Information of China (English)

    ZHAO Su-mei; LIU Yong-gang; PAN Hong-bing; ZHANG Xi; GE Chang-rong; JIA Jun-jing; GAO Shi-zheng

    2014-01-01

    Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identiifed in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1076 bp, and the cDNA fullness ratio was 86.2%. A total of 1058 unique sequences with 342 contigs (32.3%) and 716 singleton (67.7%) expressed sequence tags (EST) were obtained by clustering and assembling. Meanwhile, 826 (78.1%) ESTs were categorized as known genes, and 232 (21.9%) ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index ofSus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank (accession numbers: EU650784-EU650788, GE843306, GH228978-GH229100). The abundantly expressed genes in porcine muscle tissue were related to muscle ifber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.

  10. cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene

    Institute of Scientific and Technical Information of China (English)

    QI Zhen-yu; HUI Guo-zhen; LI Yao; ZHOU Zong-xiang; GU Shao-hua; YING Kang; XIE Yi

    2005-01-01

    Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5'RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b).Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.

  11. In-depth cDNA Library Sequencing Provides Quantitative Gene Expression Profiling in Cancer Biomarker Discovery

    Institute of Scientific and Technical Information of China (English)

    Wanling Yang; Dingge Ying; Yu-Lung Lau

    2009-01-01

    procedures may allow detection of many expres-sion features for less abundant gene variants. With the reduction of sequencing cost and the emerging of new generation sequencing technology, in-depth sequencing of cDNA pools or libraries may represent a better and powerful tool in gene expression profiling and cancer biomarker detection. We also propose using sequence-specific subtraction to remove hundreds of the most abundant housekeeping genes to in-crease sequencing depth without affecting relative expression ratio of other genes, as transcripts from as few as 300 most abundantly expressed genes constitute about 20% of the total transcriptome. In-depth sequencing also represents a unique ad-vantage of detecting unknown forms of transcripts, such as alternative splicing variants, fusion genes, and regulatory RNAs, as well as detecting mutations and polymorphisms that may play important roles in disease pathogenesis.

  12. Complete Genome Sequence of Human Respiratory Syncytial Virus from Lanzhou, China

    OpenAIRE

    Zhu, Chuanfeng; Fu, Shengfang; Zhou, Xv; Yu, Li

    2017-01-01

    ABSTRACT A complete genome of human respiratory syncytial virus was sequenced and analyzed. Phylogenetic analysis showed that the full-length human respiratory syncytial virus (HRSV) genome sequence belongs to gene type NA1. We sequenced the genome in order to create the full-length cDNA infectious clone and develop vaccines against HRSV.

  13. Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

    Science.gov (United States)

    Sawada, R; Ohashi, K; Anaguchi, H; Okazaki, H; Hattori, M; Minato, N; Naruto, M

    1990-04-01

    To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

  14. Human Vascular Endothelial Growth Factor cDNA Cloning and Expression in Osteoblasts

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Human vascular endothelial growth factor (VEGF) cDNA was amplified by nested polymerase chain reaction method from the HL60 cells. Then a pCD-hVEGF165 recombinant plasmid was constructed. Rabbit osteoblasts were transfected with pCD-hVEGF165 plasmid by lipofectin mediated gene transfer. The transient expressive results were detected by immunohistochemical method. It was observed that the expression of human VEGF gene was detected 72 h after transfecting distinctly.

  15. cDNA sequence, gene structure, and in vitro expression of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Arpagaus, M; Fedon, Y; Cousin, X; Chatonnet, A; Bergé, J B; Fournier, D; Toutant, J P

    1994-04-01

    Three genes, ace-1, ace-2, and ace-3, encode three acetylcholinesterase classes (A, B, and C) in the nematode Caenorhabditis elegans. A fragment of genomic DNA was amplified by a polymerase chain reaction (PCR) using degenerate oligonucleotides based on sequences conserved in the cholinesterase family. This fragment mapped to chromosome X at a position that perfectly matched the location of ace-1 previously determined by genetic methods. Comparison of genomic and cDNA sequences showed that the open reading frame was interrupted by eight introns. The product of ace-1 (ACE-1, 620 amino acids) presented 42% identity with Torpedo and human acetylcholinesterases, 41% with human butyrylcholinesterase, and 35% with Drosophila acetylcholinesterase. The overall structure of cholinesterases was conserved in ACE-1 as indicated by the conserved sequence positions of Ser-216, His-468, and Glu-346 (S200, H440, E327 in Torpedo (AChE) as components of the catalytic triad, of the six cysteines which form three intrachain disulfide bonds, and of Trp-99(84), a critical side chain in the choline binding site. Spodoptera Sf9 cells were infected by a recombinant baculovirus containing ace-1 cDNA. The secreted enzyme was active and existed as hydrophilic 5 and 11.5 S molecular forms. It hydrolyzed both acetylthiocholine and butyrylthiocholine and was inhibited by acetylthiocholine above 10 mM.

  16. Cloning and Sequencing of cDNA Encoding Islet Cell Autoantigen 69kD Protein from Chinese%国人 ICA69 基因 cDNA 的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: It is reported that cDNA encoding human islet cell autoantigen 69kD protein (hiICA69) has been cloned, so to confirm the nucleotide sequences from the insulinoma cells of Chinese. Methods: cDNA encoding hiICA69 has been amplificated by PCR, from the cDNA library of Chinese insulinoma cells. The PCR product was inserted into the pSPORT 1 vector, and was subcloned into the pUC18 plasmid. After the positive colony was screened by the blue/white colony and the restriction analysis, the nucleotide sequences of the full - length cDNA were analysed by means of the dideoxy chain termination method. Resalts: The results showed that the amplified fragment contained 1449bp, encoded 483 - amino acids. For the sequencing analysis of ICA69 gene from the insulinoma in Mongolian race, the nucleotide sequence of the recombinant was coincident with that reported by Miyazaki and that from EMBL data's bank in addition to one difference of only base on the codon. The change located in the 416th base (A→T), which led to the change of one amino acid (Gln→Leu) . Conclusion: The gene obtained by the method of gene engineering and identified by means of sequence analysis would be able to lay a foundation for follow - up research.%目的:克隆国人胰岛细胞自身抗原 69kD 蛋白基因 ( hiICA69 ) 并经序列分析予以确证。方法:采用聚合酶链式反应技术,从中国人胰岛细胞瘤 cDNA 文库中扩增出 hiICA69 编码序列cDNA,将基因片段插入 pSPORT 1 质粒,进一步亚克隆到 pUCl8 载体中,经蓝白斑和限制性酶谱分析得以初步筛选后,双脱氧末端终止法对其全部核苷酸序列予以确定。结果:证实了 hiICA69 基因全长为 1449bp、编码 483 个氨基酸。与 pietropaolo 等报道的序列比较,仅在编码第 139 位氨基酸的密码子由 CAA→CTA,即由谷氨酰胺→亮氨酸,其余均与文献报道和 EMBL 核酸数据库提供的序列相同。结论:这一基因的获得和

  17. Comparison of the tyrosine aminotransferase cDNA and genomic DNA sequences of normal mink and mink affected with tyrosinemia type II.

    Science.gov (United States)

    Leib, S R; McGuire, T C; Prieur, D J

    2005-01-01

    Type II tyrosinemia, designated Richner-Hanhart syndrome in humans, is a hereditary metabolic disorder with autosomal recessive inheritance characterized by a deficiency of tyrosine aminotransferase activity. Mutations occur in the human tyrosine aminotransferase gene, resulting in high levels of tyrosine and disease. Type II tyrosinemia occurs in mink, and our hypothesis was that it would also be associated with mutation(s) in the tyrosine aminotransferase gene. Therefore, the transcribed cDNA and the genomic tyrosine aminotransferase gene were sequenced from normal and affected mink. The gene extended over 11.9 kb and had 12 exons coding for a predicted 454-amino-acid protein with 93% homology with human tyrosine aminotransferase. FISH analysis mapped the gene to chromosome 8 using the Mandahl and Fredga (1975) nomenclature and chromosome 5 using the Christensen et al. (1996) nomenclature. The hypothesis was rejected because sequence analysis disclosed no mutations in either cDNA or introns that were associated with affected mink. This suggests that an unlinked gene regulatory mutation may be the cause of tyrosinemia in mink.

  18. [Cloning and sequencing of KIR2DL1 framework gene cDNA and identification of a novel allele].

    Science.gov (United States)

    Sun, Ge; Wang, Chang; Zhen, Jianxin; Zhang, Guobin; Xu, Yunping; Deng, Zhihui

    2016-10-01

    To develop an assay for cDNA cloning and haplotype sequencing of KIR2DL1 framework gene and determine the genotype of an ethnic Han from southern China. Total RNA was isolated from peripheral blood sample, and complementary DNA (cDNA) transcript was synthesized by RT-PCR. The entire coding sequence of the KIR2DL1 framework gene was amplified with a pair of KIR2DL1-specific PCR primers. The PCR products with a length of approximately 1.2 kb were then subjected to cloning and haplotype sequencing. A specific target fragment of the KIR2DL1 framework gene was obtained. Following allele separation, a wild-type KIR2DL1*00302 allele and a novel variant allele, KIR2DL1*031, were identified. Sequence alignment with KIR2DL1 alleles from the IPD-KIR Database showed that the novel allele KIR2DL1*031 has differed from the closest allele KIR2DL1*00302 by a non-synonymous mutation at CDS nt 188A>G (codon 42 GAG>GGG) in exon 4, which has caused an amino acid change Glu42Gly. The sequence of the novel allele KIR2DL1*031 was submitted to GenBank under the accession number KP025960 and to the IPD-KIR Database under the submission number IWS40001982. A name KIR2DL1*031 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee. An assay for cDNA cloning and haplotype sequencing of KIR2DL1 has been established, which has a broad applications in KIR studies at allelic level.

  19. 诸葛菜基因EPSPS的cDNA核苷酸序列%The cDNA Nucleotide Sequence of EPSPS Gene from Orychophragmus violaceus

    Institute of Scientific and Technical Information of China (English)

    刘晓军; 邓运涛; 游大慧; 李旭锋

    2002-01-01

    @@1 Source The sequence was determined using the 3′ RACE(rapid amplification cDNA ends) RT-PCR and 5′RACE RT-PCR product, which was ligated to the pMD18-T vector, from the cDNA of Orychophragmus violaceus.

  20. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D

    1990-01-01

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells...... osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb....

  1. Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6×His tag in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    ZHAO YuFeng; LEI MingKe; WU YuanXin; ZHANG ZiSheng; WANG CunWen

    2009-01-01

    Human mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation of acetaldehyde to acetic acid. Therefore, ALDH2 has therapeutic potential in detoxification of acetaldehyde. Furthermore, ALDH2 catalyzes nitroglycerin to nitrate and 1, 2-glyceryldinitrate during therapy for angina pectoris, myocardial infarction, and heart failure. Large quantities of ALDH2 will be needed for potential clinical practice. In this study, Pichia pastoris was used as a platform for expression of human ALDH2.Based on the ALDH2~*1 cDNA sequence, we designed ALDH2 cDNA by choosing the P. pastoris preferred codons and by decreasing the G + C content level. The sequence was synthesized using the overlap extension PCR method. The cDNA and 6×His tags were subcloned into the plasmid pPIC9K.The recombinant protein was expressed in P. pastoris GS115 and purified using Ni~(2+)-Sepharose affinity chromatography. The amount of secreted protein in the culture was 80 mg/L in shake-flask cultivation and 260 mglL in high-density bioreactor fermentation. Secreted ALDH2 was easily purified from the culture supernatant by using Ni2+-Sepharose affinity chromatography. After purification of the fermentation supernatant, the enzyme had a specific activity of 1.2 U/mg protein. The yield was about 16 mg/L in a shake flask culture of P. pastoris GS115 which contained the original human ALDH2~*1 cDNA.

  2. Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6×His tag in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Human mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation of acetaldehyde to acetic acid. Therefore, ALDH2 has therapeutic potential in detoxification of acetaldehyde. Further-more, ALDH2 catalyzes nitroglycerin to nitrate and 1, 2-glyceryldinitrate during therapy for angina pectoris, myocardial infarction, and heart failure. Large quantities of ALDH2 will be needed for potential clinical practice. In this study, Pichia pastoris was used as a platform for expression of human ALDH2. Based on the ALDH2*1 cDNA sequence, we designed ALDH2 cDNA by choosing the P. pastoris preferred codons and by decreasing the G + C content level. The sequence was synthesized using the overlap extension PCR method. The cDNA and 6×His tags were subcloned into the plasmid pPIC9K. The recombinant protein was expressed in P. pastoris GS115 and purified using Ni2+-Sepharose affinity chromatography. The amount of secreted protein in the culture was 80 mg/L in shake-flask cultivation and 260 mg/L in high-density bioreactor fermentation. Secreted ALDH2 was easily purified from the culture supernatant by using Ni2+-Sepharose affinity chromatography. After purification of the fermentation supernatant, the enzyme had a specific activity of 1.2 U/mg protein. The yield was about 16 mg/L in a shake flask culture of P. pastoris GS115 which contained the original human ALDH2*1 cDNA.

  3. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR ... RNA species and approximately 80 structurally distinct ... a reverse transcription kit with Oligo dT as the primers according to ... 2). On the basis of the high identity, we concluded that the cDNA isolated is ...

  4. PvuII RFLP detected by a human. beta. ADH cDNA probe

    Energy Technology Data Exchange (ETDEWEB)

    Parsian, A.; Burgess, A.K.; Khan, M.A.; Devor, E.J. (Washington Univ. School of Medicine, St. Louis, MO (USA))

    1989-12-11

    A 0.97 kb cDNA (ADH12) fragment encoding human exons 7, 8, 9 of the ADH{sub 2} gene was isolated from an adult human liver cDNA library. The insert can be excised by Pst I digestion. Pvu II identifies a two-allele polymorphism with bands at 4.4 kb (A{sub 1}) and 3.0 kb (A{sub 2}) and invariant bands at 5.1, 4.0, 2.8, and 2.3 kb. It was localized on Chromosome 4q21-q25 by in situ hybridization. Co-dominant segregation was observed in 18 informative families.

  5. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  6. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSEDIN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us-ing BLAST algorithm revealed that 22ESFs are highly homologous with the known genes and many of them play impor-tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differemiation expres-sion. The result showed that 27 EST clones are expressed at different level in control and all-traus retinoic acid treated BT-325 cells. A full-length cDNA was cloned using the EST-HGBB098.Conclusion. DDRT-PCR was a simple and effective method to segally analyze the differentially expressed genes.

  7. ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSED IN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

    Institute of Scientific and Technical Information of China (English)

    金虎林; 胡松年; 李光涛; 涂纯; 袁建刚; 强伯勤

    2000-01-01

    Objective. To investigate the differentiation process of the human glioblastoma cells. Methods. Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. Results. Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them,46 ESTs were sequenced and delivered into the GenBank. The homology comparison us ing BLAST algorithm revealed that 22ESTs are highly homologous with the known genes and many of them play impor tant roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differentiation expres sion. The result showed that 27 EST clones are expressed at different level in control and all-trans retinoi c acid treated BT-325 cells. A full-length cDNA was cloned using the ES T-HGBB098. Conclusion. DDRT-PCR was a simple and effective method to serially analyze the differentially expressed genes.

  8. Generation and analysis of expressed sequence tags from Trypanosoma cruzi trypomastigote and amastigote cDNA libraries.

    Science.gov (United States)

    Agüero, Fernán; Abdellah, Karim Ben; Tekiel, Valeria; Sánchez, Daniel O; González, Antonio

    2004-08-01

    We have generated 2771 expressed sequence tags (ESTs) from two cDNA libraries of Trypanosoma cruzi CL-Brener. The libraries were constructed from trypomastigote and amastigotes, using a spliced leader primer to synthesize the cDNA second strand, thus selecting for full-length cDNAs. Since the libraries were not normalized nor pre-screened, we compared the representation of transcripts between the two using a statistical test and identify a subset of transcripts that show apparent differential representation. A non-redundant set of 1619 reconstructed transcripts was generated by sequence clustering. This dataset was used to perform similarity searches against protein and nucleotide databases. Based on these searches, 339 sequences could be assigned a putative identity. One thousand one-hundred and sixteen sequences in the non-redundant clustered dataset (68.8%) are new expression tags, not represented in the T. cruzi epimastigote ESTs that are in the public databases. Additional information is provided online at http://genoma.unsam.edu.ar/projects/tram. To the best of our knowledge these are the first ESTs reported for the life cycle stages of T. cruzi that occur in the vertebrate host.

  9. Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

    Directory of Open Access Journals (Sweden)

    Bendahmane Abdelhafid

    2011-05-01

    Full Text Available Abstract Background Melon (Cucumis melo, an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs and 3,073 single nucleotide polymorphisms (SNPs in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but

  10. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    Institute of Scientific and Technical Information of China (English)

    Yong-Bo Liu; Zhao-Xia Wei; Li Li; Hang-Sheng Li; Hui Chen; Xiao-Wen Li

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMARTTM PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder,cDNA xProfiler, Digital GENE Expression Displayer (DGED),and Digital Differential Display (DDD) were used.RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed.CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocardnoma.

  11. [Mapping and human genome sequence program].

    Science.gov (United States)

    Weissenbach, J

    1997-03-01

    Until recently, human genome programs focused primarily on establishing maps that would provide signposts to researchers seeking to identify genes responsible for inherited diseases, as well as a basis for genome sequencing studies. Preestablished gene mapping goals have been reached. The over 7,000 microsatellite markers identified to date provide a map of sufficient density to allow localization of the gene of a monogenic disease with a precision of 1 to 2 million base pairs. The physical map, based on systematically arranged overlapping sets of artificial yeast chromosomes (YACs), has also made considerable headway during the last few years. The most recently published map covers more than 90% of the genome. However, currently available physical maps cannot be used for sequencing studies because multiple rearrangements occur in YACs. The recently developed sets of radioinduced hybrids are extremely useful for incorporating genes into existing maps. A network of American and European laboratories has successfully used these radioinduced hybrids to map 15,000 gene tags from large-scale cDNA library sequencing programs. There are increasingly pressing reasons for initiating large scale human genome sequencing studies.

  12. Characterization of the porcine carboxypeptidase E cDNA

    DEFF Research Database (Denmark)

    Hreidarsdôttir, G.E.; Cirera, Susanna; Fredholm, Merete

    2007-01-01

    the sequence of the cDNA for the porcine CPE gene including all the coding region and the 3'-UTR region was generated. Comparisons with bovine, human, mouse, and rat CPE cDNA sequences showed that the coding regions of the gene are highly conserved both at the nucleotide and at the amino acid level. A very low...

  13. A NEW METHOD TO CONSTRUCT A FULL-LENGTH cDNA LIBRARY OF HUMAN NORMAL BLADDER TISSUE

    Institute of Scientific and Technical Information of China (English)

    成瑜; 李旭; 陈葳; 杨玉琮; 赵乐

    2003-01-01

    Objective Using template-switch mechanism at the 5'-end of mRNA technique (SMART) to construct a full-length cDNA library of human normal bladder tissue. Methods The novel procedures used the template-switching activity of powerscript reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, 5 cycles of PCR were performed using a modified oligo(dT) primer and an anchor primer to enrich the full-length cDNA population with 1.0 g human normal bladder poly(A)+RNA, then double-strand cDNA was synthesized. After digestion with sfiI and size-fractionation by CHROMA SPIN-400 columns, double-strand cDNA was ligated into λTripIEx2 vector and was packaged. We determined the titer of the primary library and the percentage of recombinant clones and finally amplified the library. Results The titer of the cDNA library constructed was 2.1×106 pfu*mL-1, and the amplified cDNA library was 6×1011 pfu*mL-1, the percentage of recombination clones was 99%. Conclusion Using SMART technique helps us to construct full-length cDNA library with high efficiency and high capacity which lays solid foundation for screening target genes of bladder diseases with probes and antibodies.

  14. cDNA and genomic cloning of human palmitoyl-protein thioesterase (PPT), the enzyme defective in infantile neuronal ceroid lipofuscinosis

    Energy Technology Data Exchange (ETDEWEB)

    Schriner, J.E.; Yi, W.; Hofmann, S.L. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States)

    1996-06-15

    Palmitoyl-protein thioesterase (PPT) is a small glycoprotein that removes palmitate groups from cysteine residues in lipid-modified proteins. We recently reported mutations in PPT in patients with infantile neuronal ceroid lipofuscinosis (INCL), a severe neurodegenerative disorder. INCL is characterized by the accumulation of proteolipid storage material in brain and other tissues, suggesting that the disease is a consequence of abnormal catabolism of acylated proteins. In the current paper, we report the sequence of the human PPT cDNA and the structure of the human PPT gene. The cDNA predicts a protein of 306 amino acids that contains a 25-amino-acid signal peptide, three N-linked glycosylation sites, and consensus motifs characteristic of thioesterases. Northern analysis of a human tissue blot revealed ubiquitous expression of a single 2.5-kb mRNA, with highest expression in lung, brain, and heart. The human PPT gene spans 25 kb and is composed of seven coding exons and a large eighth exon, containing the entire 3{prime}-untranslated region of 1388 bp. An Alu repeat and promoter elements corresponding to putative binding sites for several general transcription factors were identified in the 1060 nucleotides upstream of the transcription start site. The human PPT cDNA sequence and gene structure will provide the means for the identification of further causative mutations in INCL and facilitate genetic screening in selected high-risk populations. 31 refs., 5 figs., 1 tab.

  15. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

    Science.gov (United States)

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2014-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  16. A juvenile hormone-repressible transferrin-like protein from the bean bug, Riptortus clavatus: cDNA sequence analysis and protein identification during diapause and vitellogenesis.

    Science.gov (United States)

    Hirai, M; Watanabe, D; Chinzei, Y

    2000-05-01

    We found several juvenile hormone-responsive cDNAs in the bean bug, Riptortus clavatus, by using mRNA differential display (Hirai et al., 1998). One of them, a juvenile hormone-repressible cDNA, JR-3, was cloned, sequenced, characterized and identified as a transferrin (RcTf). RcTf cDNA encoded 652 amino acids with a calculated molecular weight of 71,453 Da. The deduced amino acid sequence showed significant homology with the transferin genes of several insects, Manduca sexta (43% identity), Blaberus discoidalis (43%), Aedes aegypti (43%), Drosophila melanogaster (36%), Sarcophaga peregrina (36%) and the human (25%). Antiserum was prepared by using recombinant RcTf protein expressed in Escherichia coli as an antigen. The antiserum reacted specifically with both the recombinant protein and the native protein from the bugs, with sizes of 70 and 75 kDa, respectively. The 75 kDa protein was partially purified from hemolymph of diapausing female bugs and the first ten amino acids were found to be identical to that of RcTf cDNA, indicating that the 75 kDa protein is RcTf. The tissue distribution of RcTf in the bug was examined by Western blot analysis. In diapausing animals, RcTf was detected in the fat body, hemolymph and ovary but not in the gut. In the post-diapause stage, RcTf was also detected in eggs, in addition to the fat body and ovary. These results indicate that RcTf is incorporated into the oocytes during vitellogenesis, and suggest that it may provide iron for the developing embryos.

  17. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    Energy Technology Data Exchange (ETDEWEB)

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O' Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O.; Barrero, Roberto A.; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A.; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; de Fatima Bonaldo, Maria; Bono Hidemasa; Bromberg, Susan K.; Brookes, Anthony J.; Bruford, Elspeth; Carninci Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; Gopinath, Gopal R.; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba Rie; et al.

    2004-01-15

    The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4 percent of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5 percent of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for nonprotein-coding RNA

  18. Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

    Directory of Open Access Journals (Sweden)

    Tadashi Imanishi

    2004-06-01

    Full Text Available The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/. It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs, identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA

  19. Generation and analysis of expressed sequence tags from a cDNA library of the fruiting body of Ganoderma lucidum

    Directory of Open Access Journals (Sweden)

    Li Xiwen

    2010-03-01

    Full Text Available Abstract Background Little genomic or trancriptomic information on Ganoderma lucidum (Lingzhi is known. This study aims to discover the transcripts involved in secondary metabolite biosynthesis and developmental regulation of G. lucidum using an expressed sequence tag (EST library. Methods A cDNA library was constructed from the G. lucidum fruiting body. Its high-quality ESTs were assembled into unique sequences with contigs and singletons. The unique sequences were annotated according to sequence similarities to genes or proteins available in public databases. The detection of simple sequence repeats (SSRs was preformed by online analysis. Results A total of 1,023 clones were randomly selected from the G. lucidum library and sequenced, yielding 879 high-quality ESTs. These ESTs showed similarities to a diverse range of genes. The sequences encoding squalene epoxidase (SE and farnesyl-diphosphate synthase (FPS were identified in this EST collection. Several candidate genes, such as hydrophobin, MOB2, profilin and PHO84 were detected for the first time in G. lucidum. Thirteen (13 potential SSR-motif microsatellite loci were also identified. Conclusion The present study demonstrates a successful application of EST analysis in the discovery of transcripts involved in the secondary metabolite biosynthesis and the developmental regulation of G. lucidum.

  20. Transcription Profiling of the Model Cyanobacterium Synechococcus sp. Strain PCC 7002 by Next-Gen (SOLiD™) Sequencing of cDNA

    OpenAIRE

    Ludwig, Marcus; Bryant, Donald A.

    2011-01-01

    The genome of the unicellular, euryhaline cyanobacterium Synechococcus sp. PCC 7002 encodes about 3200 proteins. Transcripts were detected for nearly all annotated open reading frames by a global transcriptomic analysis by Next-Generation (SOLiD™) sequencing of cDNA. In the cDNA samples sequenced, ∼90% of the mapped sequences were derived from the 16S and 23S ribosomal RNAs and ∼10% of the sequences were derived from mRNAs. In cells grown photoautotrophically under standard conditions [38°C, ...

  1. Transcription profiling of the model cyanobacterium Synechococcus sp. strain PCC 7002 by NextGen (SOLiD™) Sequencing of cDNA

    OpenAIRE

    Marcus eLudwig; Bryant, Donald A.

    2011-01-01

    The genome of the unicellular, euryhaline cyanobacterium Synechococcus sp. PCC 7002 encodes about 3200 proteins. Transcripts were detected for nearly all annotated open reading frames by a global transcriptomic analysis by Next-Generation (SOLiDTM) sequencing of cDNA. In the cDNA samples sequenced, ~90% of the mapped sequences were derived from the 16S and 23S ribosomal RNAs and ~10% of the sequences were derived from mRNAs. In cells grown photoautotrophically under standard conditions (38 &#...

  2. Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties.

    Science.gov (United States)

    Temeyer, Kevin B; Brake, Danett K; Tuckow, Alexander P; Li, Andrew Y; Pérez de León, Adalberto A

    2013-02-04

    Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3'-5'-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman's assay in microplates. A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis. Recombinant P

  3. 酵母双杂交筛选胎肾上腺cDNA文库中HNP-1结合蛋白%SCREENING THE GENE SEQUENCES OF THE INTERACTION PROTEINS OF HNP-1 FROM HUMAN FETAL ADRENAL CDNA LIBRARY BY YEAST TWO HYBRID SYSTEM

    Institute of Scientific and Technical Information of China (English)

    杜润滋; 邓璐霞; 黄宁; 罗朝志

    2011-01-01

    [Objective] To screen proteins binding with α-defensin (HNP-1) mature peptide from placenta cDNA libraty by yeast two hybrid technique. [Methods] The cDNA fragment encoding HNP-1 mature peptide was amplified by polymera-sechain reaction (PCR) and constructed into pGBK-T7 vector as the bait plasmid in yeast two hybrid system 3. Subsequently , the RNA from fetal adrenal gland was obtained and then transformed into cDNA library using SMART technology. The fetal adrenal cDNA library was screened with pGBKT7-HNP-1 as bait plasmid by yeast-two hybrid system Matchmaker Lexa. Finally, the positive clone was obtained by PCR and then identified by sequence. Then the interaction between them was determined by GST pull down in vitro and coimmunoprecipitates experiments in vivo. [Results] Bait and cDNA library have been constructed successfully and transformed into yeast. Then the interaction protein was found-melanocortin 2 receptor (ACTHR), CCAAT-enhancer-binding proteins (C/EBP), Tramembrane trafficking protein (TMP21), low density lipoprotein receptor-related protein 6 (LRP6). Therefore, melanocortin 2 receptor (ACTH-R) was determined into the major subjects. And bands which can demonstrate the relationship between HNP-1 and ACTH-R was obtained in GST pull down and coimmunoprecipitates experiments. [Conclusion] ACTH-R can bind to HNP-1 we obtained from fetal adrenal cDNA library and it may play important roles in the function of HNP-1 mature peptide.%[目的]筛选胎肾上腺cDNA文库中与α防御素HNP-1成熟肽具有相互作用的蛋白分子.[方法]通过聚合酶链反应(PCR)成功获得HNP-1成熟肽基因插入酵母表达载体pGBK-T7中构建诱饵质粒,同时提取胎肾上腺RNA,SMART技术制备人胎肾上腺cDNA文库,并采用Matchmaker LexA酵母双杂交系统从胎肾上腺cDNA文库中筛选与HNP-1成熟肽相互作用的蛋白.最后通过PCR筛选获得阳性克隆并测序,而后经回转实验,GST pull down以及免疫共沉淀再次验证

  4. Amino acid and cDNA sequences of a neutral phospholipase A2 from the long-nosed viper (Vipera ammodytes ammodytes) venom.

    Science.gov (United States)

    Krizaj, I; Liang, N S; Pungercar, J; Strukelj, B; Ritonja, A; Gubensek, F

    1992-03-15

    The amino acid sequence of a non-toxic phospholipase A2, ammodytin I2, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non-maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137-amino-acid-residue precursor molecule consisting of a 16-residue signal peptide, followed by a 121-residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A2 from the long-nosed viper. Identities in the signal-peptide region are considerably higher, 96% and 100%, respectively.

  5. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1--December 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.

    1993-12-31

    The ultimate goal of this proposal is to create a cDNA map of the human genome. Mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach will generate 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  6. [The structure and specific features of the cDNA expression of the human gene MRPL37].

    Science.gov (United States)

    Levshenkova, E V; Ukraintsev, K E; Orlova, V V; Alibaeva, R A; Kovriga, I E; Zhugdernamzhilyn, O; Frolova, E I

    2004-01-01

    A 147-bp cDNA fragment was isolated from human lymphocytes activated with concanavalin A using the method of direct selection. A complete copy of the selected gene having total homology with the mitochondrial ribosomal gene MRPL37 was obtained by the RACE (rapid amplification of cDNA ends) technique. The MRPL37 gene was localized on human chromosome 1 using a DNA panel composed of somatic cellular human-hamster hybrids. The Northern blotting and RT-PCR (reverse transcription-polymerase chain reaction) demonstrated that the RNA of the human MRPL37 gene is widely represented in the lymphoma populations of Raji B cells and MT4 T cells, as well as in pancreas, liver, and lung embryonic fibroblasts WI-38 and LEH. The highest expression level of the MRPL37 mouse homologue was found in the cells of skeletal muscles, the heart, and organs of reproductive system: the uterus, ovaries, and testicles. A comparative analysis of the MRPL37 amino acid sequence with those of proteins represented in the Fasta33 and GenBank databases showed a homologous region in MRPL37 and PDCD9 (programmed cell death 9, MPRS30) proteins. The chicken homologue of PDCD9 is interesting because its overexpression causes apoptosis of the mouse fibroblasts C3H10T1/2. The existence of a common domain indicates possible similar functional peculiarities of the PDCD9 and MRPL37 genes and may imply the MRPL37 involvement in the process of apoptosis. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

  7. hSmad5 gene, a human hSmad family member: its full length cDNA, genomic structure, promoter region and mutation analysis in human tumors.

    Science.gov (United States)

    Gemma, A; Hagiwara, K; Vincent, F; Ke, Y; Hancock, A R; Nagashima, M; Bennett, W P; Harris, C C

    1998-02-19

    hSmad (mothers against decapentaplegic)-related proteins are important messengers within the Transforming Growth Factor-beta1 (TGF-beta1) superfamily signal transduction pathways. To further characterize a member of this family, we obtained a full length cDNA of the human hSmad5 (hSmad5) gene by rapid amplification of cDNA ends (RACE) and then determined the genomic structure of the gene. There are eight exons and two alternative transcripts; the shorter transcript lacks exon 2. We identified the hSmad5 promoter region from a human genomic YAC clone by obtaining the nucleotide sequence extending 1235 base pairs upstream of the 5' end of the cDNA. We found a CpG island consistent with a promoter region, and we demonstrated promoter activity in a 1232 bp fragment located upstream of the transcription initiation site. To investigate the frequency of somatic hSmad5 mutations in human cancers, we designed intron-based primers to examine coding regions by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Neither homozygous deletions or point mutations were found in 40 primary gastric tumors and 51 cell lines derived from diverse types of human cancer including 20 cell lines resistant to the growth inhibitory effects of TGF-beta1. These results suggest that the hSmad5 gene is not commonly mutated and that other genetic alterations mediate the loss of TGF-beta1 responsiveness in human cancers.

  8. Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells

    Energy Technology Data Exchange (ETDEWEB)

    Davidson, D.J.; Fraser, M.J.; Castellino, F.J. (Univ. of Notre Dame, IN (USA))

    1990-06-12

    A comparison has been made between the Asn{sup 289}-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, the authors find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate and truncated high-mannose oligosaccharide. Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. The data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, the authors conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.

  9. cDNA, genomic sequence cloning and overexpression of ribosomal protein S25 gene (RPS25) from the Giant Panda.

    Science.gov (United States)

    Hao, Yan-Zhe; Hou, Wan-Ru; Hou, Yi-Ling; Du, Yu-Jie; Zhang, Tian; Peng, Zheng-Song

    2009-11-01

    RPS25 is a component of the 40S small ribosomal subunit encoded by RPS25 gene, which is specific to eukaryotes. Studies in reference to RPS25 gene from animals were handful. The Giant Panda (Ailuropoda melanoleuca), known as a "living fossil", are increasingly concerned by the world community. Studies on RPS25 of the Giant Panda could provide scientific data for inquiring into the hereditary traits of the gene and formulating the protective strategy for the Giant Panda. The cDNA of the RPS25 cloned from Giant Panda is 436 bp in size, containing an open reading frame of 378 bp encoding 125 amino acids. The length of the genomic sequence is 1,992 bp, which was found to possess four exons and three introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos taurus, Mus musculus and Rattus norvegicus as determined by Blast analysis, 92.6, 94.4, 89.2 and 91.5%, respectively. Primary structure analysis revealed that the molecular weight of the putative RPS25 protein is 13.7421 kDa with a theoretical pI 10.12. Topology prediction showed there is one N-glycosylation site, one cAMP and cGMP-dependent protein kinase phosphorylation site, two Protein kinase C phosphorylation sites and one Tyrosine kinase phosphorylation site in the RPS25 protein of the Giant Panda. The RPS25 gene was overexpressed in E. coli BL21 and Western Blotting of the RPS25 protein was also done. The results indicated that the RPS25 gene can be really expressed in E. coli and the RPS25 protein fusioned with the N-terminally his-tagged form gave rise to the accumulation of an expected 17.4 kDa polypeptide. The cDNA and the genomic sequence of RPS25 were cloned successfully for the first time from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively, which were both sequenced and analyzed preliminarily; then the cDNA of the RPS25 gene was overexpressed in E. coli BL21 and immunoblotted, which is the first

  10. Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains.

    Science.gov (United States)

    Hohman, V S; Schluter, S F; Marchalonis, J J

    1992-01-01

    A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.

  11. Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences.

    Science.gov (United States)

    Bower, Neil I; Johnston, Ian A

    2010-11-01

    Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3' or 5' end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3' or 5' T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3' and 5' ends of numerous cDNAs from a single cDNA synthesis reaction.

  12. Analysis of xylem formation in pine by cDNA sequencing

    Science.gov (United States)

    Allona, I.; Quinn, M.; Shoop, E.; Swope, K.; St Cyr, S.; Carlis, J.; Riedl, J.; Retzel, E.; Campbell, M. M.; Sederoff, R.; hide

    1998-01-01

    Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5' ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.

  13. Mitochondrial HMG to CoA synthase (mHS): cDNA cloning in human, mouse and C. elegans, mapping to human chromosome 1p12-13 and partial human genomic cloning

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal, Quebec (Canada)]|[Kingston General Hospital, Ontario (Canada)] [and others

    1994-09-01

    mHS catalyzes the rate-limiting first step of ketogenesis in the liver. A cytoplasmic HS isozyme, encoded by another gene, catalyzes an early step in cholesterol synthesis. Starting from a rat mHS cDNA obtained by RT-PCR from the published rat cDNA sequence, we obtained and sequenced human and mouse cDNAs spanning the entire coding sequence of natural human and mouse mHS, as well as sequencing C. elegans HS-like cDNA. Consensus sequences for 3 mitochondrial and 4 cytoplasmic HSs were created and compared to invertebrate HS sequences. We found high conversation in the active site and at other regions presumably important for HS function. We mapped the mHS locus, HMGCS2 by in situ hybridization to chromosome 1P12-13, in contrast to the human cHS locus (HMGCS1) known to be on chromosome 5p13. Comparative mapping results suggest that these two chromosomal regions may be contiguous in other species, constant with a recent gene duplication event. Furthermore, we have characterized a human genomic mHS subclone containing 4 mHS exons, and found the position of all splice junctions to be identical to that of the hamster cHS gene except for one site in the 3{prime} nontranslated region. We calculate that the mHS and cHS genes were derived from a common ancestor 400-700 Myrs ago, implying that ketogenesis from fat may have become possible around the time of emergence of vertebrates ({approximately}500 Myr ago). Ketogenesis has evolved into an important pathway of energy metabolism, and we predict the mHS deficiency may prove to be responsible for some as yet explained cases of Reye-like syndromes in humans. This hypothesis can now be tested at the molecular level without the necessity of obtaining hepatic tissue.

  14. cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella (Lepidoptera: Plutellidae)

    Institute of Scientific and Technical Information of China (English)

    SHAO-LIWANG; CHENG-FASHENG; CHUAN-LINGQIAO; MIYATATADASHI

    2005-01-01

    Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.

  15. Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

    Energy Technology Data Exchange (ETDEWEB)

    Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J. (Wistar Inst. of Anatomy and Biology, Philadelphia, PA (USA))

    1990-09-01

    The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.

  16. Sequence determinants in human polyadenylation site selection

    Directory of Open Access Journals (Sweden)

    Gautheret Daniel

    2003-02-01

    Full Text Available Abstract Background Differential polyadenylation is a widespread mechanism in higher eukaryotes producing mRNAs with different 3' ends in different contexts. This involves several alternative polyadenylation sites in the 3' UTR, each with its specific strength. Here, we analyze the vicinity of human polyadenylation signals in search of patterns that would help discriminate strong and weak polyadenylation sites, or true sites from randomly occurring signals. Results We used human genomic sequences to retrieve the region downstream of polyadenylation signals, usually absent from cDNA or mRNA databases. Analyzing 4956 EST-validated polyadenylation sites and their -300/+300 nt flanking regions, we clearly visualized the upstream (USE and downstream (DSE sequence elements, both characterized by U-rich (not GU-rich segments. The presence of a USE and a DSE is the main feature distinguishing true polyadenylation sites from randomly occurring A(A/UUAAA hexamers. While USEs are indifferently associated with strong and weak poly(A sites, DSEs are more conspicuous near strong poly(A sites. We then used the region encompassing the hexamer and DSE as a training set for poly(A site identification by the ERPIN program and achieved a prediction specificity of 69 to 85% for a sensitivity of 56%. Conclusion The availability of complete genomes and large EST sequence databases now permit large-scale observation of polyadenylation sites. Both U-rich sequences flanking both sides of poly(A signals contribute to the definition of "true" sites. However, the downstream U-rich sequences may also play an enhancing role. Based on this information, poly(A site prediction accuracy was moderately but consistently improved compared to the best previously available algorithm.

  17. Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

    Institute of Scientific and Technical Information of China (English)

    Ge-Jian Zhu; Ying-Nian Yu; Xin Li; Yu-Li Qian

    2002-01-01

    AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9 ( CYP2 C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2 C9 eDNA was cloned and expressed stably in CHL cellsMETHODS: After extraction of total RNA from human livertissue, the human CYP2C9 eDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR),and cloned into cloning vector pGEM-T. The cDNA fragmentwas identified by DNA sequencing and subcloned into amammalian expression vector pREP9. A transgenic cell linewas established by transfecting the recombinant vector ofpREP9-CYP2C9 into CHL cells. The enzyme activity ofCYP2C9 catalyzing oxidation of tolbutamide to hydroxytolbutamide in S9 fraction of the cell was determined by highperformance liquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from theeDNA segment was identical to that of CYP2 C9 * 1, the wildtype CYP2 C9. However, there were two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acidsequence was the same, L7, P382. The S9 fraction of theestablished cell line metabolizes tolbutamide to hydroxytolbutamide; tolbutamide hydroxylass activity was found to be0.465 ± 0.109 μmol@ min-1 . g1 S9 protein or 8.62 ± 2.02 mol@ min 1 ~mol-1 CYP, but was undetectable in parental CHL cell.CONCLUSION: The cDNA of human CYP2C9 was successfullycloned and a cell line of CHL- CYP2C9, efficiently expressingthe protein of CYP2C9, was established.

  18. The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats

    DEFF Research Database (Denmark)

    Sarin, C T; Tack, B F; Kristensen, Torsten;

    1986-01-01

    We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and...

  19. Completion sequence and cloning of the infectious cDNA of a chb isolate of cucumber green mottle mosaic virus.

    Science.gov (United States)

    Zhong, M; Zhao, X; Liu, Y; Wang, Y; Cao, K

    2015-03-01

    Cucumber green mottle mosaic virus (CGMMV) is an important and widespread seed-borne virus that infects Cucurbitaceous plants. It is a member of the genus Tobamovirus in the family Virgaviridae with a monopartite (+) ssRNA genome. Here we report the complete genome sequence, construction and testing of the infectious clones of a chb isolate of CGMMV. Full-length CGMMV cDNA was cloned into the vector pUC19. The linearized vector containing full-length cDNA was used as template for in vitro transcription, and the synthesized capped transcript was highly infectious in Chenopodium amaranticolor and cucumber (Cucumis sativus). Inoculated plants showed symptoms typical of CGMMV infection. The infectivity was confirmed by mechanical transmission to new plants, RT-PCR and western blot. Progeny virus derived from infectious transcripts had the same biological and biochemical properties as wild-type virus. To our knowledge, this is the first detailed report of a biologically active transcript from CGMMV.

  20. Cloning and expression analysis of human reticulon 4c cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RTNs (reticulons) is a gene family related to the growth and differentiation of neuroendocrine cell. This family is composed of several members such as RTN1, RTN2 and RTN3. RTN1 and RTN2 have been proved to have 3 transcripts with different length. Because the RTN1c cDNA was involved in the sologenesis of small cell lung carcinoma (SCLC), it was selected as a bioinformatic probe to clone novel members of RTN family with the electric hybridization assistant new-gene cloning method (EHAC). A 1677-bp cDNA was identified from human brain cDNA library. The cDNA contains an intact open reading frame (ORF) which encodes a protein of 199 amino acids. This deduced protein is highly homologous to RTN1c, RTN2c and RTN3 with identities of 64.4%, 45.8% and 50.0% respectively. This new gene was named RTN4c (GenBank accession number: AF087901). Northern hybridization showed that the full length of RTN4c transcript is about 1.8 kb. It is hardly expressed in heart, placenta, lung, spleen, thymus, testis, ovary, small intestine and peripheral blood white cells; but it is highly expressed in the tissues of skeletal muscle, brain, liver and kidney, and less expressed in the pancreas, prostate and colon. Furthermore, Northern results also showed that there is a 2.3 kb transcript expressed in 14 tissues except liver and skeletal muscle; while another 5.0 kb transcript in brain, skeletal muscle and testis. By the electric hybridization walking, we obtained two full-length contigs with a length of 4632 and 2235 bp respectively. The former encodes a protein with 1192 amino acids and was defined as RTN4a; the latter encodes another protein with 373 amino acids, and was named RTN4b. The RTN4 gene was mapped to human chromosome 2p14-p13 region by the radiation hybridization (RH).

  1. Purification, amino acid sequence, and cDNA cloning of trypsin inhibitors from onion (Allium cepa L.) bulbs.

    Science.gov (United States)

    Deshimaru, Masanobu; Watanabe, Akira; Suematsu, Keiko; Hatano, Maki; Terada, Shigeyuki

    2003-08-01

    Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3 x 10(-9) M, 2.3 x 10(-7) M, and 3.1 x 10(-7) M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.

  2. Existence of homologous sequences corresponding to cDNA of the ver

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The presence of DNA homologues corresponding to verc203 (vernalization-related cDNA clone) was investigated by molecular hybridization techniques. The genes were detected in 16 plant species that cover 12 subclasses of the Takhtajan system of angiosperms classification including diverse model species. The results of Southern blot analysis showed a low copy number of this gene existed in rice, wheat, barley and Arabidopsis. The hybridization result of PCR products demonstrated the conservation of the gene corresponding to ver203 in diverse plants. The phylogenetic tree of the ver203 gene in tested plants was supported by evolution relationship of species. The ver203 gene expressed in a vernalized plumule winter wheat, instead of the root. And the endosperm before the treatment was essential for the ver203 expression during vernalization in wheat. In Arabidopsis thaliana, the pattern of expression showed that the gene corresponding to ver203 was expressed at low temperature for 14 days. Gibberellin (GA3) may accelerate the expression of ver203 gene in Arabidopsis exposed to low temperature. However, it could not replace vernalization treatment to initiate the gene expression.

  3. Antibody to a human DNA repair protein allows for cloning of a Drosophila cDNA that encodes an apurinic endonuclease.

    Science.gov (United States)

    Kelley, M R; Venugopal, S; Harless, J; Deutsch, W A

    1989-03-01

    The cDNA of a Drosophila DNA repair gene, AP3, was cloned by screening an embryonic lambda gt11 expression library with an antibody that was originally prepared against a purified human apurinic-apyrimidinic (AP) endonuclease. The 1.2-kilobase (kb) AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles were located. The cDNA clone yielded an in vitro translation product of 35,000 daltons, in agreement with the predicted size of the translation product of the only open reading frame of AP3, and identical to the molecular size of an AP endonuclease activity recovered following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Drosophila extracts. The C-terminal portion of the predicted protein contained regions of presumptive DNA-binding domains, while the DNA sequence at the amino end of AP3 showed similarity to the Escherichia coli recA gene. AP3 is expressed as an abundant 1.3-kb mRNA that is detected throughout the life cycle of Drosophila melanogaster. Another 3.5-kb mRNA also hybridized to the AP3 cDNA, but this species was restricted to the early stages of development.

  4. Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.

    Science.gov (United States)

    Schmidt, W M; Mueller, M W

    1996-05-01

    Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequence analysis of complete 5'-terminal unknown coding regions of rare RNA molecules. In contrast with standard tailing protocols using dNTPs as the substrate, ribo-tailing of cDNA ends is easily controllable, self-limited (from two to four rNMP incorporations) and highly efficient (>98%). By virtue of the homopolymeric ribo-tail, the modified cDNA is anchored to the 3' overhang of a double-stranded DNA-adaptor in a T4 DNA ligase-dependent ligation. PCR amplification, mediated by two sequence-specific primers, yields the desired unique product suitable for cloning and dideoxy-sequencing.

  5. Fiscal 1999 achievement report on the analysis of the complete sequencing of full-length cDNA; 1999 nendo dai 2 ji hosei yosan kanzencho cDNA kozo kaiseki seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    This report accommodates the results of study conducted during the period of April 1, 2000, through March 31, 2001. The study began with the partial sequence determination for cDNA (complementary deoxyribonucleic acid) terminals presented by the cDNA library, novel clones were then selected out of them, and efforts proceeded to sequence all the bases therein. In this study, partial sequences were determined for 519,000 clones, and entire sequences for 7252 clones. The obtained sequence data were subjected to a homological analysis and then converted into an amino acid sequence, and then protein function prediction and the like were performed using the SOSUI program or the like. A prototype system to isolate novel clones out of partial sequences and a system for the graphic display of cDNA-genome links were fabricated. As for expression profile databases, the iAFLP (introduced amplified fragment length polymorphism) method was used to construct a high-throughput system and a function analysis database. (NEDO)

  6. Primary structure of fox (Vulpes vulpes) proinsulin based on sequence studies of pancreatic peptides and cDNA.

    Science.gov (United States)

    Fiertek, D; Gromowska, M; Andersen, A S; Hansen, P H; Majewski, T; Izdebski, J

    2000-08-01

    Insulin and C-peptide were extracted and purified from fox (Vulpes vulpes) pancreas using gel filtration, ion-exchange chromatography and HPLC. Chromatographic data for the insulin, as well as for its oxidized and carboxymethylated chains proved it to be identical to that of polar fox (Alopex lagopus) and dog. The sequence analysis of a peptide which was assumed to be the corresponding C-peptide revealed that it comprises 23 amino acid residues and is identical to the C-peptide fragment isolated from dog pancreas: it differs from polar fox C-peptide by a single substitution (Asp-->Glu). mRNA was isolated from pancreatic tissue and cDNA was obtained by reverse transcription. A polymerase chain reaction was performed using gene-specific primers to obtain a DNA fragment corresponding to part of fox proinsulin. DNA sequencing revealed 100% identity to dog proinsulin at the protein level, although some amino acids were encoded by different codons. The total sequence of proinsulin was deduced from these results.

  7. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  8. Expressed Sequence Tags Analysis and Design of Simple Sequence Repeats Markers from a Full-Length cDNA Library in Perilla frutescens (L.

    Directory of Open Access Journals (Sweden)

    Eun Soo Seong

    2015-01-01

    Full Text Available Perilla frutescens is valuable as a medicinal plant as well as a natural medicine and functional food. However, comparative genomics analyses of P. frutescens are limited due to a lack of gene annotations and characterization. A full-length cDNA library from P. frutescens leaves was constructed to identify functional gene clusters and probable EST-SSR markers via analysis of 1,056 expressed sequence tags. Unigene assembly was performed using basic local alignment search tool (BLAST homology searches and annotated Gene Ontology (GO. A total of 18 simple sequence repeats (SSRs were designed as primer pairs. This study is the first to report comparative genomics and EST-SSR markers from P. frutescens will help gene discovery and provide an important source for functional genomics and molecular genetic research in this interesting medicinal plant.

  9. cDNA microarray reveals signaling pathways involved in hormones expression of human pituitary.

    Science.gov (United States)

    Ma, Yue-Yun; Qi, Xiao-Fei; Song, Shao-Jun; Zhao, Zhan-Yong; Zhu, Zhi-Dong; Qi, Jia; Zhang, Xin; Xiao, Hua-Sheng; Teng, Yun; Han, Ze-Guang

    2005-09-01

    Pituitary, a master gland of neuroendocrine system, secretes hormones that orchestrate many physiological processes, under the regulation of multiple signaling pathways. To investigate the genes involved in hormones expression of human pituitary, homemade cDNA microarray containing 14,800 human genes/ESTs were used to profile the gene expression in both fetal and adult pituitaries. Seven hundred and twelve known genes changed over 2-fold between the both tissues. Of which, 23 genes were changed with hormones expression in aging were confirmed by RT-PCR, not only the known regulators such as Pit1, GATA4, ESRRA, GABA-A, and EMK, but also LOC55884, DUSP3, PNN, and RCL, which had not been reported to be involved in the hormones expression. Correspondingly, the mRNAs of GH, PRL, POMC, TSH-beta, FSH-beta, and LH-beta, was increased as much as 6- to 20-fold in adult pituitary than those in fetal pituitary, by real-time quantitative RT-PCR assay. In addition, the mRNAs of signaling pathways, such as cAMP-PKA-CREB, PI3K-Akt, and PKA-ERK were further investigated. Of them, it was only cAMP-PKA-CREB pathway, but not PI3K-Akt and PKA-ERK have the same expressing pattern as hormones. It suggested that cDNA microarray is highly advantages to profile the differential expressed genes that were involved in hormones expression of human pituitary, but it might ignore some responding proteins regulated posttranscriptionally.

  10. cDNA immunization of mice with human thyroglobulin generates both humoral and T cell responses: a novel model of thyroid autoimmunity.

    Directory of Open Access Journals (Sweden)

    Eric M Jacobson

    Full Text Available Thyroglobulin (Tg represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD. Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT, are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis.

  11. Improved rapid amplification of cDNA ends (RACE) for mapping both the 5' and 3' terminal sequences of paramyxovirus genomes.

    Science.gov (United States)

    Li, Zhuo; Yu, Meng; Zhang, Hong; Wang, Hai-Yan; Wang, Lin-Fa

    2005-12-01

    Rapid amplification of cDNA ends (RACE) is a powerful PCR-based technique for determination of RNA terminal sequences. However, most of the RACE methods reported in the literature are developed specifically for the mapping of eukaryotic transcripts with 3' poly-A tail and 5' cap structure. In this study, an improved RACE strategy was developed which allows both 5' and 3' RACE of paramyxovirus genomic RNA using the same set of common molecular biology reagents without having to rely on expensive RACE kits. Mapping of RNA genome terminal sequences is an essential part of characterizing novel paramyxoviruses since these sequences contain important signals for genome replication and transcription, and are important molecular markers for studying virus evolution. The usefulness of this strategy was demonstrated by rapid characterization of both genome ends for a novel paramyxovirus recently isolated from human kidney primary cells. The RACE strategy described in this paper is simple, cost-effective and can be used to map genome ends of any RNA viruses.

  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. The cDNA sequences encoding two components of the polymeric fraction of the intracellular hemoglobin of Glycera dibranchiata.

    Science.gov (United States)

    Zafar, R S; Chow, L H; Stern, M S; Scully, J S; Sharma, P R; Vinogradov, S N; Walz, D A

    1990-12-15

    The intracellular hemoglobin of the polychaete Glycera dibranchiata consists of several components, some of which self-associate into a "polymeric" fraction. The cDNA library constructed from the poly(A+) mRNA of Glycera erythrocytes (Simons, P. C., and Satterlee, J. D. (1989) Biochemistry 28, 8525-8530) was screened with two oligodeoxynucleotide probes corresponding to the amino acid sequences MEEKVP and AMNSKV. Each of the two probes identified a full-length positive insert; these were sequenced using the dideoxynucleotide chain termination method. One clone was 630 bases long and contained 36 bases of 5'-untranslated RNA, a reading frame of 441 bases coding for the 147 amino acids of globin P2 including the residues MEEKVP, and a 3'-untranslated region of 153 bases. The other clone was 540 bases long and contained 24 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for globin P3 including the residues AMNSKV, and a 3'-untranslated region of 75 bases. The inferred amino acid sequences of the two globins were in agreement with the partial amino acid sequences obtained by chemical methods. The P2 and P3 globin sequences, together with the previously determined P1 sequence of a complete insert and partial sequences P4, P5, and P6 obtained from partial inserts (Zafar, R. S., Chow, L. H., Stern, M. S., Vinogradov, S. N., and Walz, D. A. (1990) Biochim. Biophys. Acta, in press) suggest that there are at least six components in the polymeric fraction of Glycera hemoglobin, which is in agreement with the results of polyacrylamide gel electrophoresis in Tris/glycine buffer, pH 8.3, 6 M urea. Nothern and dot blot analyses of Glycera erythrocyte poly(A+) mRNA using the foregoing two cDNA probes clearly demonstrated the presence of mature messages encoding both types of globins. Comparison of the polymeric sequences P1, P2, and P3 with the "monomeric" globins M-II and M-IV using the alignment and templates of Bashford et al. (Bashford, D., Chothia, C

  14. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    赵勇[1; 余龙[2; 高洁[3; 付强[4; 华益民[5; 张宏来[6; 赵寿元[7

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas

  15. [Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

    Science.gov (United States)

    Ramazanova, A S; Fil'kin, S Iu; Starkov, V G; Utkin, Iu N

    2011-01-01

    Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

  16. An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries

    Science.gov (United States)

    Habermann, Bianca; Bebin, Anne-Gaelle; Herklotz, Stephan; Volkmer, Michael; Eckelt, Kay; Pehlke, Kerstin; Epperlein, Hans Henning; Schackert, Hans Konrad; Wiebe, Glenis; Tanaka, Elly M

    2004-01-01

    Background The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. Results Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. Conclusions Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. PMID:15345051

  17. The venom gland transcriptome of Latrodectus tredecimguttatus revealed by deep sequencing and cDNA library analysis.

    Directory of Open Access Journals (Sweden)

    Quanze He

    Full Text Available Latrodectus tredecimguttatus, commonly known as black widow spider, is well known for its dangerous bite. Although its venom has been characterized extensively, some fundamental questions about its molecular composition remain unanswered. The limited transcriptome and genome data available prevent further understanding of spider venom at the molecular level. In the present study, we combined next-generation sequencing and conventional DNA sequencing to construct a venom gland transcriptome of the spider L. tredecimguttatus, which resulted in the identification of 9,666 and 480 high-confidence proteins among 34,334 de novo sequences and 1,024 cDNA sequences, respectively, by assembly, translation, filtering, quantification and annotation. Extensive functional analyses of these proteins indicated that mRNAs involved in RNA transport and spliceosome, protein translation, processing and transport were highly enriched in the venom gland, which is consistent with the specific function of venom glands, namely the production of toxins. Furthermore, we identified 146 toxin-like proteins forming 12 families, including 6 new families in this spider in which α-LTX-Lt1a family2 is firstly identified as a subfamily of α-LTX-Lt1a family. The toxins were classified according to their bioactivities into five categories that functioned in a coordinate way. Few ion channels were expressed in venom gland cells, suggesting a possible mechanism of protection from the attack of their own toxins. The present study provides a gland transcriptome profile and extends our understanding of the toxinome of spiders and coordination mechanism for toxin production in protein expression quantity.

  18. Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines.

    Science.gov (United States)

    Doenecke, A; Winnacker, E L; Hallek, M

    1997-10-01

    The isolation of rearranged immunoglobulin (Ig) variable region (V) genes is usually performed by PCR with consensus primers binding to conserved regions within the V sequences. However, the isolation of Ig genes by this method is hampered in 15-35% by technical difficulties, mostly mismatches of oligonucleotide primers to V sequences. In order to obtain DNA sequences from V heavy chain (VH) genes which could not be amplified with consensus primers, we used a modified PCR technique, the rapid amplification of cDNA ends (RACE) PCR in combination with new heavy chain constant region primers for the isolation of human and murine VH genes. In comparison, consensus primer PCR with different sets of previously published oligonucleotide primers was used. Both methods were applied to isolate VH genes from murine B cell lymphoma (A20 and BCL1), myeloma (NS1) and hybridoma (SP6) cell lines and from freshly isolated human chronic lymphocytic leukemia and lymphoma cells. RACE PCR allowed the amplification and subsequent cloning of the complete VH gene in all cases. In contrast, consensus primer PCR failed to isolate the VH sequence of the murine A20 cell line; this was explained by a mismatch of consensus primers with VH sequences. When both PCR methods amplified VH sequences, the DNA sequences obtained were identical. Taken together, RACE PCR represents a reliable and versatile method for the isolation of VH genes from human and murine lymphoma cells, in particular if consensus primer PCR fails.

  19. Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase. A new family of fungal peroxidases.

    Science.gov (United States)

    Baunsgaard, L; Dalbøge, H; Houen, G; Rasmussen, E M; Welinder, K G

    1993-04-01

    Sequence analysis and cDNA cloning of Coprinus peroxidase (CIP) were undertaken to expand the understanding of the relationships of structure, function and molecular genetics of the secretory heme peroxidases from fungi and plants. Amino acid sequencing of Coprinus macrorhizus peroxidase, and cDNA sequencing of Coprinus cinereus peroxidase showed that the mature proteins are identical in amino acid sequence, 343 residues in size and preceded by a 20-residue signal peptide. Their likely identity to peroxidase from Arthromyces ramosus is discussed. CIP has an 8-residue, glycine-rich N-terminal extension blocked with a pyroglutamate residue which is absent in other fungal peroxidases. The presence of pyroglutamate, formed by cyclization of glutamine, and the finding of a minor fraction of a variant form lacking the N-terminal residue, indicate that signal peptidase cleavage is followed by further enzymic processing. CIP is 40-45% identical in amino-acid sequence to 11 lignin peroxidases from four fungal species, and 42-43% identical to the two known Mn-peroxidases. Like these white-rot fungal peroxidases, CIP has an additional segment of approximately 40 residues at the C-terminus which is absent in plant peroxidases. Although CIP is much more similar to horseradish peroxidase (HRP C) in substrate specificity, specific activity and pH optimum than to white-rot fungal peroxidases, the sequences of CIP and HRP C showed only 18% identity. Hence, CIP qualifies as the first member of a new family of fungal peroxidases. The nine invariant residues present in all plant, fungal and bacterial heme peroxidases are also found in CIP. The present data support the hypothesis that only one chromosomal CIP gene exists. In contrast, a large number of secretory plant and fungal peroxidases are expressed from several peroxidase gene clusters. Analyses of three batches of CIP protein and of 49 CIP clones revealed the existence of only two highly similar alleles indicating less

  20. Cloning, characterization and heterologous expression of epoxide hydrolase-encoding cDNA sequences from yeasts belonging to the genera Rhodotorula and Rhodosporidium

    NARCIS (Netherlands)

    Visser, H.; Weijers, C.A.G.M.; Ooyen, van A.J.J.; Verdoes, J.C.

    2002-01-01

    Epoxide hydrolase-encoding cDNA sequences were isolated from the basidiomycetous yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031 in order to evaluate the molecular data and potential application of this type of enzymes. The deduce

  1. Gene expression of panaxydol-treated human melanoma cells using radioactive cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Joong Youn; Yu, Su Jin; Soh, Jeong Won; Kim, Meyoung Kon [College of Medicine, Korea Univ., Seoul (Korea, Republic of)

    2001-07-01

    Polyacetylenic alcohols derived from Panax ginseng have been studied to be an anticancer reagent previously. One of the Panax ginseng polyacetylenic alcohols, i.e., panaxydol, has been studied to possess an antiproliferative effect on human melanoma cell line (SK-MEL-1). In ths study, radioactive cDNA microarrays enabled an efficient approach to analyze the pattern of gene expression (3.194 genes in a total) simultaneously. The bioinformatics selection of human cDNAs, which is specifically designed for immunology, apoptosis and signal transduction, were arrayed on nylon membranes. Using with {sup 33}P labeled probes, this method provided highly sensitive gene expression profiles of our interest including apoptosis, cell proliferation, cell cycle, and signal transduction. Gene expression profiles were also classified into several categories in accordance with the duration of panaxydol treatment. Consequently, the gene profiles of our interest were significantly up (199 genes, > 2.0 of Z-ratio) or down-(196 genes, < 2.0 of Z-ratio) regulated in panaxydol-treated human melanoma cells.

  2. Nucleotide sequence of a cDNA clone encoding a major allergenic protein in rice seeds. Homology of the deduced amino acid sequence with members of alpha-amylase/trypsin inhibitor family.

    Science.gov (United States)

    Izumi, H; Adachi, T; Fujii, N; Matsuda, T; Nakamura, R; Tanaka, K; Urisu, A; Kurosawa, Y

    1992-05-18

    A cDNA clone of rice major allergenic protein (RAP) was isolated from a cDNA library of maturing rice seeds. The cDNA had an open reading frame (486 nucleotides) which coded a 162 amino acid residue polypeptide comprising a 27-residue signal peptide and a 135-residue mature protein of M(r) 14,764. The deduced amino acid sequence of RAP showed a considerable similarity to barley trypsin inhibitor [1983, J. Biol. Chem. 258, 7998-8003] and wheat alpha-amylase inhibitor [1981, Phytochemistry 20, 1781-1784].

  3. 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

  4. Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): application to sequencing 6.5 kb genome segment of hantavirus strain B-1.

    Science.gov (United States)

    Isegawa, Y; Sheng, J; Sokawa, Y; Yamanishi, K; Nakagomi, O; Ueda, S

    1992-12-01

    A method, referred to as cassette-ligation mediated polymerase chain reaction (PCR), has been developed to permit selective and specific amplification of cDNA sequence from total cellular RNA. This technique comprises (i) digestion of cDNA with multiple restriction enzymes, (ii) ligation of cleavage products to double-stranded DNA cassettes possessing a corresponding restriction site and (iii) amplification of cassette-ligated restriction fragments containing a short, known sequence (but not all the other ligation products) by PCR using the specific and cassette primers; the specific primer is designed to prime synthesis from the known sequence of the cDNA whereas the cassette primer anneals to one strand of the cassette. Sequencing from the cassette primer provides information to design a new primer for the next walking step. The amplified cDNA fragments are often larger than the maximum DNA fragments (500-600 bp) that can be sequenced without the need of synthesizing internal sequencing primer. Each of such large cDNA fragments is dissected into smaller DNA fragments by repeating cassette-ligation mediated PCR exploiting different restriction sites and different sets of cassette primers. This dissection process reduces the number of specific primers to a minimum, thereby increasing the speed of sequencing and minimizing the overall cost. We have successfully applied this cDNA walking and sequencing by the cassette-ligation mediated PCR to the sequencing of an entire 6.5 kb genome segment of hantavirus strain B-1.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Preparation of a subtractive cDNA library enriched in cDNAs which expressed at a high level in cultured senescent human fibroblasts.

    Science.gov (United States)

    Tahara, H; Hara, E; Tsuyama, N; Oda, K; Ide, T

    1994-03-30

    Subtracted cDNA library was prepared by subtracting [cDNA from young growing SV40-transformed human fibroblasts] from [cDNA from growing SV40-transformed fibroblasts in extended lifespan]. Isolated cDNA clones which expressed at high level in life-extended transformed cells also expressed at high level in normal senescent fibroblasts but did at low level in growing and growth-arrested young cells. Neither fibronectin nor procollagen cDNA was isolated. This cDNA library is useful for isolation of senescent-specific cDNA species which express at high level in normal senescent cells but at low level in growing and growth-arrested young cells, avoiding growth-arrest-specific cDNAs.

  6. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein.

    OpenAIRE

    Gray, P W; Barrett, K; Chantry, D; Turner, M.; Feldmann, M

    1990-01-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNF alpha with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surf...

  7. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. The GGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest that GGPPS may be one of the candidate genes for prostate cancer.

  8. Construction of a cDNA library and preliminary analysis of expressed sequence tags in Piper hainanense.

    Science.gov (United States)

    Fan, R; Ling, P; Hao, C Y; Li, F P; Huang, L F; Wu, B D; Wu, H S

    2015-10-19

    Black pepper is a perennial climbing vine. It is widely cultivated because its berries can be utilized not only as a spice in food but also for medicinal use. This study aimed to construct a standardized, high-quality cDNA library to facilitated identification of new Piper hainanense transcripts. For this, 262 unigenes were used to generate raw reads. The average length of these 262 unigenes was 774.8 bp. Of these, 94 genes (35.9%) were newly identified, according to the NCBI protein database. Thus, identification of new genes may broaden the molecular knowledge of P. hainanense on the basis of Clusters of Orthologous Groups and Gene Ontology categories. In addition, certain basic genes linked to physiological processes, which can contribute to disease resistance and thereby to the breeding of black pepper. A total of 26 unigenes were found to be SSR markers. Dinucleotide SSR was the main repeat motif, accounting for 61.54%, followed by trinucleotide SSR (23.07%). Eight primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among twenty-one piper germplasm. These results present a novel sequence information of P. hainanense, which can serve as the foundation for further genetic research on this species.

  9. Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

    Energy Technology Data Exchange (ETDEWEB)

    Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M; Butterfield, Y; Stott, J; Barber, S; Babakaiff, R; Matsuo, C; Wong, D; Yang, G; Smailus, D; Brown-John, M; Mayo, M; Beland, J; Gibson, S; Olson, T; Tsai, M; Featherstone, R; Chand, S; Siddiqui, A; Jang, W; Lee, E; Klein, S; Prange, C; Myers, R M; Green, E D; Wagner, L; Gerhard, D; Marra, M; Jones, S M; Holt, R

    2005-10-31

    Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.

  10. Cathepsin B from the white shrimp Litopenaeus vannamei: cDNA sequence analysis, tissues-specific expression and biological activity.

    Science.gov (United States)

    Stephens, A; Rojo, L; Araujo-Bernal, S; Garcia-Carreño, F; Muhlia-Almazan, A

    2012-01-01

    Cathepsin B is a cystein proteinase scarcely studied in crustaceans. Its function has not been clearly described in shrimp species belonging to the sub-order Dendrobranchiata, which includes the white shrimp Litopenaeus vannamei and other species from the Penaeidae family. Studies on vertebrates suggest that these lysosomal enzymes intracellularly hydrolize protein, as other cystein proteinases. However, the expression of the gene encoding the shrimp cathepsin B in the midgut gland was affected by starvation in a similar way as other digestive proteinases which extracellularly hydrolyze food protein. In this study the white shrimp L. vannamei cathepsin B (LvCathB) cDNA was sequenced, and characterized. Its gene expression was evaluated in various shrimp tissues, and changes in the mRNA amounts were compared with those observed on other digestive proteinases from the midgut gland during starvation. By using qRT-PCR it was found that LvCathB is expressed in most shrimp tissues except in pleopods and eye stalk. Changes on LvCathB mRNA during starvation suggest that the enzyme participates during intracellular protein hydrolysis but also, after food ingestion, it participates in hydrolyzing food proteins extracellularly as confirmed by the high activity levels we found in the gastric juice and midgut gland of the white shrimp.

  11. Sequence and characterization of cDNA encoding the motilin precursor from chicken, dog, cow and horse. Evidence of mosaic evolution in prepromotilin.

    Science.gov (United States)

    Huang, Z; Depoortere, I; De Clercq, P; Peeters, T

    1999-11-15

    Motilin is involved in the regulation of the fasting motility pattern in man and in dog, but may have a different role in other species. Immunoreactive motilin has been demonstrated in several species, but the sequence is mostly unknown. The aim of this study was to isolate and sequence the cDNA encoding the motilin precursor from several mammalian species and from chicken. Total RNA was isolated from the duodenal mucosa of the chicken, dog, cow and horse. In each case single stranded cDNA was synthesized. Motilin cDNA fragments were amplified by PCR, ligated into a plasmid and cloned. Clones which were positive after screening with an appropriate (32)P-labeled probe were sequenced. The 5'- and 3'-ends were determined by the rapid amplification of cDNA ends (RACE) method. Analysis of the cDNAs revealed an open reading frame coding for 115 (chicken and cow), or 117 (dog and horse) amino acids. It consists of a 25 amino acid signal peptide, motilin itself, and a 68 (chicken and cow) or 70 (dog and horse) amino acid motilin associated peptide (MAP). As in all motilin precursors already sequenced (man, monkey, pig and rabbit), an endoproteinase cleavage site is present at Lys(23)-Lys(24). Comparison of all known sequences shows considerable identity in amino acid and nucleotide sequence of the signal peptide and motilin. However, the MAPs differ not only in length but also, more strongly, in amino acid and nucleotide sequence. Our study demonstrates that the N- and C-terminal regions of the motilin precursor have evolved at different rates, which is evidence for 'mosaic evolution'.

  12. Expression of the human era cDNA in E.coli

    Institute of Scientific and Technical Information of China (English)

    WU Yuan-ming; CHEN Su-min; ZHANG Jun-jie; JI Zong-ling; LIU Hui-ping; CHEN Nan-chun

    2001-01-01

    To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 ( and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.

  13. Cynomolgus monkey testicular cDNAs for discovery of novel human genes in the human genome sequence

    Directory of Open Access Journals (Sweden)

    Terao Keiji

    2002-12-01

    Full Text Available Abstract Background In order to contribute to the establishment of a complete map of transcribed regions of the human genome, we constructed a testicular cDNA library for the cynomolgus monkey, and attempted to find novel transcripts for identification of their human homologues. Result The full-insert sequences of 512 cDNA clones were determined. Ultimately we found 302 non-redundant cDNAs carrying open reading frames of 300 bp-length or longer. Among them, 89 cDNAs were found not to be annotated previously in the Ensembl human database. After searching against the Ensembl mouse database, we also found 69 putative coding sequences have no homologous cDNAs in the annotated human and mouse genome sequences in Ensembl. We subsequently designed a DNA microarray including 396 non-redundant cDNAs (with and without open reading frames to examine the expression of the full-sequenced genes. With the testicular probe and a mixture of probes of 10 other tissues, 316 of 332 effective spots showed intense hybridized signals and 75 cDNAs were shown to be expressed very highly in the cynomolgus monkey testis, but not ubiquitously. Conclusions In this report, we determined 302 full-insert sequences of cynomolgus monkey cDNAs with enough length of open reading frames to discover novel transcripts as human homologues. Among 302 cDNA sequences, human homologues of 89 cDNAs have not been predicted in the annotated human genome sequence in the Ensembl. Additionally, we identified 75 dominantly expressed genes in testis among the full-sequenced clones by using a DNA microarray. Our cDNA clones and analytical results will be valuable resources for future functional genomic studies.

  14. Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

    Institute of Scientific and Technical Information of China (English)

    赵大中; 陈丹英; 杨橙; 翟中和

    2000-01-01

    Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Caucus carota L, and their partial amino acid sequences were determined. The homological analysis showed that the sequence from the 64 ku protein was highly homological to p-glucosidase, and that from the 55 ku protein had no significant homologue in GenBank. Using conservative sequence of animal IF proteins as primer, we cloned a cDNA fragment from Daucus carota L. Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves. Homological analysis revealed that it had moderate homology to a variety of a-helical proteins. Our results might shed more light on molecular characterization of IF existence in higher plant.

  15. Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Daucus carota L.,and their partial amino acid sequences were determined.The homological analysis showed that the sequence from the 64 ku protein was highly homological to b -glucosidase,and that from the 55 ku protein had no significant homologue in GenBank.Using conservative sequence of animal IF proteins as primer,we cloned a cDNA fragment from Daucus carota L.Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves.Homological analysis revealed that it had moderate homology to a variety of a -helical proteins.Our results might shed more light on molecular characterization of IF existence in higher plant.

  16. Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays

    OpenAIRE

    2004-01-01

    Abstract Background Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a s...

  17. Cloning and Sequence Analysis of Interleukin 10 (IL-10) Full-length cDNA from Cyprinus carpio L.

    Institute of Scientific and Technical Information of China (English)

    Xiangru FENG; Yilong CHEN; Xiao ZHAO; Wendong WANG; Junhui ZHANG; Zhenguo YANG SUN; Shengmei JIA; Qiang LU

    2012-01-01

    Abstract [Objective] This study aimed to obtain IL-IO (interleukin 10) full-length cD- NA of common carp (Cyprinus carpio L.) and conduct the sequence analysis. []~lethod] The differentially expressed cDNA fragment was obtained by DD-RTPCR (differential display RT-PCR). The cDNA library of peripheral blood leukocytes which were separated from common carp and stimulated by mitogen was screened with a probe labeled with DIG (digoxigenin). The IL-IO full-length cDNA was cloned from 0.8x104 pfu of recombinant phages, and the sequence analysis and homology com- parison were carried out. [Result] Sequence analysis indicated that the IL-IO full- length cDNA of common carp was 1 117 bp long, containing a.55 bp 5'-UTR, a 522 bp 3"-UTR, and a 540 bp open reading frame(ORF) encoding 179 amino acids. In addition, there were three mRNA instability motifs (ATTTA) in the 3"-untranslated region. The deduced protein sequence shared typical sequence features of the IL-IO family. Homology comparison indicated that the obtained sequence shared 89.1% homology with the carp IL-IO gene from GenBank. [Conclusion] This study laid foun- dation for further study of the expression manner, functional characteristic and regu- lation mechanism of IL-IO in vivo and the interaction mechanism in the inflammatory reaction and immune response.

  18. Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Y. Cui

    2008-05-01

    Full Text Available Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1 allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+, expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG, 267-277 (NYHAVNIVGYG and 284-303 (YWIVRNSWDTTWGDSGYGYF. Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%, an extended strand (17.13%, a ß turn (5.61%, and a random coil (43.30%. A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

  19. Presence of the delta-MSH sequence in a proopiomelanocortin cDNA cloned from the pituitary of the galeoid shark, Heterodontus portusjacksoni.

    Science.gov (United States)

    Dores, Robert M; Cameron, Erin; Lecaude, Stephanie; Danielson, Phillip B

    2003-08-01

    Since a fourth MSH sequence, delta-MSH, has been detected in the proopiomelanocortin (POMC) gene of a dogfish and a stingray, members of superorder Squalea (class Chondrichthyes), it is possible that this novel MSH sequence might be a feature common to the POMC genes of all modern sharks and rays. As an initial step towards addressing this question, a full-length POMC cDNA was cloned and sequenced from the pituitary of the Port Jackson shark, Heterodontus portusjacksoni. The Port Jackson shark represents one of the oldest lineages in superorder Galea, and this superorder together with superorder Squalea form infraclass Neoselachii (the extant sharks and rays). The Port Jackson shark POMC cDNA has an open reading frame that is 1032 nucleotides in length and encodes the deduced amino acids sequences for beta-endorphin, ACTH/alpha-MSH, beta-MSH, gamma-MSH, and delta-MSH. Port Jackson shark delta-MSH has 83% primary sequence identity with dogfish and stingray delta-MSH, and it appears that the delta-MSH sequence may have been the result of an internal domain duplication and reinsertion of the beta-MSH sequence. The presence of the delta-MSH sequence in the POMC genes of representatives of both superorders of infraclass Neoselachii would indicate that the delta-MSH sequence must have been present in the ancestral euselachian shark that gave rise to the neoselachian radiation.

  20. Cloning and expression of a cDNA covering the complete coding region of the P32 subunit of human pre-mRNA splicing factor SF2

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Rasmussen, H H

    1993-01-01

    We have cloned and expressed a cDNA encoding the 32-kDa subunit (P32) of the human pre-mRNA splicing factor, SF2. This cDNA extends beyond the 5'-end of a previously reported cDNA [Krainer et al., Cell 66 (1991) 383-394]. Importantly, our fragment includes an ATG start codon which was absent from...

  1. Cloning and Sequence Analysis of the Full-length cDNA of a Novel yp05 Gene Associated With Citrinin Production in Monascus aurantiacus

    Institute of Scientific and Technical Information of China (English)

    YON-GHUA XIONG; YANG XU; WEI-HUA LAI; YAN-PIN LI; HUA WEI

    2007-01-01

    Objective To obtain the full-length cDNA of a novel gene (named yp05) associated with citrinin production-related genes in Monascus aurantiacus. Methods Total RNA was extracted from mycelium, 3' and 5' cDNA end of yp05 gene was amplified using smartTM trace cDNA amplification kit, and the full-length cDNA of a novel gene (named yp05) was obtained from the electronic assembly of 3'-RACE and 5'- RACE products. Results This yp05 gene was 787 bp including a 597 bp open reading frame (ORF) and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription of yp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced. Conclusion The transcription of yp05 gene belongs to differential expression genes of citrinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.

  2. 虹鳟 Ndufb2基因全长 cDNA 序列的克隆与分析%Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss

    Institute of Scientific and Technical Information of China (English)

    王家庆; 边佳; 李代宗; 马爽; 王亮; 那广宁

    2013-01-01

    Summary Rainbow trout belongs to Salmonidae aerobic fish,and it is necessary for high dissolved oxygen content of living water environment.If the dissolved oxygen content of living water is less than 5 mg/L,it will cause the increase of respiratory rate,which is the so-called“aquaculture floating head”phenomenon.Because the fish lives in hypoxia environment and the 90% oxygen consumption is in the mitochondria,the transmission mechanism in composition and electronic respiratory chain may be different from the terrestrial animal.At the mitochondrial inner membrane,electrons from NADH and succinate pass through the electron transport chain to oxygen,which is reduced to water.Complex I is one of the main sites at which premature electron leakage to oxygen occurs,thus being one of the main sites of production of harmful superoxide.The first isolation of mitochondrial complex I since 1 961,its composition and structure have had a primary understanding,but the specific mechanism of its participation in respiration,especially the function of each subunit is not clear.The protein encoded by Ndufb2 gene is a subunit of the multisubunit NADH:ubiquinone oxidoreductase(complex I). Mammalian complex I is composed of 45 different subunits.This protein has NADH dehydrogenase activity and oxidoreductase activity.It plays an important role in transferring electrons from NADH to the respiratory chain. Reverse transcription PCR(RT-PCR) and rapid amplification of cDNA ends(RACE)methods were used for the isolation of the whole cDNA of Ndufb2 gene from brain of Oncorhynchus mykiss .The assembly taskes of 3' and 5'-RACE sequence were completed by DNAman program.A pair of gene specific primers were designed to amplify the full-length cDNA sequence.ClustalX 1.81 and MEGA 3.0 software were used to calculate the amino acid sequence differences,and then the phylogenetic relationships of rainbow trout Ndufb2 gene sequence with other species were analyzed.Protein phosphorylation sites and

  3. 中华蜜蜂工蜂 cDNA 文库的构建及ESTs 测序分析%Construction of cDNA libraries and ESTs sequencing of Apis cerana cerana workers

    Institute of Scientific and Technical Information of China (English)

    张卫星; 郗学鹏; 秦明; 王帅; 刘春蕾; 王红芳; 胥保华

    2016-01-01

    Objectives] To build a cDNA library to improve understanding of how honey bee workers respond to adverse conditions and analyze the quality of the resultant library. [Methods] A cDNA library of Apis cerana cerana was constructed using the SMART technique. [Results] The library’s capacity was 3.6×106 cfu/mL, the recombination rate was 97% and the average length of inserts was approximately 1 000 bp. 306 ESTs were generated by ESTs sequencing. Additionally, 234 non-repetitive sequences were formed, including 207 singletons and 27 contigs after initial assembly. Using Blastx to query, compare and annotate these sequences with those in GenBank, revealed that 141 sequences could be assigned putative functions because they were homologous to known genes. Other sequences had no obvious homology, which suggests there is potential for the discovery of new functional genes. [Conclusion] The construction of a cDNA library has important benefits for cloning, screening and gene function research in Apis cerana cerana.%【目的】为了解中华蜜蜂 Apis cerana cerana 工蜂的抗逆性,构建了中华蜜蜂工蜂的 cDNA 文库,并对文库质量进行分析。【方法】本研究利用 SMART 技术构建了中华蜜蜂工蜂的全长 cDNA 文库。【结果】文库库容为3.6×106 cfu/mL,文库重组率为97%,插入片段长度多数分布在1000 bp 左右。挑取 cDNA克隆进行 EST 测序,共进行了306个成功反应,软件拼接共得到234个单基因簇(Unigene),其中包括207个单拷贝(Singletons)序列及27个重叠群(Contigs)。使用 Blastx 将这些序列同 GenBank 等数据库进行查询、比对和注释,结果显示141条序列有相关同源性,其他序列没有明显的同源性,这也为我们发现新功能基因提供了可靠依据。【结论】此文库的构建在中华蜜蜂功能基因的分离、克隆、筛选以及基因功能研究等方面具有重要作用。

  4. 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.

  5. Efficient expression of human factor Ⅸ cDNA in livermediated by hydrodynamics-based plasmid administration

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringer's solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3-10 d. These results suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.

  6. 生物信息学在克隆去势SD大鼠cDNA消减差异片段SCn4的全长cDNA及其序列分析中的应用%Electronic cDNA library screening a full-length cDNA for human rectum adenocarcinoma SCn4 fragment of suppression subtractive hybridization and sequence anglicizing

    Institute of Scientific and Technical Information of China (English)

    王松; 沈霖; 陈国庆; 金丽

    2006-01-01

    从去势SD大鼠cDNA消减文库中抽取消减差异未知基因的cDNA部分序列,利用生物信息学的方法延伸表达序列标签(Expression Sequence Tag,EST)序列,以获得基因的部分乃至全长cDNA序列,避免或部分避免构建与筛选cDNA文库等烦琐的实验室工作.该策略体现了后基因组生物信息学的重要性,它在克隆确定致病新基因方面提供了新思路、新方法.本研究利用生物信息学"电子"克隆了去势SD大鼠cDNA消减文库中的差异片段SCn4的全长cDNA并对其可能的全基因序列作了预测.

  7. Estabishment of A Human Liver Cancer Cell Line Transfected with IL-2 cDNA and Its Biologic Activity

    Institute of Scientific and Technical Information of China (English)

    孙跃明; 王学浩; 杜竞辉

    2001-01-01

    Objective To obtain IL-2 gene transfected human liver cancer cells and study IL-2 expression and its biologic activity in vivo. Methods Human liver cancer cells SMMC-7721 were cocultured with recombinant retroviral vector LNC-IL-2,and screening was performed in G418 medium.The exogenous IL-2 cDNA at the DNA,RNA,and protein levels were determined by using dot hybridization,PR-PCR and MTT methods respectively.The tumorigenesis and antitumorigenesis of the screened liver cancer cell with subcutaneous injection in nude mice were observed. Results and Conclusion The IL-2 cDNA was successfully integrated into SMMC-7721 cell genomic DNA and continuously expressed for more than 88 days.Subcutaneous vaccination of the nude mice with transfected cells revealed an obvious suppression of its tumorigenicity,and could induce antitumor activity in vivo.

  8. cDNA cloning,sequence analysis,and recombinant expression of akitonin beta,a C-type lectin-like protein from Agkistrodon acutus

    Institute of Scientific and Technical Information of China (English)

    Xiang-dong ZHA; Jing LIU; Kang-sen XU

    2004-01-01

    AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structurefunction relationships and to achieve its recombinant production. METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template.The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBADTOPO as vector and transformed into E. coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin β was found and accepted by GenBank (accession number AF387100). Akitonin β consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin β expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro. CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.

  9. Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 (HSC70) in Fenneropenaeus chinensis

    Institute of Scientific and Technical Information of China (English)

    JIAO Chuanzhen; WANG Zaizhao; LI Fuhua; ZHANG Chengsong; XIANG Jianhai

    2004-01-01

    The cDNA encoding hsc70 of Chinese shrimp Fenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5′- and 3′-untranslated region(5′- and 3′-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1-547 bp, but they did not exist in the region of 548-2090 bp of hsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 from Oncorhynchus mykiss and HSC70 from Homo sapiens. It also exhibited 85.8% similarity with HSP70 from Mus musculu and 85.4% with HSC70 from Manduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills in Fenneropenaeus chinensis. No difference could be detected on hsc70 mRNA level in muscle between heat-shocked and control animals.

  10. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. (Vanderbilt Univ., Nashville, TN (United States))

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  11. Evolutionarily conserved sequences on human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Frazer, Kelly A.; Sheehan, John B.; Stokowski, Renee P.; Chen, Xiyin; Hosseini, Roya; Cheng, Jan-Fang; Fodor, Stephen P.A.; Cox, David R.; Patil, Nila

    2001-09-01

    Comparison of human sequences with the DNA of other mammals is an excellent means of identifying functional elements in the human genome. Here we describe the utility of high-density oligonucleotide arrays as a rapid approach for comparing human sequences with the DNA of multiple species whose sequences are not presently available. High-density arrays representing approximately 22.5 Mb of nonrepetitive human chromosome 21 sequence were synthesized and then hybridized with mouse and dog DNA to identify sequences conserved between humans and mice (human-mouse elements) and between humans and dogs (human-dog elements). Our data show that sequence comparison of multiple species provides a powerful empiric method for identifying actively conserved elements in the human genome. A large fraction of these evolutionarily conserved elements are present in regions on chromosome 21 that do not encode known genes.

  12. Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

    Science.gov (United States)

    Ding, Ling; Azam, Mark; Lin, Yu-Huei; Sheridan, James; Wei, Shuanghong; Gupta, Gigi; Singh, Rakesh K; Pauling, Michelle H; Chu, Waihei; Tran, Antares; Yu, Nai-Xuan; Hu, Jiefeng; Wang, Wei; Long, Hao; Xiang, Dong; Zhu, Li; Hua, Shao-Bing

    2010-10-01

    We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.

  13. Crustacean hyperglycemic hormones of two cold water crab species, Chionoecetes opilio and C. japonicus: isolation of cDNA sequences and localization of CHH neuropeptide in eyestalk ganglia.

    Science.gov (United States)

    Chung, J Sook; Ahn, I S; Yu, O H; Kim, D S

    2015-04-01

    Crustacean hyperglycemic hormone (CHH) is primarily known for its prototypical function in hyperglycemia which is induced by the release of CHH. The CHH release takes place as an adaptive response to the energy demands of the animals experiencing stressful environmental, physiological or behavioral conditions. Although >63 decapod CHH nucleotide sequences are known (GenBank), the majority of them is garnered from the species inhabiting shallow and warm water. In order to understand the adaptive role of CHH in Chionoecetes opilio and Chionoecetes japonicus inhabiting deep water environments, we first aimed for the isolation of the full-length cDNA sequence of CHH from the eyestalk ganglia of C. opilio (ChoCHH) and C. japonicus (ChjCHH) using degenerate PCR and 5' and 3' RACE. Cho- and ChjCHH cDNA sequences are identical in 5' UTR and ORF with 100% sequence identity of the putative 138aa of preproCHHs. The length of 3' UTR ChjCHH cDNA sequence is 39 nucleotides shorter than that of ChoCHH. This is the first report in decapod crustaceans that two different species have the identical sequence of CHH. ChoCHH expression increases during embryogenesis of C. opilio and is significantly higher in adult males and females. C. japonicus males have slightly higher ChjCHH expression than C. opilio males, but no statistical difference. In both species, the immunostaining intensity of CHH is stronger in the sinus gland than that of X-organ cells. Future studies will enable us to gain better understanding of the comparative metabolic physiology and endocrinology of cold, deep water species of Chionoecetes spp. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Screening target specificity of siRNAs by rapid amplification of cDNA ends (RACE) for non-sequenced species.

    Science.gov (United States)

    Sabirzhanov, Boris; Sabirzhanova, Inna B; Keifer, Joyce

    2011-05-01

    RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propose an approach for primary screening dsRNAs for potential off-target effects by using rapid amplification of cDNA ends. This method can be especially useful for model systems using species that have limited availability of sequence data.

  15. Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

    Science.gov (United States)

    Moguilevsky, N; Varsalona, F; Noyer, M; Gillard, M; Guillaume, J P; Garcia, L; Szpirer, C; Szpirer, J; Bollen, A

    1994-09-01

    A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

  16. Mass spectrometry-based cDNA profiling as a potential tool for human body fluid identification.

    Science.gov (United States)

    Donfack, Joseph; Wiley, Anissa

    2015-05-01

    Several mRNA markers have been exhaustively evaluated for the identification of human venous blood, saliva, and semen in forensic genetics. As new candidate human body fluid specific markers are discovered, evaluated, and reported in the scientific literature, there is an increasing trend toward determining the ideal markers for cDNA profiling of body fluids of forensic interest. However, it has not been determined which molecular genetics-based technique(s) should be utilized to assess the performance of these markers. In recent years, only a few confirmatory, mRNA/cDNA-based methods have been evaluated for applications in body fluid identification. The most frequently described methods tested to date include quantitative polymerase chain reaction (qPCR) and capillary electrophoresis (CE). However these methods, in particular qPCR, often favor narrow multiplex PCR due to the availability of a limited number of fluorescent dyes/tags. In an attempt to address this technological constraint, this study explored matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for human body fluid identification via cDNA profiling of venous blood, saliva, and semen. Using cDNA samples at 20pg input phosphoglycerate kinase 1 (PGK1) amounts, body fluid specific markers for the candidate genes were amplified in their corresponding body fluid (i.e., venous blood, saliva, or semen) and absent in the remaining two (100% specificity). The results of this study provide an initial indication that MALDI-TOF MS is a potential fluorescent dye-free alternative method for body fluid identification in forensic casework. However, the inherent issues of low amounts of mRNA, and the damage caused to mRNA by environmental exposures, extraction processes, and storage conditions are important factors that significantly hinder the implementation of cDNA profiling into forensic casework. Published by Elsevier Ireland Ltd.

  17. Studies on the isolation, structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal-specific cDNA, pG2

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Teisner, Børge; Højrup, Peter

    1993-01-01

    , prolines and amino acids (aa) with acidic side-chains indicating that fetal antigen 1 is a compactly folded, strongly hydrophilic molecule. The N-terminal amino acid sequence (37 aa) revealed no homology to other known protein sequences, implying that fetal antigen 1 is a 'novel' human protein. When the aa...... is encoded by the mRNA defined by the cDNA clone pG2, but definitive sequencing and expression studies of this mRNA have not been achieved. Udgivelsesdato: 1993-Apr...

  18. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  19. Gene expression profiling in human peripheral blood mononuclear cells using high-density filter-based cDNA microarrays.

    Science.gov (United States)

    Walker, J; Rigley, K

    2000-05-26

    Microarray technology has provided the ability to analyse the expression profiles for thousands of genes in parallel. The need for highly specialised equipment to use certain types of microarrays has restricted the application of this technology to a small number of dedicated laboratories. High-density filter-based cDNA microarrays provide a low-cost option for performing high-throughput gene expression analysis. We have used a model system in which filter-based cDNA microarrays representing over 4000 known human genes were used to monitor the kinetics of gene expression in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagluttinin (PHA). Using software-based cluster analysis, we identified 104 genes that altered in expression levels in response to PHA stimulation of PBMCs and showed that there was a considerable overlap between genes with similar temporal expression profiles and similar functional roles. Comparison of microarray quantitation with quantitative PCR showed almost identical expression profiles for a number of genes. Coupled with the fact that our findings are in agreement with a large number of independent observations, we conclude that the use of filter-based cDNA microarrays is a valid and accurate method for high-throughput gene expression profiling.

  20. cDNA sequence, gene structure, and cholinesterase-like domains of an esterase from Caenorhabditis elegans mapped to chromosome V.

    Science.gov (United States)

    Fedon, Y; Cousin, X; Toutant, J P; Thierry-Mieg, D; Arpagaus, M

    1993-01-01

    The structure of an esterase gene from Caenorhabditis elegans has been determined by comparison of the sequences in genomic and cDNA clones. The gene was mapped close to the center of chromosome V (1.7 centimorgans to the left of dpy-11) and is therefore distinct from the gut esterase gene ges-1. It possessed 7 short introns. The 5' splice site of intron 3 presented the sequence GC instead of the usual GT that was found in the other six introns. The cDNA was trans-spliced with the short leader SL1. The open reading frame indicated that a protein of 557 aminoacids was encoded. The deduced aminoacid sequence did not present a signal peptide at the N-terminal but a potential N-myristoylation site (GXXXS) provided that the initiator methionine was removed. This protein should therefore remain intracellular. Comparison of this C. elegans sequence to other protein sequences in databases, as well as the analysis of the secondary structure in the protein showed that it belongs to the subgroup of esterases in the alpha/beta hydrolase fold family.

  1. Sequence determination of cDNA clones of transcripts from the tumor-associated region of the Marek's disease virus genome.

    Science.gov (United States)

    Iwata, A; Ueda, S; Ishihama, A; Hirai, K

    1992-04-01

    The number of 132-bp tandem direct repeats within the long inverted repeat region of the Marek's disease virus type 1 (MDV1) genome increases concomitantly with the loss of oncogenicity during serial passages in cultured cells. Twelve clones carrying the 132-bp sequence were isolated from a cDNA library constructed from chicken embryo fibroblasts infected with the MDV1 Md5 strain. Through sequence analysis of a cDNA clone and primer extension analysis, the corresponding mRNA was found to be a linear transcript which included the two 132-bp tandem direct repeats. Two open reading frames were found in this transcript. One had a week homology with v-fms. The other should increase its size concomitantly with expansion of the 132-bp tandem direct repeat. PCR analysis of both cDNA clones and RNA gave amplified products which were as large as that produced from the genomic clone, indicating that a majority of mRNA from this region is composed of unspliced transcripts.

  2. Detection of germline mutations of hMLH1 and hMSH2 based on cDNA sequencing in China

    Institute of Scientific and Technical Information of China (English)

    Chao-Fu Wang; Xiao-Yan Zhou; Tai-Ming Zhang; Meng-Hong Sun; Da-Ren Shi

    2005-01-01

    AIM: To detect the germline mutations of hMLH1 and hMSH2based on mRNA sequencing to identify hereditary nonpolyposis colorectal cancer (HNPCC) families.METHODS: Total RNA was extracted from peripheral blood of 14 members from 12 different families fulfilling Amsterdam criteria Ⅱ. mRNA of hMLH1 and hMSH2 was reversed with special primers and heat-resistant reverse transcriptase. cDNA was amplified with expand long template PCR and cDNA sequendng analysis was followed.RESULT: Seven germline mutations were found in 6families (6/12, 50%), in 4 hMLH1 and 3 hMSH2 mutations (4/12, 33.3%); (3/12, 25%). The mutation types involved 4 missense, 1 silent and 1 frame shift mutations as well as 1 mutation in the non-coding area. Four out of the seven mutations have not been reported previously. The 4 hMLH1mutations were distributed in exons 8, 12, 16, and 19. The 3hMSH2 mutations were distributed in exons 1 and 2. Six out of the 7 mutations were pathological, which were distributed in 5 HNPCC families.CONCLUSION: Germline mutations of hMLH1 and hMSH2 can be found based on cDNA sequencing so as to identify HNPCC family, which is highly sensitive and has the advantages of cost and time saving.

  3. Global Identification of Significantly Expressed Genes in Developing Endosperm of Rice by Expression Sequence Tags and cDNA Array Approaches

    Institute of Scientific and Technical Information of China (English)

    Qichao Tu; Haitao Dong; Haigen Yao; Yongqi Fang; Cheng'en Dai; Hongmei Luo; Jian Yao; Dong Zhao; Debao Li

    2008-01-01

    Rice endosperm plays a very important role in seedling germination and determines the qualities of fice grain.Although studies on specific gene categories in endosperm have been carried out,global view of gene expression at a transcription level in rice endosperm is still limited.To gain a better understanding of the global and tissue-specific gene expression profiles in rice endosperm,a cDNA library from rice endosperm of immature seeds was sequenced.A cDNA array was constructed based on the tentative unique transcripts derived from expression sequence tag (EST) assembling results and then hybridized with cONAs from five different tissues or organs including endosperm,embryo,leaf,stem and root of rice.Significant redundancy was found for genes encoding prolamin,glutelin,allergen,and starch synthesis proteins,accounting for~34% of the total ESTs obtained.The cDNA array revealed 87 significantly expressed genes in endosperm compared with the other four organs or tissues.These genes included 13 prolamin family proteins,17 glutelin family proteins,12 binding proteins,nine catalytic proteins and four ribosomal proteins,indicating a complicated biological processing in rice endosperm.In addition,Northern verification of 1,4-alpha-glucan branching enzyme detected two isoforms in rice endosperm,the larger one of which only existed in endosperm.

  4. cDNA Cloning, Sequence Analysis of the Porcine LIM and Cysteine-rich Domain 1 Gene

    Institute of Scientific and Technical Information of China (English)

    Jun WANG; Chang-Yan DENG; Yuan-Zhu XIONG; Bo ZUO; Lei XING; Feng-E LI; Ming-Gang LEI; Rong ZHENG; Si-Wen JIANG

    2005-01-01

    LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton by their interaction with various structural proteins, kinases and transcriptional regulators. Using molecular biology combined with in silico cloning, we have cloned the complete coding sequence of pig LIM and the cysteine-rich domain 1 gene (LMCD1) which encodes a 363 amino acid protein. The estimated molecular weight of the LMCD1 protein is 40,788 Da with a pI of 8.39. It was found to be highly expressed in both skeletal muscle and cardiac muscle. Alignment analysis revealed that the deduced protein sequence shares 86%, 91% and 93% homology with that of its human, mouse and rat counterparts, respectively. The LMCD1 protein was predicted by bioinformatics software to contain a novel cysteine-rich domain in the N-terminal region, two LIM domains in the C-terminal region, nine potential protein kinase C phosphorylation sites, seven casein kinase Ⅱ phosphorylation sites, a tyrosine kinase phosphorylation site, seven N-glycosylation and N-myristoylation sites and a single potential N-glycosylation site, which is similar to the protein's human counterpart. Phylogenetic tree was constructed by aligning the amino acid sequences of the LIM domain from different species. In addition, four base mutations were detected by comparing the sequences of Large White pigs with those of Chinese Meishan pigs. The G294A mutation site was confirmed by polymerase chain reaction-single-strand conformation polymorphism analysis. Its allele frequencies were studied in five pig breeds.

  5. 人胎儿骨骼和关节RACE cDNA文库的构建%The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint

    Institute of Scientific and Technical Information of China (English)

    梁晓媛; 龚瑶琴; 刘奇迹; 李江夏; 陈丙玺; 郭辰虹

    2001-01-01

    目的 建立人胎儿骨骼和关节快速扩增cDNA末端(rapid amplification of cDNA ends,RACE cDNA)文库,为分离骨骼和关节发育相关基因奠定基础。方法 采用改进的异硫氰酸胍-酚-氯仿-异戊醇一步法提取骨骼和关节总RNA,用TaKaRa公司生产的cDNA合成试剂盒合成平末端的双链cDNA,然后与衔接子连接。再用位于双链cDNA末端的通用引物扩增全部cDNA。结果 建立了从骨骼和关节构建RACE cDNA文库的方法,并用该方法成功地构建了人胎儿骨骼和关节RACE cDNA文库。结论 所构建的的利用少量总RNA构建RACE cDNA文库方法切实可行,所构建的文库适用于用RACE方法从中分离骨骼和关节发育相关基因。%Objective To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint- specific development-related genes.Methods Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers.Results A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed.Conclusion The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.

  6. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Science.gov (United States)

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

    Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  7. IN VITRO STUDY ON THE CLONING AND TRANSDUCTION OF HUMAN O6-METHYLGUANINE-DNA-METHYLTRANSFERASE CDNA INTO HUMAN UMBILICAL CORD BLOOD CD34+ CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) gene could increase resistance to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT-PCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into G1Na retrovirus vector. Then the G1Na-MGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8′ 105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT-cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

  8. Nucleotide sequence of a cDNA coding for the barley seed protein CMa: an inhibitor of insect α-amylase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Johansson, A.

    1992-01-01

    The primary structure of the insect alpha-amylase inhibitor CMa of barley seeds was deduced from a full-length cDNA clone pc43F6. Analysis of RNA from barley endosperm shows high levels 15 and 20 days after flowering. The cDNA predicts an amino acid sequence of 119 residues preceded by a signal...... peptide of 25 amino acids. Ala and Leu account for 55% of the signal peptide. CMa is 60-85% identical with alpha-amylase inhibitors of wheat, but shows less than 50% identity to trypsin inhibitors of barley and wheat. The 10 Cys residues are located in identical positions compared to the cereal inhibitor...

  9. Immunological responses of turbot (Psetta maxima) to nodavirus infection or polyriboinosinic polyribocytidylic acid (pIC) stimulation, using expressed sequence tags (ESTs) analysis and cDNA microarrays.

    Science.gov (United States)

    Park, Kyoung C; Osborne, Jane A; Montes, Ariana; Dios, Sonia; Nerland, Audun H; Novoa, Beatriz; Figueras, Antonio; Brown, Laura L; Johnson, Stewart C

    2009-01-01

    To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-infected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified. The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those stimulated with pIC. Within the nodavirus-infected fish we observed the highest number of differentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have important roles in immune responses to nodavirus infection and dsRNA stimulation.

  10. A subtractive cDNA library from an identified regenerating neuron is enriched in sequences up-regulated during nerve regeneration.

    Science.gov (United States)

    Korneev, S; Fedorov, A; Collins, R; Blackshaw, S E; Davies, J A

    1997-01-01

    We have constructed a subtractive cDNA library from regenerating Retzius cells of the leech, Hirudo medicinalis. It is highly enriched in sequences up-regulated during nerve regeneration. Sequence analysis of selected recombinants has identified both novel sequences and sequences homologous to molecules characterised in other species. Homologies include alpha-tubulin, a calmodulin-like protein, CAAT/enhancer-binding protein (C/EBP), protein 4.1 and synapsin. These types of proteins are exactly those predicted to be associated with axonal growth and their identification confirms the quality of the library. Most interesting, however, is the isolation of 5 previously uncharacterised cDNAs which appear to be up-regulated during regeneration. Their analysis is likely to provide new information on the molecular mechanisms of neuronal regeneration.

  11. 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.

  12. Identification of "pathologs" (disease-related genes from the RIKEN mouse cDNA dataset using human curation plus FACTS, a new biological information extraction system

    Directory of Open Access Journals (Sweden)

    Socha Luis A

    2004-04-01

    Full Text Available Abstract Background A major goal in the post-genomic era is to identify and characterise disease susceptibility genes and to apply this knowledge to disease prevention and treatment. Rodents and humans have remarkably similar genomes and share closely related biochemical, physiological and pathological pathways. In this work we utilised the latest information on the mouse transcriptome as revealed by the RIKEN FANTOM2 project to identify novel human disease-related candidate genes. We define a new term "patholog" to mean a homolog of a human disease-related gene encoding a product (transcript, anti-sense or protein potentially relevant to disease. Rather than just focus on Mendelian inheritance, we applied the analysis to all potential pathologs regardless of their inheritance pattern. Results Bioinformatic analysis and human curation of 60,770 RIKEN full-length mouse cDNA clones produced 2,578 sequences that showed similarity (70–85% identity to known human-disease genes. Using a newly developed biological information extraction and annotation tool (FACTS in parallel with human expert analysis of 17,051 MEDLINE scientific abstracts we identified 182 novel potential pathologs. Of these, 36 were identified by computational tools only, 49 by human expert analysis only and 97 by both methods. These pathologs were related to neoplastic (53%, hereditary (24%, immunological (5%, cardio-vascular (4%, or other (14%, disorders. Conclusions Large scale genome projects continue to produce a vast amount of data with potential application to the study of human disease. For this potential to be realised we need intelligent strategies for data categorisation and the ability to link sequence data with relevant literature. This paper demonstrates the power of combining human expert annotation with FACTS, a newly developed bioinformatics tool, to identify novel pathologs from within large-scale mouse transcript datasets.

  13. Cloning and Sequencing of a Full-Length cDNA Encoding the RuBPCase Small Subunit (RbcS)in Tea (Camellia sinensis)

    Institute of Scientific and Technical Information of China (English)

    YE Ai-hua; JIANG Chang-jun; ZHU Lin; YU Mei; WANG Zhao-xia; DENG Wei-wei; WEI Chao-lin

    2009-01-01

    This study was aimed to isolate ribulose-l,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) from tea plant [Camellia sinensis (L.) O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP (cDNA amplified fragment length polymorphism), we have isolated some transcript-derived fragments (TDFs) occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp (EF011075) cDNA was obtained via rapid amplification of cDNA ends (RACE). It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.

  14. 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.

  15. Identification and characterization of a novel legume-like lectin cDNA sequence from the red marine algae Gracilaria fisheri.

    Science.gov (United States)

    Suttisrisung, Sukanya; Senapin, Saengchan; Withyachumnarnkul, Boonsirm; Wongprasert, Kanokpan

    2011-12-01

    A legume-type lectin (L-Lectin) gene of the red algae Gracilaria fisheri (GFL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of GFL was 1714 bp and contained a 1542 bp open reading frame encoding 513 amino acids with a predicted molecular mass of 56.5 kDa. Analysis of the putative amino acid sequence with NCBI-BLAST revealed a high homology (30-68%) with legume-type lectins (L-lectin) from Griffithsia japonica, Clavispora lusitaniae, Acyrthosiphon pisum, Tetraodon nigroviridis and Xenopus tropicalis. Phylogenetic relationship analysis showed the highest sequence identity to a glycoprotein of the red algae Griffithsia japonica (68%) (GenBank number AAM93989). Conserved Domain Database analysis detected an N-terminal carbohydrate recognition domain (CRD), the characteristic of L-lectins, which contained two sugar binding sites and a metal binding site. The secondary structure prediction of GFL showed a beta-sheet structure, connected with turn and coil. The most abundant structural element of GFL was the random coil, while the alpha-helixes were distributed at the N- and C-termini, and 21 beta-sheets were distributed in the CRD. Computer analysis of three-dimensional structure showed a common feature of L-lectins of GFL, which included an overall globular shape that was composed of a beta-sandwich of two anti-parallel beta-sheets, monosaccharide binding sites, were on the top of the structure and in proximity with a metal binding site. Northern blot analysis using a DIG-labelled probe derived from a partial GFL sequence revealed a hybridization signal of (approx.) 1.7 kb consistent with the length of the full-length GFL cDNA identified by RACE. No detectable band was observed from control total RNA extracted from filamentous green algae.

  16. Identification and characterization of a novel legume-like lectin cDNA sequence from the red marine algae Gracilaria fisheri

    Indian Academy of Sciences (India)

    Sukanya Suttisrisung; Saengchan Senapin; Boonsirm Withyachumnarnkul; Kanokpan Wongprasert

    2011-12-01

    A legume-type lectin (L-lectin) gene of the red algae Gracilaria fisheri (GFL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of GFL was 1714 bp and contained a 1542 bp open reading frame encoding 513 amino acids with a predicted molecular mass of 56.5 kDa. Analysis of the putative amino acid sequence with NCBI-BLAST revealed a high homology (30–68%) with legume-type lectins (L-lectin) from Griffithsia japonica, Clavispora lusitaniae, Acyrthosiphon pisum, Tetraodon nigroviridis and Xenopus tropicalis. Phylogenetic relationship analysis showed the highest sequence identity to a glycoprotein of the red algae Griffithsia japonica (68%) (GenBank number AAM93989). Conserved Domain Database analysis detected an N-terminal carbohydrate recognition domain (CRD), the characteristic of L-lectins, which contained two sugar binding sites and a metal binding site. The secondary structure prediction of GFL showed a -sheet structure, connected with turn and coil. The most abundant structural element of GFL was the random coil, while the -helixes were distributed at the N- and C-termini, and 21 -sheets were distributed in the CRD. Computer analysis of three-dimensional structure showed a common feature of L-lectins of GFL, which included an overall globular shape that was composed of a -sandwich of two anti-parallel -sheets, monosaccharide binding sites, were on the top of the structure and in proximity with a metal binding site. Northern blot analysis using a DIG-labelled probe derived from a partial GFL sequence revealed a hybridization signal of ∼1.7 kb consistent with the length of the full-length GFL cDNA identified by RACE. No detectable band was observed from control total RNA extracted from filamentous green algae.

  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. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells.

    Science.gov (United States)

    Tokunaga, T; Yano, O; Kuramoto, E; Kimura, Y; Yamamoto, T; Kataoka, T; Yamamoto, S

    1992-01-01

    Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.

  19. A combined de novo protein sequencing and cDNA library approach to the venomic analysis of Chinese spider Araneus ventricosus.

    Science.gov (United States)

    Duan, Zhigui; Cao, Rui; Jiang, Liping; Liang, Songping

    2013-01-14

    In past years, spider venoms have attracted increasing attention due to their extraordinary chemical and pharmacological diversity. The recently popularized proteomic method highly improved our ability to analyze the proteins in the venom. However, the lack of information about isolated venom proteins sequences dramatically limits the ability to confidently identify venom proteins. In the present paper, the venom from Araneus ventricosus was analyzed using two complementary approaches: 2-DE/Shotgun-LC-MS/MS coupled to MASCOT search and 2-DE/Shotgun-LC-MS/MS coupled to manual de novo sequencing followed by local venom protein database (LVPD) search. The LVPD was constructed with toxin-like protein sequences obtained from the analysis of cDNA library from A. ventricosus venom glands. Our results indicate that a total of 130 toxin-like protein sequences were unambiguously identified by manual de novo sequencing coupled to LVPD search, accounting for 86.67% of all toxin-like proteins in LVPD. Thus manual de novo sequencing coupled to LVPD search was proved an extremely effective approach for the analysis of venom proteins. In addition, the approach displays impeccable advantage in validating mutant positions of isoforms from the same toxin-like family. Intriguingly, methyl esterifcation of glutamic acid was discovered for the first time in animal venom proteins by manual de novo sequencing.

  20. Construction of genome-wide physical BAC contigs using mapped cDNA as probes: Toward an integrated BAC library resource for genome sequencing and analysis. Annual report, July 1995--January 1997

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, S.C.; Bocskai, D.; Cao, Y. [and others

    1997-12-31

    The goal of human genome project is to characterize and sequence entire genomes of human and several model organisms, thus providing complete sets of information on the entire structure of transcribed, regulatory and other functional regions for these organisms. In the past years, a number of useful genetic and physical markers on human and mouse genomes have been made available along with the advent of BAC library resources for these organisms. The advances in technology and resource development made it feasible to efficiently construct genome-wide physical BAC contigs for human and other genomes. Currently, over 30,000 mapped STSs and 27,000 mapped Unigenes are available for human genome mapping. ESTs and cDNAs are excellent resources for building contig maps for two reasons. Firstly, they exist in two alternative forms--as both sequence information for PCR primer pairs, and cDoreen genomic libraries efficiently for large number of DNA probes by combining over 100 cDNA probes in each hybridization. Second, the linkage and order of genes are rather conserved among human, mouse and other model organisms. Therefore, gene markers have advantages over random anonymous STSs in building maps for comparative genomic studies.

  1. Construction of a full-length enriched cDNA library and preliminary analysis of expressed sequence tags from Bengal Tiger Panthera tigris tigris.

    Science.gov (United States)

    Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2013-05-24

    In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

  2. 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.

  3. 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.

  4. Isolation, cDNA sequence analysis and tissue expression profile of a novel swine gene differentially expressed in the Longissimus dorsi muscle tissues from Large White × Meishan cross combination

    Institute of Scientific and Technical Information of China (English)

    LIU Yonggang; LEI Minggang; XIONG Yuanzhu; DENG Changyan

    2005-01-01

    In order to study the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences in gene expression in the Longissimus dorsi muscle tissues from Large White × Meishan cross combination.One novel gene differentially expressed between the hybrids and the purebreds was isolated and subsequently identified using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and its complete cDNA sequence was obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction revealed that the open reading frame of this gene encodes a protein of 188 amino acids that contains the putative conserved domain of the PRA1 family protein and this protein has high homology with the PRA1 family protein 3 of three species-rat (88 % ), human(88 % ), and mouse (87 % ), -so that it can be defined as swine PRA1 family protein 3. The phylogenetic tree analysis revealed that the swine PRA1 family protein 3 has a closer genetic relationship with the human PRA1 family protein 3 than with those of mouse and rat.The tissue expression analysis indicated that swine PRA1family protein 3 gene is highly-expressed in muscle and fat, moderately in spleen,weakly in heart, kidney, ovary, lung, and almost not expressed in small intestine and liver. The function of this gene and the relationship between this gene and heterosis are also discussed.

  5. Isolation of the gene and hypothalamic of cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat

    Energy Technology Data Exchange (ETDEWEB)

    Adelman, J.P.; Mason, A.J.; Hayflick, J.S.; Seeburg, P.H.

    1986-01-01

    Cloned cDNAs encoding the precursor protein for gonadotropin-releasing hormone (Gn-RH) and prolactin release-inhibiting factor (PIF) were isolated from libraries derived from human and rat hypothalamic mRNA. Nucleotide sequence analyses predict precursor proteins of 92 amino acids for both species and show identity between the human placental and human hypothalamic precursor proteins. Whereas the Gn-RH peptide structure is completely conserved in human and rat, the PIF domain of the precursor displays 70% interspecies homology. Genomic analyses revealed the presence of a single Gn-RH-PIF gene in human and rat containing sequences corresponding to the cDNA distributed across four exons.

  6. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Institute of Scientific and Technical Information of China (English)

    QI Fei; GUO Huarong; WANG Jian

    2008-01-01

    Reversible protein phosphorylation,catalyzed by protein kinases and phosphatases,is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes.Protein phosphatase 1(PP1) is the first and well-characterized member of the protein serine/threoninephosphatase family.In the present study.a full-length cDNA encoding the beta isolorm of the catalytic subunit of protein phosphatase 1(PP1cb).was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus,designated SmPP1cb,by the rapid amplification of cDNA ends (RACE) technique.The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame(ORF),flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region.The ORF encodes a putative 327 amino acid protein.and the N-terminal section of this protein iS highly acidic,Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp.a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B(PP2B).And its calculated molecular mass is 37 193 Da and pI 5.8.Sequence analysis indicated that,SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates.and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXXATGG,which is different from mammalian in two positions A-6 and G-3,indicating the possibility of different initiation of translation in turbot,and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals.especially zebrafish.The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  7. Comparative analysis of gene expression at early seedling stage between a rice hybrid and its parents using a cDNA microarray of 9198 uni-sequences

    Institute of Scientific and Technical Information of China (English)

    HUANG; Yi; LI; Lihua; CHEN; Ying; LI; Xianghua; XU; Caiguo; WANG; Shiping; ZHANG; Qifa

    2006-01-01

    Using a cDNA microarray consisting of 9198 expressed sequence tags, we surveyed the gene expression profiles in shoots and roots of a rice hybrid, Liangyoupei 9 and its parents Peiai 64s and 93-11 at 72 h after germination. A total of 8587 sequences had detectable signals in both shoots and roots of the three genotypes. A total of 1571 sequences exhibited significant (P<0.01) expression differences in shoots or roots among the three genotypes, of which 121 showed expression polymorphisms in both shoots and roots, and 870 revealed significant expression differences between the hybrid and one of the parents. The expression polymorphism of the sequences was associated with the functional categories of the sequences. They occurred more frequently in categories of carbohydrate, energy and lipid metabolisms and stress response than expected, while less frequently in categories of amino acid metabolism, transcription and translation regulation, and signal transduction. A total of 214 sequences exhibited significant (P<0.05) mid-parent heterosis in expression, of which 117 had homology to genes with known functions, assigned in the categories of basic metabolism, genetic information processing, cell growth and death, signal transduction, transportation and stress response. The results may provide useful information for exploring the relationship between gene expression polymorphism and phenotypic variation, and for characterizing the molecular mechanism of seedling development and heterosis in rice.

  8. Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Gibbs, R.A. (Baylor College of Medicine, Houston, TX (USA)); Nguyen, Phinga (Howard Hughes Medical Institute, Houston, TX (USA)); McBride, L.J.; Koepf, S.M. (Applied Biosystems, Foster City, CA (USA)); Caskey, C.T. (Baylor College of Medicine, Houston, TX (USA) Howard Hughes Medical Institute, Houston, TX (USA))

    1989-03-01

    The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. The authors have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. They also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.

  9. Functional genomics of 5- to 8-cell stage human embryos by blastomere single-cell cDNA analysis.

    Directory of Open Access Journals (Sweden)

    Amparo Galán

    Full Text Available Blastomere fate and embryonic genome activation (EGA during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM (n = 120, stemness (n = 190 and Trophectoderm (TE (n = 45, were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1, stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT, and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR. The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92 such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2 and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4, as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

  10. Functional Genomics of 5- to 8-Cell Stage Human Embryos by Blastomere Single-Cell cDNA Analysis

    Science.gov (United States)

    Galán, Amparo; Montaner, David; Póo, M. Eugenia; Valbuena, Diana; Ruiz, Verónica; Aguilar, Cristóbal; Dopazo, Joaquín; Simón, Carlos

    2010-01-01

    Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented. PMID:21049019

  11. 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.

  12. Transcription profiling of the model cyanobacterium Synechococcus sp. strain PCC 7002 by NextGen (SOLiD™ Sequencing of cDNA

    Directory of Open Access Journals (Sweden)

    Marcus eLudwig

    2011-03-01

    Full Text Available The genome of the unicellular, euryhaline cyanobacterium Synechococcus sp. PCC 7002 encodes about 3200 proteins. Transcripts were detected for nearly all annotated open reading frames by a global transcriptomic analysis by Next-Generation (SOLiDTM sequencing of cDNA. In the cDNA samples sequenced, ~90% of the mapped sequences were derived from the 16S and 23S ribosomal RNAs and ~10% of the sequences were derived from mRNAs. In cells grown photoautotrophically under standard conditions (38 °C, 1% (v/v CO2 in air, 250 µmol photons m-2 s-1, the highest transcript levels (up to 2% of the total mRNA for the most abundantly transcribed genes (e. g., cpcAB, psbA, psaA were generally derived from genes encoding structural components of the photosynthetic apparatus. High light exposure for one hour caused changes in transcript levels for genes encoding proteins of the photosynthetic apparatus, Type-1 NADH dehydrogenase complex and ATP synthase, whereas dark incubation for one hour resulted in a global decrease in transcript levels for photosynthesis-related genes and an increase in transcript levels for genes involved in carbohydrate degradation. Transcript levels for pyruvate kinase and the pyruvate dehydrogenase complex decreased sharply in cells incubated in the dark. Under dark anoxic (fermentative conditions, transcript changes indicated a global decrease in transcripts for respiratory proteins and suggested that cells employ an alternative phosphoenolpyruvate degradation pathway via phosphoenolpyruvate synthase (ppsA and the pyruvate:ferredoxin oxidoreductase (nifJ. Finally, the data suggested that an apparent operon involved in tetrapyrrole biosynthesis and fatty acid desaturation, acsF2-ho2-hemN2-desF, may be regulated by oxygen concentration.

  13. The enzyme and the cDNA sequence of a thermolabile and double-strand specific DNase from Northern shrimps (Pandalus borealis.

    Directory of Open Access Journals (Sweden)

    Inge W Nilsen

    Full Text Available BACKGROUND: We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies. METHODOLOGY/PRINCIPAL FINDINGS: A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis. The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65 degrees C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN. Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases. CONCLUSIONS/SIGNIFICANCE: The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature

  14. Assessment of adaptive evolution between wheat and rice as deduced from full-length common wheat cDNA sequence data and expression patterns

    Directory of Open Access Journals (Sweden)

    Hayashizaki Yoshihide

    2009-06-01

    Full Text Available Abstract Background Wheat is an allopolyploid plant that harbors a huge, complex genome. Therefore, accumulation of expressed sequence tags (ESTs for wheat is becoming particularly important for functional genomics and molecular breeding. We prepared a comprehensive collection of ESTs from the various tissues that develop during the wheat life cycle and from tissues subjected to stress. We also examined their expression profiles in silico. As full-length cDNAs are indispensable to certify the collected ESTs and annotate the genes in the wheat genome, we performed a systematic survey and sequencing of the full-length cDNA clones. This sequence information is a valuable genetic resource for functional genomics and will enable carrying out comparative genomics in cereals. Results As part of the functional genomics and development of genomic wheat resources, we have generated a collection of full-length cDNAs from common wheat. By grouping the ESTs of recombinant clones randomly selected from the full-length cDNA library, we were able to sequence 6,162 independent clones with high accuracy. About 10% of the clones were wheat-unique genes, without any counterparts within the DNA database. Wheat clones that showed high homology to those of rice were selected in order to investigate their expression patterns in various tissues throughout the wheat life cycle and in response to abiotic-stress treatments. To assess the variability of genes that have evolved differently in wheat and rice, we calculated the substitution rate (Ka/Ks of the counterparts in wheat and rice. Genes that were preferentially expressed in certain tissues or treatments had higher Ka/Ks values than those in other tissues and treatments, which suggests that the genes with the higher variability expressed in these tissues is under adaptive selection. Conclusion We have generated a high-quality full-length cDNA resource for common wheat, which is essential for continuation of the

  15. POSSIBLE REASONS FOR TP53 ACCUMULATION IN NASO- PHARYNGEAL CARCINOMA USING ATLAS HUMAN CANCER cDNA EXPRESSION ARRAY

    Institute of Scientific and Technical Information of China (English)

    李虹; 韩为农; 张玲; 冯湘玲; 姚开泰

    2002-01-01

    Objective: To compare gene expression profiles of nasopharyngeal carcinoma (NPC) tissue with that of control tissue by cDNA Array and to discuss possible reasons of TP53 accumulation in NPC tissue. Methods: (1) hybridization of Atlas Human Cancer cDNA Expression Array 7742-1; (2) analysis of Atlas Arrays using Atlasimage 1.01a; (3) verification of results of array by RT-PCR; (4) verification of protein expression alterations by immuno- histochemistry. Results: (1) Of 588 tumor-related genes, 134 genes were upregulated, 88 downregulated; (2) Of 32 TP53-regulated genes, 13 genes were shown differential expression, 11 upregulated, 2 downregulated; (3) ATM and JNK2 were upregulated; (4) mRNA expression of ubiquitin-conjugating enzyme E2 (M74524) and ubiquitin- conjugating enzyme E2 (L22005) has no evident changes; Conclusion: (1) TP53 dysfunction exists in NPC tissues; (2) ATM and JNK might be the important causes of TP53 accumulation.

  16. cDNA microarray analysis of human keratinocytes cells of patients submitted to chemoradiotherapy and oral photobiomodulation therapy: pilot study.

    Science.gov (United States)

    Antunes, Heliton S; Wajnberg, Gabriel; Pinho, Marcos B; Jorge, Natasha Andressa Nogueira; de Moraes, Joyce Luana Melo; Stefanoff, Claudio Gustavo; Herchenhorn, Daniel; Araújo, Carlos M M; Viégas, Celia Maria Pais; Rampini, Mariana P; Dias, Fernando L; de Araujo-Souza, Patricia Savio; Passetti, Fabio; Ferreira, Carlos G

    2017-08-24

    Oral mucositis is an acute toxicity that occurs in patients submitted to chemoradiotherapy to treat head and neck squamous cell carcinoma. In this study, we evaluated differences in gene expression in the keratinocytes of the oral mucosa of patients treated with photobiomodulation therapy and tried to associate the molecular mechanisms with clinical findings. From June 2009 to December 2010, 27 patients were included in a randomized double-blind pilot study. Buccal smears from 13 patients were obtained at days 1 and 10 of chemoradiotherapy, and overall gene expression of samples from both dates were analyzed by complementary DNA (cDNA) microarray. In addition, samples from other 14 patients were also collected at D1 and D10 of chemoradiotherapy for subsequent validation of cDNA microarray findings by qPCR. The expression array analysis identified 105 upregulated and 60 downregulated genes in our post-treatment samples when compared with controls. Among the upregulated genes with the highest fold change, it was interesting to observe the presence of genes related to keratinocyte differentiation. Among downregulated genes were observed genes related to cytotoxicity and immune response. The results indicate that genes known to be induced during differentiation of human epidermal keratinocytes were upregulated while genes associated with cytotoxicity and immune response were downregulated in the laser group. These results support previous clinical findings indicating that the lower incidence of oral mucositis associated with photobiomodulation therapy might be correlated to the activation of genes involved in keratinocyte differentiation.

  17. Large scale full-length cDNA sequencing reveals a unique genomic landscape in a lepidopteran model insect, Bombyx mori.

    Science.gov (United States)

    Suetsugu, Yoshitaka; Futahashi, Ryo; Kanamori, Hiroyuki; Kadono-Okuda, Keiko; Sasanuma, Shun-ichi; Narukawa, Junko; Ajimura, Masahiro; Jouraku, Akiya; Namiki, Nobukazu; Shimomura, Michihiko; Sezutsu, Hideki; Osanai-Futahashi, Mizuko; Suzuki, Masataka G; Daimon, Takaaki; Shinoda, Tetsuro; Taniai, Kiyoko; Asaoka, Kiyoshi; Niwa, Ryusuke; Kawaoka, Shinpei; Katsuma, Susumu; Tamura, Toshiki; Noda, Hiroaki; Kasahara, Masahiro; Sugano, Sumio; Suzuki, Yutaka; Fujiwara, Haruhiko; Kataoka, Hiroshi; Arunkumar, Kallare P; Tomar, Archana; Nagaraju, Javaregowda; Goldsmith, Marian R; Feng, Qili; Xia, Qingyou; Yamamoto, Kimiko; Shimada, Toru; Mita, Kazuei

    2013-09-01

    The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

  18. Opsin cDNA sequences of a UV and green rhodopsin of the satyrine butterfly Bicyclus anynana

    NARCIS (Netherlands)

    Vanhoutte, Kürt; Eggen, BJL; Janssen, JJM; Stavenga, DG

    2002-01-01

    The cDNAs of an ultraviolet (UV) and long-wavelength (LW) (green) absorbing rhodopsin of the bush brown Bicyclus anynana were partially identified. The UV sequence, encoding 377 amino acids, is 76-79% identical to the UV sequences of the papilionids Papilio glaucus and Papilio xuthus and the moth Ma

  19. Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs.

    Science.gov (United States)

    Handley-Gearhart, P M; Stephen, A G; Trausch-Azar, J S; Ciechanover, A; Schwartz, A L

    1994-12-30

    The ubiquitin-activating enzyme E1 catalyzes the first step in the ubiquitin conjugation pathway. Previously, we have cloned and sequenced the cDNA for human E1. Expression of the E1 cDNA in the ts20 cell line, which harbors a thermolabile E1, abrogated the phenotypic defects associated with this line. However, little is known of the cell biology of the E1 protein or the nature of the E1 doublet. Thus, we constructed epitope-tagged E1 cDNAs in which the HA monoclonal antibody epitope tag sequence (from influenza hemagglutinin and recognized by the 12CA5 monoclonal antibody) was fused to the amino terminus of E1. Because the amino-terminal amino acid sequence of E1 is unknown, three constructs were made in which the HA tag was placed at each of the first three ATGs in the open reading frame (HA-1E1, HA-2E1, and HA-3E1). Western analysis of HeLa cells transfected with the constructs revealed that HA-1E1 closely comigrated with the upper band of the E1 doublet, and HA-2E1 comigrated with the lower band of the E1 doublet; HA-3E1 appeared smaller than either of the E1 bands. Metabolic labeling with 32P and immunoprecipitation with anti-HA antibody revealed that only the HA-1E1 protein product is phosphorylated; polyclonal anti-E1 antibody showed that only the upper band of the endogenous E1 doublet is phosphorylated. Each of the constructs was able to rescue the mutant phenotype of the ts20 cell line. Immunofluorescence studies showed that HA-2E1 and HA-3E1 were distributed in the cytoplasm with both negative and positive nuclei. This pattern of distribution has also been observed when immunostaining with a monoclonal antibody to E1 (1C5). However, the staining pattern associated with a polyclonal anti-E1 antibody (JJJ) is characterized by positive staining cytoplasm and nuclei in all cells. The HA-1E1 construct exhibited apparently exclusive nuclear distribution in HeLa cells. The difference between the staining patterns of the polyclonal and monoclonal anti-E1

  20. Gene discovery from Jatropha curcas by sequencing of ESTs from normalized and full-length enriched cDNA library from developing seeds

    Directory of Open Access Journals (Sweden)

    Sugantham Priyanka Annabel

    2010-10-01

    Full Text Available Abstract Background Jatropha curcas L. is promoted as an important non-edible biodiesel crop worldwide. Jatropha oil, which is a triacylglycerol, can be directly blended with petro-diesel or transesterified with methanol and used as biodiesel. Genetic improvement in jatropha is needed to increase the seed yield, oil content, drought and pest resistance, and to modify oil composition so that it becomes a technically and economically preferred source for biodiesel production. However, genetic improvement efforts in jatropha could not take advantage of genetic engineering methods due to lack of cloned genes from this species. To overcome this hurdle, the current gene discovery project was initiated with an objective of isolating as many functional genes as possible from J. curcas by large scale sequencing of expressed sequence tags (ESTs. Results A normalized and full-length enriched cDNA library was constructed from developing seeds of J. curcas. The cDNA library contained about 1 × 106 clones and average insert size of the clones was 2.1 kb. Totally 12,084 ESTs were sequenced to average high quality read length of 576 bp. Contig analysis revealed 2258 contigs and 4751 singletons. Contig size ranged from 2-23 and there were 7333 ESTs in the contigs. This resulted in 7009 unigenes which were annotated by BLASTX. It showed 3982 unigenes with significant similarity to known genes and 2836 unigenes with significant similarity to genes of unknown, hypothetical and putative proteins. The remaining 191 unigenes which did not show similarity with any genes in the public database may encode for unique genes. Functional classification revealed unigenes related to broad range of cellular, molecular and biological functions. Among the 7009 unigenes, 6233 unigenes were identified to be potential full-length genes. Conclusions The high quality normalized cDNA library was constructed from developing seeds of J. curcas for the first time and 7009 unigenes coding

  1. The molecular basis for UV vision in birds: spectral characteristics, cDNA sequence and retinal localization of the UV-sensitive visual pigment of the budgerigar (Melopsittacus undulatus).

    Science.gov (United States)

    Wilkie, S E; Vissers, P M; Das, D; Degrip, W J; Bowmaker, J K; Hunt, D M

    1998-02-15

    Microspectrophotometric (msp) studies have shown that the colour-vision system of many bird species is based on four pigments with absorption peaks in the red, green, blue and UV regions of the spectrum. The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour as mate selection and detection of food. Avian visual pigments are composed of an opsin protein covalently bound via a Schiff-base linkage to the chromophore 11-cis-retinal. Here we report the cDNA sequence of a UV opsin isolated from an avian species, Melopsittacus undulatus (budgerigar or small parakeet). This sequence has been expressed using the recombinant baculovirus system; the pigment generated from the expressed protein on addition of 11-cis-retinal yielded an absorption spectrum typical of a UV photopigment, with lambdamax 365+/-3 nm. This is the first UV opsin from an avian species to be sequenced and expressed in a heterologous system. In situ hybridization of this sequence to budgerigar retinas selectively labelled a sub-set of UV cones, representing approx. 9% of the total cone population, that are distributed in a semi-regular pattern across the entire retina.

  2. Construction and high expression of retroviral vector with human clotting factor IX cDNA in vitro

    Institute of Scientific and Technical Information of China (English)

    卢大儒; 邱信芳; 郑冰; 邱晓赟; 薛京伦

    1995-01-01

    The construction of the high liter and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCTX, LIXSN and LCTXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporalion. Human dolling factor IX was delected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNC1X was 800000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3μg per 106 cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5μg per 106 cells in 24 h in hemophilia B patients’ skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral liter and expression of human FIX were increased, and the construction of retroviral vector backbone was improved

  3. Production of Recombinant Vector Containing the Coding Sequence of Human Hepcidin

    Directory of Open Access Journals (Sweden)

    Keyhanvar, N.

    2013-01-01

    Full Text Available Background and objective: Hepcidin is a cystein-rich antimicrobial peptide,which is secreted by the liver. It fights against wide spectrum of bacteria, virusesand fungi and it is a major regulator of iron homeostasis. Today, scientists havemade many efforts on the production of hepcidin. Baculovirus expression systemis one of the best eukaryotic expression systems for production of recombinanthepcidin and production of the recombinant vector is one of the most importantsteps in this expression system.Material & Methods: First, the total RNA was separated from HepG2 cell lineas a source of hepcidin expression. Then, after synthesis of total cDNA, humanhepcidin sequence was amplified, using specific primers by PCR method. Next,hepcidin sequence was cloned into pTZ57R/T vector. After digestion ofrecombinant vector using ECoRI and BamHI restriction enzymes, recombinantpFastBac HT B vector containing human hepcidin cDNA was produced.Results: Coding sequence of human hepcidin is correctly cloned into pTZ57R/Tvector and sub cloning into pFastBac HT B vector is performed successfully. Thepresence of a clear band near 274 bp resulted from PCR amplification andrestriction enzyme are the confirmation of the cloning of human hepcidin.Conclusion: According to our knowledge, the present study is the first work thatfocuses on recombinant vector containing coding sequence of human prohepcidin.This recombinant vector can be used for human hepcidin production.Keywords: Vector; Hepcidin; Iron

  4. cDNA Cloning and Sequence Analysis of ADH Gene in Delia antiqua%葱蝇ADH基因的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    陈春露; 陈斌; 司风玲; 何正波

    2012-01-01

    [ Objective ] The aim was to clone the ADH gene of Delia antiqun, and carry out a sequence analysis. [ Method ] The cDNA sequence of ADH gene was cloned with the method of RACE, and then studied with homology analysis, comparison of amino acid sequence and phylogenetic analysis. [Result] The full length of cDNA obtained was 1 088 bp, among which there were 771 bp of ORF, encoding a protein of 256 amino acids with a calculated molecular weight of 30.80 kKa and a theoretical isolectric point of 8.22. The deduced amino acid sequence had the highest identity with that of Glossina morsitans based on homological analysis,and a phylogenic tree was inferred with homological ADH sequences from other insects. [ Conclusion ] The study provides a basis for the further research of ADH gene.%[目的]对葱蝇(Delia antiqua)ADH基因进行克隆,并对其进行序列分析.[方法]通过RACE的方法克隆葱蝇ADH基因的cDNA序列,同时对该序列进行同源性分析、氨基酸序列比对和系统发育分析.[结果]试验获得的cDNA全长1 088 bp,其中ORF 771 bp,编码256个氨基酸,推测其相对分子质量为30.80 kDa,等电点为8.22;通过该基因推导的氨基酸序列与其他物种的ADH进行相似性比较和系统发育分析,发现葱蝇与刺舌蝇(Glossina morsuans)氨基酸序列的同源性最高.[结论]该研究为ADH基因的进一步研究提供了基础.

  5. Characterization of gamma-crystallin from a catfish: structural characterization of one major isoform with high methionine by cDNA sequencing.

    Science.gov (United States)

    Pan, F M; Chang, W C; Lin, C H; Hsu, A L; Chiou, S H

    1995-04-01

    gamma-Crystallin is the major and most abundant lens protein present in the eye lens of most teleostean fishes. To facilitate structural characterization of gamma-crystallins isolated from the lens of the catfishes (Clarias fuscus), a cDNA mixture was synthesized from the poly(A)+mRNA isolated from fresh eye lenses, and amplification by polymerase chain reaction (PCR) was adopted to obtain cDNAs encoding various gamma-crystallins. Plasmids of transformed E. coli strain JM109 containing amplified gamma-crystallin cDNAs were purified and prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing more than five clones containing DNA inserts of 0.52 kb revealed the presence of one major isoform with a complete reading frame of 534 base pairs, covering a gamma-crystallin (gamma M1) with a deduced protein sequence of 177 amino acids excluding the initiating methionine. It was of interest to find that this crystallin of pI 9.1 contains a high-methionine content of 15.3% in contrast to those gamma-crystallins of low-methionine content from most mammalian lenses. Sequence comparisons of catfish gamma M1-crystallin with those published sequences of gamma-crystallins from carp, bovine and mouse lenses indicate that there is approx. an 82% sequence homology between the catfish and the carp species of piscine class whereas only 51-58% homology is found between mammals and the catfish. Moreover the differences in the hydropathy profiles for these two groups of gamma-crystallins, i.e. one with a high-methionine content from teleostean fishes and the other with a low-methionine content from mammalian species, reflect a distinct variance in the polarity distributions of surface amino acids in these crystallins.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Active Expression of Human Tissue Plasminogen Activator (t-PA) c-DNA from Pulmonary Metastases in the Methylotrophic Yeast Pichia Pastoris KM71H Strain

    Science.gov (United States)

    Mohseni, Amir Hossein; Soleimani, Mohammad; Majidzadeh-A, Keivan; Taghinezhad-S, Sedigheh; Keyvani, Hossein

    2017-08-27

    Background: Human tissue-type plasminogen activator (t-PA) is a key protease of the trypsin family. It catalyzes the activation of zymogen plasminogen to the fibrin-degrading proteinase, plasmin, leading to digestion of fibrin clots. The recombinant enzyme produced by recombinant technology issued to dissolve blood clots in treatment of various human diseases such as coronary artery thrombosis, pulmonary embolism, acute ischemic stroke (AIS). Pichia pastoris expression system is a unique system for the production of high level of recombinant proteins. GS115 and KM71H are two kinds of Pichia pastoris strains whilst production of recombinant proteins in these strains is not predictable. The aim of the study was evaluation of t-PA expression in KM71H strains. Methods: In this study, the cDNA of the t-PA gene was amplified by PCR, sequenced and cloned into Pichia pastoris KM71H host strain using pPICZalphaA expression vector that allows methanol-induced expression and secretion of the protein. Results: Dot blotting results confirmed the presence oft-PA in the cell supernatant. Western blotting test revealed the approximate size of 70 KDa for recombinant t-PA. Quantitative ELISA experiment showed 810 μg/L of t-PA in the supernatant samples. Zymography analysis confirmed the proteolytic activity and biological function of the expressed recombinant t-PA. Conclusions: Correspondingly, Pichia pastoris KM71H is an appropriate strain for production of active recombinant protein. Creative Commons Attribution License

  7. A human cellular sequence implicated in trk oncogene activation is DNA damage inducible

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Ishai, R.; Scharf, R.; Sharon, R.; Kapten, I. (Technion-Israel Institute of Technology, Haifa (Israel))

    1990-08-01

    Xeroderma pigmentosum cells, which are deficient in the repair of UV light-induced DNA damage, have been used to clone DNA-damage-inducible transcripts in human cells. The cDNA clone designated pC-5 hybridizes on RNA gel blots to a 1-kilobase transcript, which is moderately abundant in nontreated cells and whose synthesis is enhanced in human cells following UV irradiation or treatment with several other DNA-damaging agents. UV-enhanced transcription of C-5 RNA is transient and occurs at lower fluences and to a greater extent in DNA-repair-deficient than in DNA-repair-proficient cells. Southern blot analysis indicates that the C-5 gene belongs to a multigene family. A cDNA clone containing the complete coding sequence of C-5 was isolated. Sequence analysis revealed that it is homologous to a human cellular sequence encoding the amino-terminal activating sequence of the trk-2h chimeric oncogene. The presence of DNA-damage-responsive sequences at the 5' end of a chimeric oncogene could result in enhanced expression of the oncogene in response to carcinogens.

  8. Profiling of differentially expressed chemotactic-related genes in MCP-1 treated macrophage cell line using human cDNA arrays

    Institute of Scientific and Technical Information of China (English)

    Guang-Xing Bian; Hong Miao; Lei Qiu; Dong-Mei Cao; Bao-Yu Guo

    2005-01-01

    AIM: To study the global gene expression of chemotactic genes in macrophage line U937 treated with human monocyte chemoattractant protein-1 (MCP-1) through the use of ExpreeChipTMHO2 cDNA array.METHODS: Total RNA was extracted from MCP-1 treated macrophage line U937 and normal U937 cells, reversely transcribed to cDNA, and then screened in parallel with HO2 human cDNA array chip. The scanned result was additionally validated using RT-PCR.RESULTS: The result of cDNA array showed that one chemotactic-related gene was up-regulated more than two-fold (RANTES) and seven chemotactic-related genes were down-regulated more than two-fold (CCR1, CCR5,ccl16, GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2) in MCP-1 treated U937 cells at mRNA level.RT-PCR analysis of four of these differentially expressed genes gave results consistent with cDNA array findings.CONCLUSION: MCP-1 could influence some chemokine and receptor expressions in macrophages in vitro. MCP-1mainly down-regulates the expression of chemotactic genes influencing neutrophilic granulocyte expression (GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2), and the mRNA level of CCR5, which plays a critical role in many disorders and illnesses.

  9. Comparative analysis of protein coding sequences from human, mouse and the domesticated pig

    DEFF Research Database (Denmark)

    Jørgensen, Frank Grønlund; Hobolth, Asger; Hornshøj, Henrik

    2005-01-01

    rubrices in order to investigate 1) the relationships between three major lineages of mammals: rodents, artiodactys and primates, and 2) the rate of evolution and the occurrence of positive Darwinian selection using codon based models of sequence evolution. Results: We provide evidence......Background: The availability of abundant sequence data from key model organisms has made large scale studies of mulecular evolution an exciting possibility. Here we use full length cDNA alignments comprising more than 700,000 nucleotides from human, mouse, pig and the Japanese pufferfish Fugu...

  10. Comparative analysis of protein coding sequences from human, mouse, and the domesticated pig  

    DEFF Research Database (Denmark)

    Jørgensen, Frank Grønlund; Hobolth, Asger; Hornshøj, H.

    2005-01-01

    rubrices in order to investigate 1) the relationships between three major lineages of mammals: rodents, artiodactyls and primates, and 2) the rate of evolution and the occurrence of positive Darwinian selection using codon based models of sequence evolution. Results We provide evidence......Background The availability of abundant sequence data from key model organisms has made large scale studies of molecular evolution an exciting possibility. Here we use full length cDNA alignments comprising more than 700,000 nucleotides from human, mouse, pig and the Japanese pufferfish Fugu...

  11. Human Genome Sequencing in Health and Disease

    Science.gov (United States)

    Gonzaga-Jauregui, Claudia; Lupski, James R.; Gibbs, Richard A.

    2013-01-01

    Following the “finished,” euchromatic, haploid human reference genome sequence, the rapid development of novel, faster, and cheaper sequencing technologies is making possible the era of personalized human genomics. Personal diploid human genome sequences have been generated, and each has contributed to our better understanding of variation in the human genome. We have consequently begun to appreciate the vastness of individual genetic variation from single nucleotide to structural variants. Translation of genome-scale variation into medically useful information is, however, in its infancy. This review summarizes the initial steps undertaken in clinical implementation of personal genome information, and describes the application of whole-genome and exome sequencing to identify the cause of genetic diseases and to suggest adjuvant therapies. Better analysis tools and a deeper understanding of the biology of our genome are necessary in order to decipher, interpret, and optimize clinical utility of what the variation in the human genome can teach us. Personal genome sequencing may eventually become an instrument of common medical practice, providing information that assists in the formulation of a differential diagnosis. We outline herein some of the remaining challenges. PMID:22248320

  12. Normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  13. cDNA cloning, characterization and expression analysis of DTX2, a human WWE and RING-finger gene, in human embryos.

    Science.gov (United States)

    Yi, Zhengfang; Yi, Tingfang; Wu, Zirong

    2006-06-01

    The WWE domain is a conserved globular domain in several proteins and predicted to mediate specificprotein-protein interactions in ubiquitin and ADP ribose conjugation systems. The RING domain is a conserved and specialized zinc-finger motif with 40-60 residues binding to two zinc atoms, which is also probably involved in mediating protein-protein interactions. Here, from human fetal heart cDNA library, we identified DTX2, a human WWE & RING-finger gene, with high similarity with its homologues. Evaluation of full-length cDNA obtained by RACE indicated it encodes a protein composed of two WWE domains and a RING-finger region. The DTX2 gene located in human chromosome 7q11.23 spanning approximately 44.3 kb on the genome and the deduced protein is 622 amino acids. Northern analysis revealed DTX2 was expressed in the 18-week, 22.5-week human embryo hearts and adult hearts, especially with high levels in the 18-week and adult hearts. Taken together, these results indicate that DTX2 is a gene encoding a WWE-RING-finger protein and involved in regulating heart development and heart functions.

  14. Human cDNA mapping using fluorescence in situ hybridization. Final progress report, April 1, 1994--July 31, 1997

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.

    1997-12-31

    The ultimate goal of this research is to generate and apply novel technologies to speed completion and integration of the human genome map and sequence with biomedical problems. To do this, techniques were developed and genome-wide resources generated. This includes a genome-wide Mapped and Integrated BAC/PAC Resource that has been used for gene finding, map completion and anchoring, breakpoint definition and sequencing. In the last period of the grant, the Human Mapped BAC/PAC Resource was also applied to determine regions of human variation and to develop a novel paradigm of primate evolution through to humans. Further, in order to more rapidly evaluate animal models of human disease, a BAC Map of the mouse was generated in collaboration with the MTI Genome Center, Dr. Bruce Birren.

  15. Efficient cDNA cloning by direct phenotypic correction of a mutant human cell line (HPRT-) using an Epstein-Barr virus derived cDNA expression vector.

    NARCIS (Netherlands)

    P.B.G.M. Belt; W. Jongmans; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude); P. van de Putte (Pieter)

    1991-01-01

    textabstractHuman cells are, in general, poor recipients of foreign DNA, which has severely hampered the cloning of genes by direct phenotypic correction of deficient human cell lines after DNA mediated gene transfer. In this communication a methodology is presented which largely circumvents this pr

  16. 羊驼垂体催乳素(PRL)基因全长cDNA的克隆及序列分析%Cloning and Sequence Analysis of the Full-length cDNA of PRL Gene from Alpaca Pituitary

    Institute of Scientific and Technical Information of China (English)

    薛霖莉; 董常生; 赫晓燕; 范瑞文; 王海东; 曹靖; 郝欢庆

    2011-01-01

    In order to provide theoretical basis for studying biological function and application of alpaca prolactin (PRL), the alpaca PRL cDNA sequence were cloned and analyzed.According to the known cDNA sequences from mammals, alpaca PRL primers was designed and the full-length cDNA of PRL from alpaca pituitary was cloned by RT-PCR and RACE techniques.The size of full-length cDNA of PRL from alpaca pituitary was 959 bp and it contained an open reading frame (ORF) of 687 bp which encoded PRL precursor protein with 229 AA.PRL precursor protein was a single-chain polypeptide composed of 30-AA signal peptide and 199-AA mature peptide.The spatial structure of alpaca PRL protein was similar to human GH.The result of the sequence alignment showed that the amino acids composition of alpaca PRL was similar to most mammals, but the methionine at 81-AA (51-AA for mature peptide) might lead to different spatial structure which might impact functions of alpaca PRL.A phylogenetic tree constructed basing on the amino acid sequences of alpaca PRL and other organisms showed that the relationships between alpaca PRL and camel PRL were closest and that the evolution speed of alpaca was very slow with no ' episodic' evolution pattern as most mammals such as primates, rodents and ruminant.%获得并分析羊驼PRL基因cDNA全序列结构,为研究羊驼催乳素(PRL)的各种生物学作用和生产应用提供理论依据.根据已知的不同哺乳动物的PRL基因cDNA序列,设计羊驼PRL引物,运用RT-PCR方法和cDNA末端快速扩增(RACE)技术获得羊驼PRL基因cDNA全序列.羊驼PRL基因cDNA序列全长959 bp,编码区为687bp,编码229个氨基酸的PRL碰前体蛋白.预测羊驼PRL蛋白质的空间结构类似人生长激素(GH),但在81位(成熟肽为51位)为蛋氨酸可能导致蛋白空间结构的不同而影响羊驼PRL的功能;序列比对结果表明,羊驼PRL的cDNA序列与大多数哺乳动物相似.构建的基因进化树分析结果显示,羊驼PRL与骆

  17. Expression of human interleukin-11 cDNA in E.coli

    Institute of Scientific and Technical Information of China (English)

    苗继红; 王嘉玺; 彭善云; 唐佩弦; 邹民吉; 段聚宝; 赵春文; 马贤凯

    1995-01-01

    A 551-bp hIL-11 gene fragment that includes no nucleotide sequences encoding signalpolypeptide and the initial 8 amino acids of the mature protein was cloned into a high-level expression vectorpEx31B of E.coli.The authors identified the recombinant plasmid,designated pEx31-IL11,by restriction endonu-cleases digestion and DNA sequencing.The resulting recombinant plasmids were then used to transform E.colistrain HB101,and expression in the PL promoter system,which is temperature-regulated,was achieved.The ex-pressed fusion protein amounts to 50% of total bacterial proteins.The hIL-11 protein expressed in E.coliwas fused to the N-terminal 99 amino acids of the MS2 polymerase to form the inclusion body.Theserecombinant proteins can be purified to about 80% by extracting inclusion body with urea.OneIL-6-dependent cell line 7 TD1 was used for bioassay.The recombinant hIL-11 protein was preliminarily puri-fied and renatured to a specific activity of 10~5U/mg,even in the presence of an excess of a neutralizing an-ti-IL-6 antibody.

  18. cDNA Cloning and Sequence Analysis of ADH Gene in Delia antiqua%葱蝇ADH基因的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    陈春露; 陈斌; 司风玲; 何正波

    2012-01-01

    【目的】对葱蝇(De如antiqua)ADH基因进行克隆,并对其进行序列分析。【方法】通过RACE的方法克隆葱蝇ADH基因的cDNA序列,同时对该序列进行同源性分析、氨基酸序列比对和系统发育分析。[结果]试验获得的cDNA全长1088bp,其中ORF771bp,编码256个氨基酸,推测其相对分子质量为30.80kDa,等电点为8.22;通过该基因推导的氨基酸序列与其他物种的ADH进行相似性比较和系统发育分析,发现葱蝇与刺舌蝇(Glossina morsitans morsitoas)氨基酸序列的同源性最高。【结论】该研究为ADH基因的进一步研究提供了基础。%[Objective] This study aims to conduct cloning and sequence analysis of ADH gene in D. Antiqua. [Method] Full-length cDNA of ADH gene in D. antiqua was cloned by using RACE technology (GenBank access number: JQ666006). Analysis of the homology, characteristics and functional domains of ADH sequence and the phy- Iogenetic relationship to other dipteran ADH were conducted. [Result] The full length of ADH cDNA is 1 088 bp containing a 771 bp of ORF, encoding 256 amino acids, with a calculated relative molecular weight of 30.80 kDa and a theoretical isoelectric point of 8.22. The deduced amino acid sequence shares the highest homology with Glossina morsitans morsitans based on homological analysis and phylogenetic analysis. [Conclusion] This study provides basis for further research of ADH gene.

  19. Cloning and sequence of cDNA encoding 1-aminocyclo- propane-1-carboxylate oxidase in Vanda flowers

    Directory of Open Access Journals (Sweden)

    Pattana Srifah Huehne

    2013-08-01

    Full Text Available The 1-aminocyclopropane-1-carboxylate oxidase (ACO gene in the final step of ethylene biosynthesis was isolated from ethylene-sensitive Vanda Miss Joaquim flowers. This consists of 1,242 base pairs (bp encoding for 326 amino acid residues. To investigate the specific divergence in orchid ACO sequences, the deduced Vanda ACO was aligned with five other orchid ACOs. The results reveal that the ACO sequences within Doritaenopsis, Phalaenopsis and Vanda show highly conserved and almost 95% identical homology, while the ACOs isolated from Cymbidium, Dendrobium and Cattleya are 8788% identical to Vanda ACO. In addition, the 2-oxoglutarate- Fe(II_oxygenase (Oxy domain of orchid ACOs consists of a higher degree of amino acid conservation than that of the non-haem dioxygenase (DIOX_N domain. The overall homology regions of Vanda ACO are commonly folded into 12 α-helices and 12 β-sheets similar to the three dimensional template-structure of Petunia ACO. This Vanda ACO cloned gene is highly expressed in flower tissue compared with root and leaf tissues. In particular, there is an abundance of ACO transcript accumulation in the column followed by the lip and the perianth of Vanda Miss Joaquim flowers at the fully-open stage.

  20. cDNA sequence and Fab crystal structure of HL4E10, a hamster IgG lambda light chain antibody stimulatory for γδ T cells.

    Directory of Open Access Journals (Sweden)

    Petra Verdino

    Full Text Available Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αβ T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer.

  1. cDNA sequence and Fab crystal structure of HL4E10, a hamster IgG lambda light chain antibody stimulatory for γδ T cells.

    Science.gov (United States)

    Verdino, Petra; Witherden, Deborah A; Podshivalova, Katie; Rieder, Stephanie E; Havran, Wendy L; Wilson, Ian A

    2011-01-01

    Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αβ T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML) protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer.

  2. Cloning and expression analysis of MBLL cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The mbl (muscleblind) gene of Drosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila's photoreceptors. Primers have been designed according to human EST086139, which is highly homologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLL is a widely expressed gene, but the expression amounts differ in these tissues.

  3. Analysis of expression sequence tags from a full-length-enriched cDNA library of developing sesame seeds (Sesamum indicum

    Directory of Open Access Journals (Sweden)

    Ke Tao

    2011-12-01

    Full Text Available Abstract Background Sesame (Sesamum indicum is one of the most important oilseed crops with high oil contents and rich nutrient value. However, genetic improvement efforts in sesame could not get benefit from molecular biology technology due to poor DNA and RNA sequence resources. In this study, we carried out a large scale of expressed sequence tags (ESTs sequencing from developing sesame seeds and further conducted analysis on seed storage products-related genes. Results A normalized and full-length enriched cDNA library from 5 ~ 30 days old immature seeds was constructed and randomly sequenced, leading to generation of 41,248 expressed sequence tags (ESTs which then formed 4,713 contigs and 27,708 singletons with 44.9% uniESTs being putative full-length open reading frames. Approximately 26,091 of all these uniESTs have significant matches to the counterparts in Nr database of GenBank, and 21,628 of them were assigned to one or more Gene ontology (GO terms. Homologous genes involved in oil biosynthesis were identified including some conservative transcription factors regulating oil biosynthesis such as LEAFY COTYLEDON1 (LEC1, PICKLE (PKL, WRINKLED1 (WRI1 and majority of them were found for the first time in sesame seeds. One hundred and 17 ESTs were identified possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin. In total, 9,347 putative functional genes from developing seeds were identified, which accounts for one third of total genes in the sesame genome. Further analysis of the uniESTs identified 1,949 non-redundant simple sequence repeats (SSRs. Conclusions This study has provided an overview of genes expressed during sesame seed development. This collection of sesame full-length cDNAs covered a wide variety of genes in seeds, in particular, candidate genes involved in biosynthesis of sesame oils and lignans. These EST sequences enriched with full length will contribute to comparative genomic studies on sesame and

  4. Analysis of expression sequence tags from a full-length-enriched cDNA library of developing sesame seeds (Sesamum indicum).

    Science.gov (United States)

    Ke, Tao; Dong, Caihua; Mao, Han; Zhao, Yingzhong; Chen, Hong; Liu, Hongyan; Dong, Xuyan; Tong, Chaobo; Liu, Shengyi

    2011-12-24

    Sesame (Sesamum indicum) is one of the most important oilseed crops with high oil contents and rich nutrient value. However, genetic improvement efforts in sesame could not get benefit from molecular biology technology due to poor DNA and RNA sequence resources. In this study, we carried out a large scale of expressed sequence tags (ESTs) sequencing from developing sesame seeds and further conducted analysis on seed storage products-related genes. A normalized and full-length enriched cDNA library from 5 ~ 30 days old immature seeds was constructed and randomly sequenced, leading to generation of 41,248 expressed sequence tags (ESTs) which then formed 4,713 contigs and 27,708 singletons with 44.9% uniESTs being putative full-length open reading frames. Approximately 26,091 of all these uniESTs have significant matches to the counterparts in Nr database of GenBank, and 21,628 of them were assigned to one or more Gene ontology (GO) terms. Homologous genes involved in oil biosynthesis were identified including some conservative transcription factors regulating oil biosynthesis such as LEAFY COTYLEDON1 (LEC1), PICKLE (PKL), WRINKLED1 (WRI1) and majority of them were found for the first time in sesame seeds. One hundred and 17 ESTs were identified possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin. In total, 9,347 putative functional genes from developing seeds were identified, which accounts for one third of total genes in the sesame genome. Further analysis of the uniESTs identified 1,949 non-redundant simple sequence repeats (SSRs). This study has provided an overview of genes expressed during sesame seed development. This collection of sesame full-length cDNAs covered a wide variety of genes in seeds, in particular, candidate genes involved in biosynthesis of sesame oils and lignans. These EST sequences enriched with full length will contribute to comparative genomic studies on sesame and other oilseed plants and serve as an abundant

  5. Toward a better knowledge of the molecular evolution of phosphoenolpyruvate carboxylase by comparison of partial cDNA sequences.

    Science.gov (United States)

    Gehrig, H H; Heute, V; Kluge, M

    1998-01-01

    To get deeper insight into the evolution of phosphoenolpyruvate carboxylase we have identified PEPC fragments (about 1,100 bp) of another 12 plants species not yet investigated in this context. The selected plants include one Chlorophyta, two Bryophyta, four Pteridophyta, and five Spermatophyta species. The obtained phylogenetic trees on PEPC isoforms are the most complete ones up to now available. Independent of their manner of construction, the resulting dendrograms are very similar and fully consistent with the main topology as it is postulated for the evolution of the higher terrestrial plants. We found a distinct clustering of the PEPC sequences of the prokaryotes, the algae, and the spermatophytes. PEPC isoforms of the archegoniates are located in the phylogenetic trees between the algae and spermatophytes. Our results strengthen the view that the PEPC is a very useful molecular marker with which to visualize phylogenetic trends both on the metabolic and organismic levels.

  6. cDNA cloning and sequence analysis of genome segments S8 from rice black-streaked dwarf virus

    Institute of Scientific and Technical Information of China (English)

    张恒木; 陈剑平; 薛庆中; 雷娟利

    2002-01-01

    Genome segments S8 of two Chinese isolates of rice black-streaked dwarf virus (RBSDV), one from Zhejiang Province and another from Hebei Province, were amplified by RT-PCR and sequenced. Both segments consisted of 1936 nts in full length (EMBL accession numbers were AJ297431 and AJ297432, respectively) and contained only one big open reading frame which encoded a polypeptide with molecular weight of 68kD. The two Chinese isolates shared 94.0% and 96.5% identity at nucleotide and amino acid level, respectively. They shared 94.5-94.9% and 92.5-92.9% homology with S8 of RBSDV Japanese isolate at nucleotide and amino acid level, respectively; shared 85.1-87.6% and 91.7-91.9% homology with S7 of Italian MRDV (maize rough dwarf virus).

  7. Human mitochondrial HMG CoA synthase: Liver cDNA and partial genomic cloning, chromosome mapping to 1p12-p13, and possible role in vertebrate evolution

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal (Canada)] [and others

    1994-10-01

    Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) is the first enzyme of ketogenesis, whereas the cytoplasmic HS isozyme (cHS) mediates an early step in cholersterol synthesis. We here report the sequence of human and mouse liver mHS cDNAs, the sequence of an HS-like cDNA from Caenorhabditis elegans, the structure of a partial human mHS genomic clone, and the mapping of the human mHS gene to chromosome 1p12-p13. the nucleotide sequence of the human mHS cDNA encodes a mature mHS peptide of 471 residues, with a mean amino acid identity of 66.5% with cHS from mammals and chicken. Comparative analysis of all known mHS and cHS protein and DNA sequences shows a high degree of conservation near the N-terminus that decreases progressively toward the C-terminus and suggests that the two isozymes arose from a common ancestor gene 400-900 million years ago. Comparison of the gene structure of mHS and cHS is also consistant with a recent duplication event. We hypothesize that the physiologic result of the HS gene duplication was the appearance of HS within the mitochondria around the time of emergence of early vertebrates, which linked preexisting pathways of beta oxidation and leucine catabolism and created the HMG CoA pathway of ketogenesis, thus providing a lipid-derived energy source for the vertebrate brain. 56 refs., 4 figs., 2 tabs.

  8. cDNA sequence and tissue distribution of the mRNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (calpactin I)

    DEFF Research Database (Denmark)

    Saris, Chris J M; Kristensen, Torsten; D’Eustachio, Peter

    1987-01-01

    We have isolated and sequenced cDNA clones of bovine nd murine pl 1 mRNAs. The nonpolyadenylated mRNAs are predicted to be 614 and 600 nucleotides, respectively. The p l l mRNAs both contain a 291 nucleotide open reading frame, preceded by a 5”untranslated region of 73 nucleotides in bovine p l l...

  9. Targeted rapid amplification of cDNA ends (T-RACE)—an improved RACE reaction through degradation of non-target sequences

    OpenAIRE

    Neil I Bower; Johnston, Ian A

    2010-01-01

    Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-contain...

  10. Sequence determinants of human microsatellite variability

    Directory of Open Access Journals (Sweden)

    Jakobsson Mattias

    2009-12-01

    Full Text Available Abstract Background Microsatellite loci are frequently used in genomic studies of DNA sequence repeats and in population studies of genetic variability. To investigate the effect of sequence properties of microsatellites on their level of variability we have analyzed genotypes at 627 microsatellite loci in 1,048 worldwide individuals from the HGDP-CEPH cell line panel together with the DNA sequences of these microsatellites in the human RefSeq database. Results Calibrating PCR fragment lengths in individual genotypes by using the RefSeq sequence enabled us to infer repeat number in the HGDP-CEPH dataset and to calculate the mean number of repeats (as opposed to the mean PCR fragment length, under the assumption that differences in PCR fragment length reflect differences in the numbers of repeats in the embedded repeat sequences. We find the mean and maximum numbers of repeats across individuals to be positively correlated with heterozygosity. The size and composition of the repeat unit of a microsatellite are also important factors in predicting heterozygosity, with tetra-nucleotide repeat units high in G/C content leading to higher heterozygosity. Finally, we find that microsatellites containing more separate sets of repeated motifs generally have higher heterozygosity. Conclusions These results suggest that sequence properties of microsatellites have a significant impact in determining the features of human microsatellite variability.

  11. Chromosomal mapping of 18S-28S rRNA genes and 10 cDNA clones of human chromosome 1 in the musk shrew (Suncus murinus).

    Science.gov (United States)

    Kuroiwa, A; Matsubara, K; Nagase, T; Nomura, N; Seong, J K; Ishikawa, A; Anunciado, R V; Tanaka, K; Yamagata, T; Masangkay, J S; Dang, V B; Namikawa, T; Matsuda, Y

    2001-01-01

    The direct R-banding fluorescence in situ hybridization (FISH) method was used to map 18S-28S ribosomal RNA genes and 10 human cDNA clones on the chromosomes of the musk shrew (Suncus murinus). The chromosomal locations of 18S-28S ribosomal RNA genes were examined in the five laboratory lines and wild animals captured in the Philippines and Vietnam, and the genes were found on chromosomes 5, 6, 9, and 13 with geographic variation. The comparative mapping of 10 cDNA clones of human chromosome 1 demonstrated that human chromosome 1 consisted of at least three segments homologous to Suncus chromosomes (chromosomes 7, 10, and 14). This approach with the direct R-banding FISH method is useful for constructing comparative maps between human and insectivore species and for explicating the process of chromosomal rearrangements during the evolution of mammals.

  12. 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

  13. Mapping and Sequencing the Human Genome

    Science.gov (United States)

    1988-01-01

    Numerous meetings have been held and a debate has developed in the biological community over the merits of mapping and sequencing the human genome. In response a committee to examine the desirability and feasibility of mapping and sequencing the human genome was formed to suggest options for implementing the project. The committee asked many questions. Should the analysis of the human genome be left entirely to the traditionally uncoordinated, but highly successful, support systems that fund the vast majority of biomedical research. Or should a more focused and coordinated additional support system be developed that is limited to encouraging and facilitating the mapping and eventual sequencing of the human genome. If so, how can this be done without distorting the broader goals of biological research that are crucial for any understanding of the data generated in such a human genome project. As the committee became better informed on the many relevant issues, the opinions of its members coalesced, producing a shared consensus of what should be done. This report reflects that consensus.

  14. The DNA sequence, annotation and analysis of human chromosome 3

    DEFF Research Database (Denmark)

    Muzny, Donna M; Scherer, Steven E; Kaul, Rajinder

    2006-01-01

    After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chr...

  15. Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

    Directory of Open Access Journals (Sweden)

    Holt Robert A

    2010-04-01

    Full Text Available Abstract Background Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar, but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. Results From existing expressed sequence tag (EST resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. Conclusions 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate.

  16. Molecular cloning, sequencing, and expression analysis of cDNA encoding metalloprotein II (MP II) induced by single and combined metals (Cu(II), Cd(II)) in polychaeta Perinereis aibuhitensis.

    Science.gov (United States)

    Yang, Dazuo; Zhou, Yibing; Zhao, Huan; Zhou, Xiaoxiao; Sun, Na; Wang, Bin; Yuan, Xiutang

    2012-11-01

    We amplified and analyzed the complete cDNA of metalloprotein II (MP II) from the somatic muscle of the polychaete Perinereis aibuhitensis, the full length cDNA is 904 bp encoding 119 amino acids. The MP II cDNA sequence was subjected to BLAST searching in NCBI and was found to share high homology with hemerythrin of other worms. MP II expression of P. aibuhitensis exposed to single and combined metals (Cu(II), Cd(II)) was analyzed using real time-PCR. MP II mRNA expression increased at the start of Cu(II) exposure, then decreased and finally return to the normal level. Expression pattern of MP II under Cd(II) exposure was time- and dose-dependent. MP II expression induced by a combination of Cd(II) and Cu(II) was similar to that induced by Cd(II) alone.

  17. Expression of angiostatin cDNA in human gallbladder carcinoma cell line GBC-SD and its effect on endothelial proliferation and growth

    Institute of Scientific and Technical Information of China (English)

    Ding-Zhong Yang; Jing He; Ji-Cheng Zhang; Zuo-Ren Wang

    2006-01-01

    AIM: To explore the influence of angiostatin up-regulation on the biologic behavior of gallbladder carcinoma cells in vitro and in vitro, and the potential value of angiostatin gene therapy for gallbladder carcinoma.METHODS: A eukaryotic expression vector of pcDNA3.1(+) containing murine angiostatin was constructed and identified by restriction endonuclease digestion and sequencing. The recombinant vector pcDNA3.1-angiostatin was transfected into human gallbladder carcinoma cell line GBC-SD with Lipofectamine 2000, and paralleled with the vector and mock control. The resistant clone was screened by G418 filtration. Angiostatin transcription and protein expression were examined by RT-PCR,immunofluorescence and Western-blot. The supernatant was collected to treat endothelial cells. Cell proliferation and growth in vitro were observed under microscope.RESULTS: Murine angiostatin Cdna was successfully cloned into the eukaryotic expression vector pcDNA3.1(+). After 14 d of transfection and selection with G418,macroscopic resistant cell cloning was formed in the experimental group transfected with pcDNA 3.1(+)-angiostatin and vector control. But untreated cells died in the mock control. Angiostatin was detected by RT-PCR and protein expression was detected in the experimental group by immunofluorescence and Western-blot. Cell proliferation and growth in vitro in the three groups were observed respectively under microscope. No significant difference was observed in the growth speed of GBCSD cells between groups that were transfected with and without angiostatin. After treatment with supernatant,significant differences were observed in endothelial cell (ECV-304) growth in vitro. The cell proliferation and growth were inhibited.CONCLUSION: Angiostatin does not directly inhibit human gallbladder carcinoma cell proliferation and growth in vitro, but the secretion of angiostatin inhabits endothelial cell proliferation and growth.

  18. Exome sequencing of a multigenerational human pedigree.

    Directory of Open Access Journals (Sweden)

    Dale J Hedges

    Full Text Available Over the next few years, the efficient use of next-generation sequencing (NGS in human genetics research will depend heavily upon the effective mechanisms for the selective enrichment of genomic regions of interest. Recently, comprehensive exome capture arrays have become available for targeting approximately 33 Mb or approximately 180,000 coding exons across the human genome. Selective genomic enrichment of the human exome offers an attractive option for new experimental designs aiming to quickly identify potential disease-associated genetic variants, especially in family-based studies. We have evaluated a 2.1 M feature human exome capture array on eight individuals from a three-generation family pedigree. We were able to cover up to 98% of the targeted bases at a long-read sequence read depth of > or = 3, 86% at a read depth of > or = 10, and over 50% of all targets were covered with > or = 20 reads. We identified up to 14,284 SNPs and small indels per individual exome, with up to 1,679 of these representing putative novel polymorphisms. Applying the conservative genotype calling approach HCDiff, the average rate of detection of a variant allele based on Illumina 1 M BeadChips genotypes was 95.2% at > or = 10x sequence. Further, we propose an advantageous genotype calling strategy for low covered targets that empirically determines cut-off thresholds at a given coverage depth based on existing genotype data. Application of this method was able to detect >99% of SNPs covered > or = 8x. Our results offer guidance for "real-world" applications in human genetics and provide further evidence that microarray-based exome capture is an efficient and reliable method to enrich for chromosomal regions of interest in next-generation sequencing experiments.

  19. Exome sequencing of a multigenerational human pedigree.

    Science.gov (United States)

    Hedges, Dale J; Hedges, Dale; Burges, Dan; Powell, Eric; Almonte, Cherylyn; Huang, Jia; Young, Stuart; Boese, Benjamin; Schmidt, Mike; Pericak-Vance, Margaret A; Martin, Eden; Zhang, Xinmin; Harkins, Timothy T; Züchner, Stephan

    2009-12-14

    Over the next few years, the efficient use of next-generation sequencing (NGS) in human genetics research will depend heavily upon the effective mechanisms for the selective enrichment of genomic regions of interest. Recently, comprehensive exome capture arrays have become available for targeting approximately 33 Mb or approximately 180,000 coding exons across the human genome. Selective genomic enrichment of the human exome offers an attractive option for new experimental designs aiming to quickly identify potential disease-associated genetic variants, especially in family-based studies. We have evaluated a 2.1 M feature human exome capture array on eight individuals from a three-generation family pedigree. We were able to cover up to 98% of the targeted bases at a long-read sequence read depth of > or = 3, 86% at a read depth of > or = 10, and over 50% of all targets were covered with > or = 20 reads. We identified up to 14,284 SNPs and small indels per individual exome, with up to 1,679 of these representing putative novel polymorphisms. Applying the conservative genotype calling approach HCDiff, the average rate of detection of a variant allele based on Illumina 1 M BeadChips genotypes was 95.2% at > or = 10x sequence. Further, we propose an advantageous genotype calling strategy for low covered targets that empirically determines cut-off thresholds at a given coverage depth based on existing genotype data. Application of this method was able to detect >99% of SNPs covered > or = 8x. Our results offer guidance for "real-world" applications in human genetics and provide further evidence that microarray-based exome capture is an efficient and reliable method to enrich for chromosomal regions of interest in next-generation sequencing experiments.

  20. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1, 1992--December 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  1. Cloning of cDNA encoding steroid 11. beta. -hydroxylase (P450c11)

    Energy Technology Data Exchange (ETDEWEB)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-10-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11..beta..-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage lambda vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11..beta..-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.

  2. 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.

  3. Exon-specific northern analysis and rapid amplification of cDNA ends (RACE) reveal that the proximal promoter II (PII) is responsible for aromatase cytochrome P450 (CYP19) expression in human ovary.

    Science.gov (United States)

    Jenkins, C; Michael, D; Mahendroo, M; Simpson, E

    1993-11-01

    Estrogens are synthesized from C19 steroids by a unique form of cytochrome P450, aromatase cytochrome P-450 (P-450AROM; the product of the CYP19 gene). We have shown that tissue-specific expression of human P-450AROM is determined, in part, by the use of alternative promoters. Previous methods of analysis for determining the specific 5'-termini of the different transcripts included S1 nuclease protection, primer extension, and Northern analysis. In the present study we have used the RACE procedure (rapid amplification of cDNA ends) to amplify and clone the 5' termini of P-450AROM transcripts expressed in human corpus luteum (CL). Sequencing of the resulting clones supports the results of the previously performed studies. Specifically, the proximal promoter, PII, is the predominant promoter utilized in CL, such that the start of transcription occurs 26 bp downstream of the putative TATA sequence. A minority of the clones possess an alternative 5'-end, namely I.3. Exon-specific Northern analysis confirms that the majority of the P-450AROM transcripts in CL tissue contain sequence specific for promoter II. Similarly, exon-specific Northern analysis indicates that transcripts in human follicles, as well as granulosa cells in culture, contain primarily sequence specific for promoter II.

  4. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    Science.gov (United States)

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  5. 蓝太阳鱼生长激素全长cDNA的克隆与序列分析%Cloning and sequencing of full length growth hormone cDNA from Lepomis cyanellus

    Institute of Scientific and Technical Information of China (English)

    曹运长; 李文笙; 叶卫; 林浩然

    2004-01-01

    The full length cDNA encoding growth hormone of a freshwater fish, Lepomis cyanellus, (LcGH) was cloned from pituitary RNA with RT-PCR, 3' and 5' RACE (rapid amplification of cDNA ends). The LcGH cDNA (Genbank No. AY530822), about 989nt (nucleotide) long, consisted of a open reading frame with 615nt long, 5'and 3'untranslated regions with 93nt and 224nt long respectively, and a 57nt poly (A) tail. The DNA sequence analysis showed that there are typical Kozak sequence and polyadenylation signal. The pregrowth hormone peptide of 204aa deduced from LcGH cDNA included a putative signal peptide (17aa) locating in its Nterminal. There exist a Asn-Cys-Thr glycosylation site at amino acid 201, and 4 cysteine residues (No. 69, 177, 194, 202) that are essential to construct two S-S bonds in this pregrowth hormone peptide. Homological comparision among LcGH and other species growth hormones showed that There is high homology (more than 85%) between growth hormone of Lepomis cyanellus and that of most perciformes fish, but low homology (less than 70%) in comparison with other species such as Siluriformes and Cypriniformes fish.

  6. Generation and analysis of expressed sequence tags from a normalized cDNA library of young leaf from Ma bamboo (Dendrocalamus latiflorus Munro).

    Science.gov (United States)

    Gao, Z M; Li, C L; Peng, Z H

    2011-11-01

    Ma bamboo (Dendrocalamus latiflorus Munro) belongs to Dendrocalamus genus, Bambusease tribe, Bambusoideae subfamily, Poaceae family. It is a representative species of clumping bamboo, and a principal commercial species for various construction purposes using mature culms and for human consumption using young shoots. A normalized cDNA library was constructed from young leaves of Ma bamboo and 9,574 high-quality ESTs were generated, from which 5,317 unigenes including 1,502 contigs and 3,815 singletons were assembled. The unigenes were assigned into different gene ontology (GO) categories and summarized into 13 broad biologically functional groups according to similar functional characteristics or cellular roles by BLAST search against public databases. Eight hundred and ninety-one unigenes were assigned by KO identifiers and mapped to six KEGG biochemical pathways. The transcripts involved in biosynthesis of secondary metabolites such as cytochrome 450, flavonol synthase/flavanone 3-hydroxylase, and dihydroflavonol-4-reductase were well represented by 14 unigenes in the unigene set. The candidate genes involved in phytohormone metabolism, signal transduction and encoding cell wall-associated receptor kinases were also identified. Sixty-seven unigenes related to plant resistance (R) genes, including RPP genes, RGAs and RDL/RF genes, were discovered. These results will provide genome-wide knowledge about the molecular physiology of Ma bamboo young leaves and tools for advanced studies of molecular mechanism underlying leaf growth and development.

  7. Sequence variability of a human pseudogene.

    Science.gov (United States)

    Martínez-Arias, R; Calafell, F; Mateu, E; Comas, D; Andrés, A; Bertranpetit, J

    2001-06-01

    We have obtained haplotypes from the autosomal glucocerebrosidase pseudogene (psGBA) for 100 human chromosomes from worldwide populations, as well as for four chimpanzee and four gorilla chromosomes. In humans, in a 5420-nucleotide stretch analyzed, variation comprises 17 substitutions, a 3-bp deletion, and a length polymorphism at a polyadenine tract. The substitution rate on the pseudogene (1.23 +/- 0.22 x 10(-9) per nucleotide and year) is within the range of previous estimates considering phylogenetic estimations. Recombination within the pseudogene was recognized, although the low variability of this locus prevented an accurate measure of recombination rates. At least 13% of the psGBA sequence could be attributed to gene conversion from the contiguous GBA gene, whereas the reciprocal event has been shown to lead to Gaucher disease. Human psGBA sequences showed a recent coalescence time (approximately 200,000 yr ago), and the most ancestral haplotype was found only in Africans; both observations are compatible with the replacement hypothesis of human origins. In a deeper timeframe, phylogenetic analysis showed that the duplication event that created psGBA could be dated at approximately 27 million years ago, in agreement with previous estimates.

  8. Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Haiming [Geneva Univ. Medical School (Switzerland); Morris, M.A.; Rossier, C. [Cantonal Hospital, Geneva (Switzerland)] [and others

    1995-08-10

    Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F{sub 1}F{sub 0}-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). 39 refs., 5 figs.

  9. Isolation of expressed sequences encoded by the human Xq terminal portion using microclone probes generated by laser microdissection

    Energy Technology Data Exchange (ETDEWEB)

    Yokoi, H.; Kang, X. (Univ. of Ottawa Faculty of Medicine, Ontario (Canada)); Hadano, Shinji; Kogi, Mieko; Wakasa, Kyo (Tokai Univ. School of Medicine, Kanagawa (Japan)); Ikeda, J.E. (Univ. of Ottawa Faculty of Medicine, Ontario (Canada) Tokai Univ. School of Medicine, Kanagawa (Japan) National Institute of Agrobiological Resources, Ibaraki (Japan))

    1994-04-01

    The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region. 36 refs., 6 figs., 2 tabs.

  10. 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.

  11. Transcribed sequences in the human genome to be held in San Francisco, November 7 and 8, 1992. Final report, September 1, 1992--August 31, 1993

    Energy Technology Data Exchange (ETDEWEB)

    Gardiner, K.

    1993-11-01

    The Second International Workshop on the Identification of Transcribed Sequences was held in San Francisco on November 7--8, 1992. The purpose of the workshop was to discuss and evaluate techniques for developing a complete transcriptional map of the human genome. Such a map requires the positions, sequences, and expression patterns of all genes. This goal is being approached from two different directions, each with strengths and weaknesses. One method is to identify the transcribed sequences from genomic DNA of a given region; the other is to systematically sequence and map cDNAs. The cDNA approach yields sequence information rapidly, but mapping each cDNA is a technical challenge. In the first approach, the map locations of genomic sequences are known at the outset, and the challenge is to identify exons. The efficient construction of a transcriptional map will require a diverse array of techniques.

  12. Elastic sequence correlation for human action analysis.

    Science.gov (United States)

    Wang, Li; Cheng, Li; Wang, Liang

    2011-06-01

    This paper addresses the problem of automatically analyzing and understanding human actions from video footage. An "action correlation" framework, elastic sequence correlation (ESC), is proposed to identify action subsequences from a database of (possibly long) video sequences that are similar to a given query video action clip. In particular, we show that two well-known algorithms, namely approximate pattern matching in computer and information sciences and dynamic time warping (DTW) method in signal processing, are special cases of our ESC framework. The proposed framework is applied to two important real-world applications: action pattern retrieval, as well as action segmentation and recognition, where, on average, its run time speed (in matlab) is about 3.3 frames per second. In addition, comparing with the state-of-the-art algorithms on a number of challenging data sets, our approach is demonstrated to perform competitively.

  13. The characterization of twenty sequenced human genomes.

    Directory of Open Access Journals (Sweden)

    Kimberly Pelak

    2010-09-01

    Full Text Available We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten "case" genomes from individuals with severe hemophilia A and ten "control" genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.

  14. Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1994-01-01

    We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa...

  15. Molecular cloning and sequence analysis of a cDNA encoding pituitary thyroid stimulating hormone beta-subunit of the Chinese soft-shell turtle Pelodiscus sinensis and regulation of its gene expression.

    Science.gov (United States)

    Chien, Jung-Tsun; Chowdhury, Indrajit; Lin, Yao-Sung; Liao, Ching-Fong; Shen, San-Tai; Yu, John Yuh-Lin

    2006-04-01

    A cDNA encoding thyroid stimulating hormone beta-subunit (TSHbeta) was cloned from pituitary of the Chinese soft-shell turtle, Pelodiscus sinensis, and its regulation of mRNA expression was investigated for the first time in reptile. The Chinese soft-shell turtle TSHbeta cDNA was cloned from pituitary RNA by reverse transcription and polymerase chain reaction (RT-PCR), and rapid amplification cDNA end (RACE) methods. The Chinese soft-shell turtle TSHbeta cDNA consists of 580-bp nucleotides, including 67-bp nucleotides of 5'-untranslated region (UTR), 402-bp of the open reading frame, and 97-bp of 3'-UTR followed by a 14 poly (A) trait. It encodes a precursor protein molecule of 133 amino acids with a putative signal peptide of 19 amino acids and a putative mature protein of 114 amino acids. The number and position of 12 cysteine residues, presumably forming six disulfide bonds, one putative asparagine-linked glycosylation site, and six proline residues that are found at positions for changing the backbone direction of the protein have been conserved in the turtle as in other vertebrate groups. The deduced amino acid sequence of the Chinese soft-shell turtle TSHbeta mature protein shares identities of 82-83% with birds, 71-72% with mammals, 49-57% with amphibians, and 44-61% with fish. The Chinese soft-shell turtle pituitaries were incubated in vitro with synthetic TRH (TSH-releasing hormone), thyroxine and triiodothyronine at doses of 10(-10) and 10(-8)M. TRH stimulated, while thyroid hormones suppressed, TSHbeta mRNA levels in dose-related manner. The sequences of cDNA and its deduced peptide of TSHbeta as well as the regulation of its mRNA level were reported for the first time in reptile.

  16. Interleukin-1 stimulates the expression of type I and type II interleukin-1 receptors in the rat insulinoma cell line Rinm5F; sequencing a rat type II interleukin-1 receptor cDNA.

    Science.gov (United States)

    Bristulf, J; Gatti, S; Malinowsky, D; Bjork, L; Sundgren, A K; Bartfai, T

    1994-01-01

    The insulin secreting rat Rinm5F cells are often used to study the cytotoxic actions of interleukin-1 (IL-1) on pancreatic beta-cells. We demonstrate here that Rinm5F insulinoma cells express both type I and type II interleukin-1 receptor (IL-1R) mRNAs and gene products. IL-1R agonists, recombinant murine IL-1 alpha (rmIL-1 alpha, 10 ng/ml) and recombinant rat IL-1 beta (rrIL-1 beta, 100 pg/ml or 10 ng/ml) induce the upregulation of mRNA expression for both types of IL-1 receptors (IL-1Rs). This effect of rrIL-1 beta is antagonised by preincubation with recombinant human interleukin 1 receptor antagonist protein (rhIL-1ra, 5 micrograms/ml). Furthermore, this rrIL-1 beta induced upregulation of IL-1R mRNAs is blocked by actinomycin D (7.5 micrograms/ml), whereas cycloheximide (20 micrograms/ml) has no effect. The phorbol ester PMA (20 nM) upregulates the expression of mRNAs both IL-1 receptors, whereas glucose (50 mM) upregulates the expression of the type I IL-1R mRNA only. Pretreatment of cells with pertussis toxin (100 ng/ml) partially blocks the rrIL-1 beta induced expression of mRNA for the type I and, to a lesser extent, the type II IL-1R. Incubation of the cells with rrIL-1 beta also induces a time-dependent expression of c-fos, interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) mRNAs. Binding studies with 125I-recombinant human IL-1 beta (125I-rhIL-1 beta) indicate that IL-1R gene products, with the ligand binding characteristics of the type I IL-1R, are constitutively present on Rinm5F cells. Treatment with rrIL-1 beta (6h) increases the number of 125I-rhIL-1 beta binding sites on Rinm5F cells. We have also demonstrated that the number of type II IL-1R binding sites increases after induction with rrIL-1 beta (6h), by indirect immunofluorescence using a monoclonal antibody (ALVA 42) raised against the human type II IL-1R. Furthermore, we have sequenced the type II IL-1R cDNA in the rat insulinoma Rinm5F cells. The comparison of the amino acid

  17. 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.

  18. 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

  19. 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.

  20. Screening a Novel Human Breast Cancer-Associated Antigen from a cDNA Expression Library of Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Shuhua Yang; Lin Zhang; Ruifang Niu; Defa Wang; Yurong Shi; Xiyin Wei; Yi Yang

    2005-01-01

    OBJECTIVE The aim of this research was to clone and express the antigen of the previously prepared monoclonal antibody named M4G3.METHODS Western blots were used to screen a breast cancer cell line that overexpresses the M4G3-associated antigen. A λ zap cDNA expression library of breast cancer cells was constructed and screened using M4G3 as a probe to clone the antigen. The positive clones were subcloned and identified by homologous comparison using BLAST.RESULTS The λ zap cDNA expression library had 1.0x106 independent clones. Fifteen positive clones were isolated following 3 rounds of immunoscreening and identified as being from Mycoplasma pulmonis.CONCLUSION The specific antigen that matched the monoclonal M4G3 antibody is an unknown protein of M. pulmonis. This work is helpful for the further study of the association of M. pulmonis infection with breast cancer.

  1. 家蝇防御素基因的cDNA克隆及序列分析%Cloning and sequence analysis of the full-length cDNA encoding defensin, an antimicrobial peptide from the housefly (Musca domestica)

    Institute of Scientific and Technical Information of China (English)

    王来城; 王金星; 王来元; 赵小凡

    2003-01-01

    Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.

  2. Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens

    Directory of Open Access Journals (Sweden)

    Olivier Loudig

    2017-03-01

    Full Text Available Formalin-fixed paraffin-embedded (FFPE specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases and women who did not develop invasive breast cancer within the same time interval (control. Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

  3. The tetramethylammonium chloride method for screening of cDNA libraries using highly degenerate oligonucleotides obtained by backtranslation of amino-acid sequences

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Leffers, H

    1993-01-01

    We describe a method for screening of cDNA libraries with highly degenerate oligonucleotides using tetramethylammonium chloride (TMAC). This method is a convenient alternative to using probes generated by the polymerase chain reaction (PCR), especially when these cannot easily be made. Nylon filt...

  4. End Sequencing and Finger Printing of Human & Mouse BAC Libraries

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, C

    2005-09-27

    This project provided for continued end sequencing of existing and new BAC libraries constructed to support human sequencing as well as to initiate BAC end sequencing from the mouse BAC libraries constructed to support mouse sequencing. The clones, the sequences, and the fingerprints are now an available resource for the community at large. Research and development of new metaodologies for BAC end sequencing have reduced costs and increase throughput.

  5. The complete sequence of human chromosome 5

    Energy Technology Data Exchange (ETDEWEB)

    Schmutz, Jeremy; Martin, Joel; Terry, Astrid; Couronne, Olivier; Grimwood, Jane; Lowry, State; Gordon, Laurie A.; Scott, Duncan; Xie, Gary; Huang, Wayne; Hellsten, Uffe; Tran-Gyamfi, Mary; She, Xinwei; Prabhakar, Shyam; Aerts, Andrea; Altherr, Michael; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caoile, Chenier; Challacombe, Jean F.; Chan, Yee Man; Denys, Mirian; Detter, Chris; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Goodstenin, David; Grigoriev, Igor; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimbal, Heather; Kobayashi, Arthur; Lopez, Frederick; Lou, Yunian; Martinez, Diego; Medina, Catherine; Morgan, Jenna; Nandkeshwar, Richard; Noonan, James P.; Pitluck, Sam; Pollard, Martin; Predki, Paul; Priest, James; Ramirez, Lucia; Rash, Sam; Retterer, James; Rodriguez, Alex; Rogers, Stephanie; Salamov, Asaf; Salazar, Angelica; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wheeler, Jeremy; Wu, Kevin; Yang, Joan; Dickson, Mark; Cheng, Jan-Fang; Eichler, Evan E.; Olsen, Anne; Pennacchio, Len A.; Rokhsar, Daniel S.; Richardson, Paul; Lucas, Susan M.; Myers, Richard M.; Rubin, Edward M.

    2004-04-15

    Chromosome 5 is one of the largest human chromosomes yet has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding and syntenic conservation with non-mammalian vertebrates, suggesting they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-encoding genes including the protocadherin and interleukin gene families and the first complete versions of each of the large chromosome 5 specific internal duplications. These duplications are very recent evolutionary events and play a likely mechanistic role, since deletions of these regions are the cause of debilitating disorders including spinal muscular atrophy (SMA).

  6. Expression of the human fast-twitch skeletal muscle troponin I cDNA in a human ovarian carcinoma suppresses tumor growth

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To explore the efficiency and mechanism of ovarian carcinoma gene therapy with the human fast-twitch skeletal muscle troponin I gene (TnI-fast), TnI-fast cDNA was transferred into human ovarian adeno-carcinoma cell-line SK-OV-3. In vitro, the cell growth and cell cycle of TnI-fast-, vector-, and mock-transfected cells were determined by MTT and flow cytometry assay, respectively. The condi-tioned media of TnI-fast-, vector-, and mock-transfected SK-OV-3 cells were collected, and the cell pro-liferation inhibiting rates of human umbilical cord venous endothelial cells (HUVECs) by the three conditioned media were assayed. All the three cell lines were implanted into node mice, and the tumor growth, cell apoptosis, angiogenesis, and expression of TnI-fast were observed or analyzed, respec-tively. In vitro, expression of TnI-fast protein had no inhibiting effect on the growth of the dominant and stable transfectant cells, but endothelium, when compared with vector-transfected cells and nontrans-fected parental SK-OV-3 cells. Implantation of stable clone expressing TnI-fast in the female BALB/c nude mice inhibits primary tumor growth by an average of 73%. The nude mice grafts expressing TnI-fast exhibit a significant decrease of microvascular density, a higher rate of tumor cells apoptosis and a comparable proliferation rate as control. Our study, to our knowledge, shows the slowed down growth of the primary ovarian carcinoma, suggested that grafts were self-inhibitory by halting angio-genesis. Our data might also provide a novel useful strategy for cancer therapy by antiangiogenic gene therapy with a specific angiogenesis inhibitor TnI-fast.

  7. cDNA2Genome: A tool for mapping and annotating cDNAs

    Directory of Open Access Journals (Sweden)

    Suhai Sandor

    2003-09-01

    Full Text Available Abstract Background In the last years several high-throughput cDNA sequencing projects have been funded worldwide with the aim of identifying and characterizing the structure of complete novel human transcripts. However some of these cDNAs are error prone due to frameshifts and stop codon errors caused by low sequence quality, or to cloning of truncated inserts, among other reasons. Therefore, accurate CDS prediction from these sequences first require the identification of potentially problematic cDNAs in order to speed up the posterior annotation process. Results cDNA2Genome is an application for the automatic high-throughput mapping and characterization of cDNAs. It utilizes current annotation data and the most up to date databases, especially in the case of ESTs and mRNAs in conjunction with a vast number of approaches to gene prediction in order to perform a comprehensive assessment of the cDNA exon-intron structure. The final result of cDNA2Genome is an XML file containing all relevant information obtained in the process. This XML output can easily be used for further analysis such us program pipelines, or the integration of results into databases. The web interface to cDNA2Genome also presents this data in HTML, where the annotation is additionally shown in a graphical form. cDNA2Genome has been implemented under the W3H task framework which allows the combination of bioinformatics tools in tailor-made analysis task flows as well as the sequential or parallel computation of many sequences for large-scale analysis. Conclusions cDNA2Genome represents a new versatile and easily extensible approach to the automated mapping and annotation of human cDNAs. The underlying approach allows sequential or parallel computation of sequences for high-throughput analysis of cDNAs.

  8. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

    Energy Technology Data Exchange (ETDEWEB)

    Matviw, Yu, G.; Young, D. (Univ. of Calgary Health Science Centre, Alberta (Canada))

    1992-11-01

    The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: The N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expressions of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. 42 refs., 5 figs.

  9. Tibetan antelope cystathionine γ -lyase:complete cDNA sequences%高原藏羚羊胱硫醚-γ-裂解酶基因克隆与全序列测定

    Institute of Scientific and Technical Information of China (English)

    李肃; 格日力

    2013-01-01

    Objective To identify the Cystathionine-γ-Lyase(CSE) genes coding sequences molecular cloning , exam the tissues expression spectrums and discuss the hypoxic adaptations mechanisms in Tibetan antelope .Methods The total RNA was extracted ,and the cDNA was captured by reverse transcription RT-PCR ,then identified ,se-quenced and cloned .Results There was 96 .47% homology between the Tibetan antelope gene fragment containing the purpose gene and the cattle gene in gen banks ,thus the result mean the gene which cloned before was CSE gene protein .The length of the CSE gene protein was been detected by designing primers according to the human ,mous , wild boar ,cattle CSEcDNA sequences ,and the CSE gene primers of tibetan antelope which tesied by Pnaman .Conclu-sion CSE gene protein might play an important role in the body of the Tibetan anteplope ,which provide experiment basis to the gene study about adaptation in high altitude hypoxia environment .in the future .%目的:探讨克隆高原藏羚羊胱硫醚-γ-裂解酶(CSE)基因编码区并检测其在成年高原藏羚羊组织中的表达,同时探讨高原藏羚羊低氧适应的分子生物学机制。方法从高原藏羚羊组织中提取总 RNA ,通过逆转录聚合酶链反应(RT-PCR)获得高原藏羚羊 cDNA ,并进行鉴定和测序。结果将含有目的片段克隆后经测序和 Blast分析,结果显示其部分编码序列与 GenBank 中牛 CSE 蛋白基因序列同源性96.47%,表明本实验所克隆的序列为CSE 蛋白基因。根据已知人、褐家鼠、小鼠、野猪、食蟹猴、家牛 CSEcDNA 序列和 Pnaman 软件设计高原藏羚羊 cse基因的引物。结论 CSE mRNA 可能在高原藏羚羊机体较为广泛的区域中发挥着作用,同时为高原低氧适应相关基因的研究提供了实验依据。

  10. CLONING AND ANALYSIS OF THE FULL-LENGTH cDNA SEQUENCE OF SEPIELLA MAINDRONI SCD GENE%曼氏无针乌贼(Sepiella maindroni)SCD基因全长cDNA的克隆和序列分析

    Institute of Scientific and Technical Information of China (English)

    马明华; 刘慧慧; 迟长凤; 吴常文

    2013-01-01

    硬脂酰辅酶A去饱和酶(SCD)是脂肪代谢的关键酶.本研究采用RT-PCR和RACE技术克隆了曼氏无针乌贼(Sepiella maindroni)SCD cDNA的全序列,序列全长1513bp,由261bp的5′非翻译区、编码306个氨基酸的921bp开放阅读框和331bp的3′非翻译区组成.在线翻译所得多肽理论分子量为34.92kDa,等电点为8.95,是疏水性蛋白,含有丰富的螺旋结构(45.10%),存在4个跨膜区.其氨基酸序列与真蛸(Octopus vulgaris)和长牡蛎(Crassostrea gigas)相似性达到91%,与其它非软体动物也表现为50%以上的相似性,说明SCD结构相对保守;系统进化树结果表明曼氏无针乌贼和真蛸及牡蛎进化关系最近,与鱼类稍远,与人及大鼠等哺乳动物亲缘关系最远.SCD基因是改善曼氏无针乌贼肉质的重要候选基因,其成功克隆及相关分析对于深入探讨软体动物脂肪酸代谢相关基因在生物体内作用机制及调控机理具有重要意义.%Strearyl coenzyme A desaturation enzyme is the key enzyme of fatty acid desaturation.In this paper,a 1513bp full-length cDNA of SCD gene from Sepiella maindroni was obtained with RT-PCR and rapid amplification of cDNA ends (RACE) techniques,which consisted of a 261bp 5′untranslated region (UTR),a 921bp open reading frame (ORF),and a 349bp 3′UTR.The molecular weight of deduced protein was 34.92kDa and its pI was 8.95.The SCD protein was hydrophobic protein and contained four transmembrane regions with rich spiral structures (45.10%).The deduced amino acid sequence aligned with those of SCD genes from different species showed high degree of sequence homology.The similarity of amino acid sequence of SCD protein was 91% among S.maindroni,Octopus vulgaris and Crassostrea gigas,and the homology was also more than 50% between S.maindroni and other animals.The result indicated that the structure of SCD protein was conserved.The SCD in S.maindroni was clustered with O.vulgaris and C.gigas,and further

  11. 家蚕铜锌超氧化物歧化酶cDNA的克隆与测序%Cloning and sequencing of the silkworm (Bombyx mori) copper zinc-superoxide dismutase cDNA

    Institute of Scientific and Technical Information of China (English)

    唐云明; 鲁成; 向仲怀; 许禾声

    2003-01-01

    The total RNA of the silkworm (Bombyx mori ) was obtained with chlorinated caesium density gradient cen-trifugation, mRNA was converted to cDNA by reverse transcription. This cDNA was used as a template for two PCR am-plifications by three primers. These were DP1: 5′-ATGGT (GT) GT (GT) AA (AG) GC (TC) GT-3′; DP2: 5′-ATGGT (GT) GT (GT) AA (AG) GC (TC) GT (GT) (CT) T-3′ and PA: 5′-GAGGACTCGAGCTCAAGC-3′. Northern blot identification of poly A+ mRNA extracted from silkworms by hybridization with the above amplified product detected a single mRNA transcript of approximate 500 bp. This showed that the amplified product was from silk-worm mRNA. Amplified products were separated by agarose gel electrophoresis and purified. The purified products were ligated to pUC19-T or pUCm-T vector and transformed into E. coli DHSct competent cells. Recombinant colonies were screened by X-gal. A clone of silkworm CuZn-SOD cDNA was thus obtained. DNA sequencing was done using the Sanger dideoxy method. The result of recombinant plasmid DNA sequencing using a DNA sequencer was 591 bps cloned. Se-quencing with DNAStar and DNAClub revealed that l - 3 bp of the 5′-extremity was the initiation codon ATG. 459 bp of the 5′-extremity translated 153 amino acids, 460 - 462 bp was the termination codon TAA which included the degenera-tion primer (DP2) derived from the N-terminal amino acid of the 5′end, PA of the 3′end and 73 bp poly A+ tail at the front of PA in the 3′end and so on.

  12. cDNA2Genome: A tool for mapping and annotating cDNAs

    OpenAIRE

    Suhai Sandor; Glatting Karl-Heinz; del Val Coral

    2003-01-01

    Abstract Background In the last years several high-throughput cDNA sequencing projects have been funded worldwide with the aim of identifying and characterizing the structure of complete novel human transcripts. However some of these cDNAs are error prone due to frameshifts and stop codon errors caused by low sequence quality, or to cloning of truncated inserts, among other reasons. Therefore, accurate CDS prediction from these sequences first require the identification of potentially problem...

  13. Expression of Human Vascular Endothelial Growth Factor Receptor Flt-1 Extracellular Domain 1-3 Loop cDNA in Pichia pastoris, Purification of the Expressed Product and Detection of Its Biological Activity

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To express human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia. Pastroris, and to purify the expressed product and detect its biological activity. Methods By inserting human Flt-1 (1-3 loop) cDNA coding 316 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of α secreting signal peptides, a recombinant expression plasmid pPIC9K/Flt-1 (1-3) was constructed and transformed to yeast host strain GS115, then His+ Muts phenotype transformant was screened out and cultured in flasks, and Flt-1 (1-3) was expressed under the induction of 1% methanol. Results SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, the purity of the expressed product reached above 90%. Biological assay proved that the expressed product could bind to hVEGF165 and inhibit the proliferation of HUVEC stimulated by hVEGF165. Conclusion Human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop was successfully expressed. The study lays a foundation for further application of the expressed product in the treatment of vasoformation related diseases, such as tumor and diabetic retinopathy.

  14. ASSOCIATION OF DIFFERENTIALLY EXPRESSED cDNA FRAGMENT OF FGG WITH HEPATOCELLULAR CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    范秉琳; 朱武凌; 邹国林; 段芳龄

    2002-01-01

    Objective: To identify a cDNA clone from the subtracted library of human hepatocellular carcinoma (HCC). Methods: Suppression subtractive hybridization was used to isolated a panel of genes that are differentially expressed in hepatocellular carcinoma as compared with cirrhotic liver. T/A cloning method was used to construct a subtracted cDNA library. DNA sequencing analysis and Northern blot analysis were also utilized. Results: The cloned cDNA is 787 nucleotides in length and contains an open reading frame of 230 amino acids, which is a cDNA fragment of reported human fibrinogen, gamma polypeptide (FGG). Northern analysis revealed that this gene was overexpressed in two hepatocellular carcinoma cell lines, SMMC-7721 and HepG2. Conclusion: Sequence identity proved the cDNA clone fragment of as FGG gene. Differential expression of the cDNA fragment in HCC suggested that FGG is related to HCC, indicating a new clue for developing a novel diagnostic and prognostic marker.

  15. Initial sequencing and analysis of the human genome.

    Science.gov (United States)

    Lander, E S; Linton, L M; Birren, B; Nusbaum, C; Zody, M C; Baldwin, J; Devon, K; Dewar, K; Doyle, M; FitzHugh, W; Funke, R; Gage, D; Harris, K; Heaford, A; Howland, J; Kann, L; Lehoczky, J; LeVine, R; McEwan, P; McKernan, K; Meldrim, J; Mesirov, J P; Miranda, C; Morris, W; Naylor, J; Raymond, C; Rosetti, M; Santos, R; Sheridan, A; Sougnez, C; Stange-Thomann, Y; Stojanovic, N; Subramanian, A; Wyman, D; Rogers, J; Sulston, J; Ainscough, R; Beck, S; Bentley, D; Burton, J; Clee, C; Carter, N; Coulson, A; Deadman, R; Deloukas, P; Dunham, A; Dunham, I; Durbin, R; French, L; Grafham, D; Gregory, S; Hubbard, T; Humphray, S; Hunt, A; Jones, M; Lloyd, C; McMurray, A; Matthews, L; Mercer, S; Milne, S; Mullikin, J C; Mungall, A; Plumb, R; Ross, M; Shownkeen, R; Sims, S; Waterston, R H; Wilson, R K; Hillier, L W; McPherson, J D; Marra, M A; Mardis, E R; Fulton, L A; Chinwalla, A T; Pepin, K H; Gish, W R; Chissoe, S L; Wendl, M C; Delehaunty, K D; Miner, T L; Delehaunty, A; Kramer, J B; Cook, L L; Fulton, R S; Johnson, D L; Minx, P J; Clifton, S W; Hawkins, T; Branscomb, E; Predki, P; Richardson, P; Wenning, S; Slezak, T; Doggett, N; Cheng, J F; Olsen, A; Lucas, S; Elkin, C; Uberbacher, E; Frazier, M; Gibbs, R A; Muzny, D M; Scherer, S E; Bouck, J B; Sodergren, E J; Worley, K C; Rives, C M; Gorrell, J H; Metzker, M L; Naylor, S L; Kucherlapati, R S; Nelson, D L; Weinstock, G M; Sakaki, Y; Fujiyama, A; Hattori, M; Yada, T; Toyoda, A; Itoh, T; Kawagoe, C; Watanabe, H; Totoki, Y; Taylor, T; Weissenbach, J; Heilig, R; Saurin, W; Artiguenave, F; Brottier, P; Bruls, T; Pelletier, E; Robert, C; Wincker, P; Smith, D R; Doucette-Stamm, L; Rubenfield, M; Weinstock, K; Lee, H M; Dubois, J; Rosenthal, A; Platzer, M; Nyakatura, G; Taudien, S; Rump, A; Yang, H; Yu, J; Wang, J; Huang, G; Gu, J; Hood, L; Rowen, L; Madan, A; Qin, S; Davis, R W; Federspiel, N A; Abola, A P; Proctor, M J; Myers, R M; Schmutz, J; Dickson, M; Grimwood, J; Cox, D R; Olson, M V; Kaul, R; Raymond, C; Shimizu, N; Kawasaki, K; Minoshima, S; Evans, G A; Athanasiou, M; Schultz, R; Roe, B A; Chen, F; Pan, H; Ramser, J; Lehrach, H; Reinhardt, R; McCombie, W R; de la Bastide, M; Dedhia, N; Blöcker, H; Hornischer, K; Nordsiek, G; Agarwala, R; Aravind, L; Bailey, J A; Bateman, A; Batzoglou, S; Birney, E; Bork, P; Brown, D G; Burge, C B; Cerutti, L; Chen, H C; Church, D; Clamp, M; Copley, R R; Doerks, T; Eddy, S R; Eichler, E E; Furey, T S; Galagan, J; Gilbert, J G; Harmon, C; Hayashizaki, Y; Haussler, D; Hermjakob, H; Hokamp, K; Jang, W; Johnson, L S; Jones, T A; Kasif, S; Kaspryzk, A; Kennedy, S; Kent, W J; Kitts, P; Koonin, E V; Korf, I; Kulp, D; Lancet, D; Lowe, T M; McLysaght, A; Mikkelsen, T; Moran, J V; Mulder, N; Pollara, V J; Ponting, C P; Schuler, G; Schultz, J; Slater, G; Smit, A F; Stupka, E; Szustakowki, J; Thierry-Mieg, D; Thierry-Mieg, J; Wagner, L; Wallis, J; Wheeler, R; Williams, A; Wolf, Y I; Wolfe, K H; Yang, S P; Yeh, R F; Collins, F; Guyer, M S; Peterson, J; Felsenfeld, A; Wetterstrand, K A; Patrinos, A; Morgan, M J; de Jong, P; Catanese, J J; Osoegawa, K; Shizuya, H; Choi, S; Chen, Y J; Szustakowki, J

    2001-02-15

    The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

  16. Molecular Cloning and Sequence Analysis on cDNA of Cystatin Gene from Tea Leaves%茶树巯基蛋白酶抑制剂基因的cDNA克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    王朝霞; 李叶云; 江昌俊; 余有本

    2005-01-01

    对多种已知植物巯基蛋白酶抑制剂(cystatin)基因的氨基酸序列进行比对分析,根据其高度保守的氨基酸序列设计一对简并引物,并从茶树品种龙井43鲜叶中提取总RNA,用RT-PCR法扩增出-204 bp的cDNA特异片段,然后通过3'/5'RACE的方法,分别扩增出3'端和5'端的序列,从而获得茶树巯基蛋白酶抑制剂基因的cDNA全长序列,所得序列全长627 bp,编码101个氨基酸,分子量约11.062 KDa.该基因在推测的氨基酸序列中含有巯基蛋白酶抑制剂家族中高度保守的、与其活性有关的QXVXG结构,且经Blast分析表明,该基因序列与其他植物巯基蛋白酶抑制剂基因的氨基酸序列同源性为54%~77%.%Two degenerate primers were designed according to the conserved region among the known plant cystatins. A cDNA fragment of 204 bp was amplified by RT-PCR (reverse transcription polymerase chain reaction)of total RNA extracted from fresh leaves of Tea plant (Camellia sinensis cv Longjing43). A full-length cDNA of the cystatin gene was obtained by 3'/5'RACE (rapid amplification of cDNA ends). The cDNA sequence of this 627 bp clone contained an open reading frame encoding a polypeptide of 101 amino acid residues with a predicable molecular mass of 11.026 KDa. The deduced amino acid sequence contained the motif QXVXG conserved among most members of the cystatin superfamily. By using the program of Blast on GenBank database, the sequence presented a high match with the cystatin genes from other plants, such as European chestnut, Cassava, Cowpea,Tomato, Soybean et al. All researched out sequences were all cystatins, so we can conclude that the cloned sequence is a member of cystatin gene from Tea plant.

  17. Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    Yan-Ping Huang; Xue-Song Gao; Dong Ji; Shu-Mei Lin; Yan-Wei Zhong; Qing Shao; Shu-Lin Zhang; Jun Cheng; Lin Wang; Jiang Guo; Yan Liu; Yuan Yang; Li-Ying Zhang; Gui-Qin Bai

    2005-01-01

    AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes.METHODS: We constructed p7 protein bait plasmid by doning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (α type) containing liver cDNA library plasmid, pACT2 in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics.RESULTS: Fifty colonies were selected and sequenced.Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens.CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.

  18. Mesotocin gene expression in the diencephalon of domestic fowl: cloning and sequencing of the MT cDNA and distribution of MT gene expressing neurons in the chicken hypothalamus.

    Science.gov (United States)

    Barth, S W; Bathgate, R A; Mess, A; Parry, L J; Ivell, R; Grossmann, R

    1997-10-01

    This study focuses on the structure and expression of the mesotocin (MT) gene in the chicken hypothalamus. Using an anchored and nested RT-PCR strategy, combined with circular RACE-PCR, the full length sequence of the chicken MT cDNA was obtained. The cDNA and derived amino acid sequences conformed to the structure of the oxytocin-like gene family. However, unlike most mammalian species, there does not appear to be frequent gene conversion between the MT and AVT cDNA sequences. A single specific hybridization signal of 1.2 kb was detected by Southern analysis of chicken genomic DNA, indicating only a single gene copy in the chicken genome. Northern analysis of hypothalamic RNA revealed a single band at approximately 0.6 kb. Using the same probe for in situ hybridization histochemistry, MT-mRNA was demonstrated to be predominantly localized in the parvocellular, magnocellular and periventricular subgroups of the paraventricular nucleus and, when compared to the distribution of neurons containing arginine-vasotocin (AVT)-mRNA in the same region, with far fewer neurons expressing the MT gene in the lateral subgroups. Only few and scattered neurons expressing the MT gene were found in the ventral and external subgroups of the supraoptic nucleus in which many neurons contain AVT transcripts, as demonstrated in consecutive sections. In all nuclei investigated, the intensity of AVT and MT hybridization signals per cell was approximately equal. No specific labelling for MT-mRNA was found in the bed nucleus of the stria terminalis, nor the nucleus accumbens. Using immunocytochemical detection of AVT and in situ hybridization for neurons expressing MT-mRNA, some neurons were found to contain both AVT and MT gene products in the paraventricular nucleus but not in the supraoptic nucleus.

  19. 梅花鹿卵泡刺激素α-亚基cDNA的分子克隆与序列分析%Nucleotide sequence of cloned cDNA for α-subunit of sika follicle stimulating hormone

    Institute of Scientific and Technical Information of China (English)

    关洪斌; 李庆章; 张莉

    2002-01-01

    从新屠宰的母梅花鹿脑垂体中提取总RNA,反转录获得cDNA,以此cDNA为模板用PCR法扩增目的片段,获得长为380 bp的梅花鹿卵泡刺激素α--亚基cDNA片段,将它克隆至pMD-18-T-Verctor.随机挑选3个阳性重组子进行测序,并将测序结果与绵羊、牛、猪等多种哺乳动物该基因的核苷酸序列及相应氨基酸序列进行比较.结果表明,梅花鹿卵泡刺激素α--亚基基因编码的氨基酸序列与绵羊、水牛的该基因同源性最高,达97%,只有4个氨基酸不同;与牛的该基因同源性达96%.与人的该基因氨基酸序列同源性较低,为75%.其编码的核苷酸序列与绵羊、水牛、牛的同源性最高,达96%,只有14~16个碱基不同;与人的该基因核苷酸同源性最低,为84%.总的来说,哺乳动物的卵泡刺激素α-亚基具有很高的同源性.%Total RNA was prepared from pituitary gland of new butchered sika.cDNA was synthesized by RT-PCRreaction and this cDNA was used as model in PCR amplification for α-subunit of sika follicle stimulating hormone.The PCR product was 380bp in 1.2% agarose gel electro-phoresis which just was the target fragment of predictedFSHα-subunit. It was cloned it to pMD-18-T vector. 3 positive recombinant was selected at random to analyze itssequence by DNA analysis apparatus. Its amino acid sequence was compared with some other mammalian. The resultshows that it has the highest homology with sheep and buffalo,which it reaches 97%. There are only 4 amino acidsdifference among sika ,sheep and buffalo. It has lower homology in amino acid with human, its homology is 75%. Ithas the highest homology among sika ,sheep, buffalo and bovine in nucleotide sequence, which it reaches 96%.There are 14-16 nucleotides difference among them. It has lower homology in nucleotide sequence with human, it isonly 84%. It was found that the nucleotide sequence of the o-subunit in these mammalian species are highly con-servative. According to our

  20. Identification of the gene encoding the human mitochondrial RNA polymerase (h-mtRPOL) by cyberscreening of the Expressed Sequence Tags database.

    Science.gov (United States)

    Tiranti, V; Savoia, A; Forti, F; D'Apolito, M F; Centra, M; Rocchi, M; Zeviani, M

    1997-04-01

    A gene cloning strategy based on the screening of the Expressed Sequence Tags database (dbEST) using sequences of mitochondrial housekeeping proteins of yeast was employed to identify the cDNA encoding the precursor of the human mitochondrial RNA polymerase (h-mtRPOL). The 3831 bp h-mtRPOL cDNA is located on chromosome 19p13.3 and encodes a protein of 1230 amino acid residues. The protein sequence shows significant homologies with sequences corresponding to mitochondrial RNA polymerases from lower eukaryotes, and to RNA polymerases from several bacteriophages. The mitochondrial RNA polymerase carries out the central activity of mitochondrial gene expression and, by providing the RNA primers for replication-initiation, is also implicated in the maintenance and propagation of the mitochondrial genome. Genes involved in the control of mtDNA replication and gene expression are attractive candidates for human disorders due to abnormalities of nucleo-mitochondrial intergenomic signalling. The availability of the h-mtRPOL cDNA will allow us to test its role in mitochondrial pathology. In addition, we propose the 'cyberscreening' of dbEST, based on yeast/human cross-species comparison, as a powerful, simple, rapid and inexpensive method, that may accelerate several-fold the molecular dissection of the human mitochondrial proteome.

  1. Identification of transcribed sequences in the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Gardiner, K.

    1992-12-01

    The workshop was held at the National Institutes of Mental Health, Bethesda, Maryland, on October 4 and 5, 1991. Twenty-four investigators attended from England, Germany and the United States. The topics discussed included: Genome sequence analysis using computer assisted detection of open reading frames, splice sites and hexamer patterns, direct exon identification using trapping of internal and 3' exons, and a recombination based system, cDNA library construction and screening, including the use of normalization and subtraction procedures, Alu and splice donor site PCR from hybrid cell lines, and microdissection clones as probes, use of labeled CDNAS as probes to screen lambda and cosmid libraries, and sequencing of random cDNAs.

  2. 猪Mx1基因cDNA的克隆及序列分析%Cloning and Sequence Analysis of Porcine Mx1 Gene cDNA

    Institute of Scientific and Technical Information of China (English)

    李想; 沈学文; 李文贵; 毕峻龙; 杨贵树; 尹革芬

    2011-01-01

    为获得云南本地猪Mx1基因cDNA序列,根据GenBank中猪Mx1基因的参考序列,设计合成3对引物.以云南本地猪外周淋巴细胞为样本,采用RT - PCR方法进行分段扩增,对扩增片段进行克隆,测序分析及序列拼接.结果表明,扩增的云南本地猪Mx1基因cDNA全长2546 bp,开放阅读框1992 bp,编码663个氨基酸,与GenBank中他猪种Mx1基因序列对比,核苷酸序列的同源性为99.4%~99.8%,氨基酸序列的同源性为98.8%~99.5%.成功获得云南本地猪Mx1基因的cDNA序列,为研究云南本地猪Mx1蛋白的抗病毒活性及作用机制奠定了基础.%In order to obtain the cDNA sequence of Mxl gene of Yunnan local pig, primers were designed according to the sequences of porcine Mxl gene in GenBank. Fragments were amplified using RT-PCR from peripheral lymphocytes of Yunnan local pigs, and the fragment sequences were analyzed and spliced. The results demonstrated that the cDNA length of Mxl gene amplified from Yunnan local pigs was 2 546 bp, the open reading frame was 1 992 bp with encoding 663 amino acids. Compared with other Mxl gene sequences in GenBank, the homologies of nucleotide sequences were from 99. 4% to 99. 8% , those of amino acid sequences were from 98. 8% to 99. 5%. The cDNA sequence of Mxl gene in Yunnan local pigs was successfully obtained, which may facilitate the study on the antiviral activity and mechanism of Mxl protein in Yunnan local pigs.

  3. 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.

  4. Construction of Midgut Tissue-Specific cDNA Library of Bombyx mandarina M. and Isolation and Sequence Analysis of Serine Protease Gene Fragment%野桑蚕中肠组织cDNA文库的构建及丝氨酸蛋白酶基因片段的克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    王燕红; 李兵; 王东; 朱莎; 赵华强; 卫正国; 沈卫德

    2008-01-01

    [Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.

  5. HMGA1a recognition candidate DNA sequences in humans.

    Directory of Open Access Journals (Sweden)

    Takayuki Manabe

    Full Text Available High mobility group protein A1a (HMGA1a acts as an architectural transcription factor and influences a diverse array of normal biological processes. It binds AT-rich sequences, and previous reports have demonstrated HMGA1a binding to the authentic promoters of various genes. However, the precise sequences that HMGA1a binds to remain to be clarified. Therefore, in this study, we searched for the sequences with the highest affinity for human HMGA1a using an existing SELEX method, and then compared the identified sequences with known human promoter sequences. Based on our results, we propose the sequences "-(G/A-G-(A/T-(A/T-A-T-T-T-" as HMGA1a-binding candidate sequences. Furthermore, these candidate sequences bound native human HMGA1a from SK-N-SH cells. When candidate sequences were analyzed by performing FASTAs against all known human promoter sequences, 500-900 sequences were hit by each one. Some of the extracted genes have already been proven or suggested as HMGA1a-binding promoters. The candidate sequences presented here represent important information for research into the various roles of HMGA1a, including cell differentiation, death, growth, proliferation, and the pathogenesis of cancer.

  6. Functional expression of human NKCC1 from a synthetic cassette-based cDNA: introduction of extracellular epitope tags and removal of cysteines.

    Science.gov (United States)

    Somasekharan, Suma; Monette, Michelle Y; Forbush, Biff

    2013-01-01

    The Na-K-Cl cotransporter (NKCC) couples the movement of Na(+), K(+), and Cl(-) ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl(-) -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully "cys-less" NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.

  7. Functional expression of human NKCC1 from a synthetic cassette-based cDNA: introduction of extracellular epitope tags and removal of cysteines.

    Directory of Open Access Journals (Sweden)

    Suma Somasekharan

    Full Text Available The Na-K-Cl cotransporter (NKCC couples the movement of Na(+, K(+, and Cl(- ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl(- -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully "cys-less" NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.

  8. 岩栖蝮蛇类凝血酶纯化、cDNA克隆和序列分析%Purification, cDNA cloning and sequence analysis of thrombin-like enzyme from Gloydius saxatilis

    Institute of Scientific and Technical Information of China (English)

    孙德军; 杨春伟; 杨同书; 颜炜群; 王伟

    2003-01-01

    Thrombin-like enzyme has great medical application in treating thrombus. A thrombin-like enzyme from Gloydius saxatilis snake venom was isolated and purified to homogeneity by a rapid and effective method using ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on heparin-sepharose.SDS-polyacrylamide electrophoresis under reducing condition revealed that the purified enzyme had a single protein band and its molecular weight was 32000 dalton.Total RNAs were extracted from the venom gland of the G.saxatilis snake.Using degenerate primers,we amplified the cDNA of the thrombin-like enzyme gene in the venom gland of G.saxatilis using the reverse transcription-polymerase chainreaction (RT-PCR) method.The cDNA fragment was inserted into pGEMT vector,cloned and its nucleotide sequence was determined.Its open reading frame is composed of 774 nucleotides and codes a protein prezymogen of 258 amino acids,including a putative secretory signal peptide of 18 amino acids and a proposed pro-peptide of 6 amino acid residues.It contains 12 cysteine residues.The sequence analysis indicates that the deduced amino acid sequence of the cDNA fragment shares high identity with the thrombin-like enzyme genes of other snakes in the gene bank.The query sequence exhibits strong amino acid sequence homology of 88%,88% and 86% to the serine proteas of T.gramineus,thrombin-like defibrase Ⅰ of D.acutus and serine protease catroxase Ⅱ of C.atrox respectively.Based on the amino acid sequences of other thrombin-like enzymes,the catalytic residues and disulfide bridges of this thrombin-like enzyme are deduced as follows:catalytic residues,His65,Asp110,Ser%204;and six disulfide bridges Cys31-Cys163,Cys50-Cys66,Cys98-Cys256,Cys142-Cys210,Cys174-Cys189 and Cys200-Cys225.According to the possible linked glycosylation sites N-X-T (Asn-X-Thr) or N-X-S (Asn-X-Ser),its possible glycosylation sites are N44-S45-T46 and N251-T252-T253 residues [Acta Zoologica Sinica 49(6):878-882,2003].

  9. Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

    Energy Technology Data Exchange (ETDEWEB)

    Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.

    1987-12-01

    Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.

  10. cDNA cloning of the beta subunit of teleost thyrotropin.

    OpenAIRE

    Ito, M.; Koide, Y.; Takamatsu, N; Kawauchi, H.; Shiba, T.

    1993-01-01

    cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human fo...

  11. Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus: probing a human leukocyte cDNA library using the yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-kang; ZHAO Long-feng; CHENG Jun; GUO Jiang; LUN Yong-zhi; HONG Yuan

    2006-01-01

    Background The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS 1TP5 protein.Methods The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS 1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization.After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH 109 with Y 187 containing a leukocyte cDNA library plasmid.The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes

  12. 沙田柚无机焦磷酸酶基因的 cDNA 克隆及序列分析%Cloning and sequence analysis of inorganic pyrophosphatase gene from Citrus grandis var.shatianyu

    Institute of Scientific and Technical Information of China (English)

    秦新民; 万珊; 李惠敏; 覃屏生; 张渝

    2015-01-01

    related)and non-self(genetically unrelated)pollen.Inorganic pyrophosphatase(IPPase)play important roles in regu-lating the growth and development in plants.In order to better understand the mechanism of IPP gene in the self-in-compatibility of Citrus grandis var.shatianyu ,the inorganic pyrophosphatase gene of C .grandis var.shatianyu was cloned and physicochemical properties of pyrophosphatase were analyzed.The total RNA was isolated from style of C .grandis var.shatianyu used the total RNA Purification System(Invitrogen)and following the manufacture′s pro-tocols.According to the EST sequence(internal fragment of inorganic pyrophosphatase gene)in suppression subtrac-tive hybridization libraries of C .grandis var.shatianyu style,4 specific primers 5′-GSP1,5′-nGSP1,3′-GSP2 and 3′-nGSP2 were designed for amplifying 3-RACE and 5-RACE of the gene.The full-length sequences of cDNA of inor-ganic pyrophosphatase gene were obtained from suppression subtractive hybridization libraries of C .grandis var.shatianyu style by the SMART-RACE PCR method.A comparison of the similarity of the full-length cDNA se-quence of the inorganic pyrophosphatase gene was performed in the GenBank database used the BLAST program.DNAman software was used for mino acid sequence and homology analysis.Prediction of molecular weight, isoeletric point(pI)and hydrophobicity were performed by using on line software ExpASy and DNAman.A 150 bp band in 3-RACE,and a 900 bp band in 5-RACE were cloned by nested and non nested PCR method.For the full-length cDNA sequences,the middle sequence,3-RACE and 5-RACE sequence of the inorganic pyrophosphatase gene were spliced and formed the full-length cDNA sequences.As a result,the cDNA of inorganic pyrophosphatase was 1 136 bp in length containing an 654 bp open reading frame(ORF),which encoded a protein of 217 amino acids with a 170 bp 5 untranslated region(5 UTR)and a 321 bp 3 UTR.The sequence of the cloned cDNA of the inorganic pyro-phosphatase from C .grandis

  13. Sequencing and analysis of an Irish human genome.

    LENUS (Irish Health Repository)

    Tong, Pin

    2010-01-01

    Recent studies generating complete human sequences from Asian, African and European subgroups have revealed population-specific variation and disease susceptibility loci. Here, choosing a DNA sample from a population of interest due to its relative geographical isolation and genetic impact on further populations, we extend the above studies through the generation of 11-fold coverage of the first Irish human genome sequence.

  14. Complete Genome Sequence of the Human Gut Symbiont Roseburia hominis

    DEFF Research Database (Denmark)

    Travis, Anthony J.; Kelly, Denise; Flint, Harry J;

    2015-01-01

    We report here the complete genome sequence of the human gut symbiont Roseburia hominis A2-183(T) (= DSM 16839(T) = NCIMB 14029(T)), isolated from human feces. The genome is represented by a 3,592,125-bp chromosome with 3,405 coding sequences. A number of potential functions contributing to host-...

  15. Sulforaphane-induced apoptosis in human leukemia HL-60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray.

    Science.gov (United States)

    Shang, Hung-Sheng; Shih, Yung-Luen; Lee, Ching-Hsiao; Hsueh, Shu-Ching; Liu, Jia-You; Liao, Nien-Chieh; Chen, Yung-Liang; Huang, Yi-Ping; Lu, Hsu-Feng; Chung, Jing-Gung

    2017-01-01

    Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN-induced cytotoxic effects and whether or not they went through cell-cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL-60 cells. Cell viability, cell-cycle distribution, sub-G1 (apoptosis), reactive oxygen species (ROS) and Ca(2+) production, levels of mitochondrial membrane potential (ΔΨm ), and caspase-3, -8, and -9 activities were assayed by flow cytometry. Apoptosis-associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub-G1 phase development. Furthermore, SFN increased ROS and Ca(2+) production and decreased the levels of ΔΨm and activated caspase-3, -8, and -9 activities in HL-60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas-L, caspase-8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl-x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase -8, -3, -4, -6, and -7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL-60 cells via Fas- and mitochondria-dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311-328, 2017.

  16. The preparation of an infectious full-length cDNA clone of Saffold virus

    OpenAIRE

    Okuwa Takako; Shimizu Hiroyuki; Asif Naeem; Hosomi Takushi; Himeda Toshiki; Muraki Yasushi; Ohara Yoshiro

    2011-01-01

    Abstract The pathogenicity of Saffold virus (SAFV) among humans still remains unclear, although it was identified as a novel human cardiovirus in 2007. In order to encourage the molecular pathogenetic studies of SAFV, we generated an infectious cDNA clone of SAFV type 3 (SAFV-3). The present study demonstrated that the synthesis of the full-length infectious RNA by T7 RNA polymerase was terminated by a homologous sequence motif with the human preproparathyroid hormone (PTH) signal in the SAFV...

  17. GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP

    NARCIS (Netherlands)

    Qin Ling,; Prins, P.; Jones, J.T.; Popeijus, H.; Smant, G.; Bakker, J.; Helder, J.

    2001-01-01

    The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power

  18. Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

    Science.gov (United States)

    Herzner, Anna-Maria; Hagmann, Cristina Amparo; Goldeck, Marion; Wolter, Steven; Kübler, Kirsten; Wittmann, Sabine; Gramberg, Thomas; Andreeva, Liudmila; Hopfner, Karl-Peter; Mertens, Christina; Zillinger, Thomas; Jin, Tengchuan; Xiao, Tsan Sam; Bartok, Eva; Coch, Christoph; Ackermann, Damian; Hornung, Veit; Ludwig, Janos; Barchet, Winfried; Hartmann, Gunther; Schlee, Martin

    2015-10-01

    Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

  19. EXPRESSION OF cDNA FOR RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR IN ESCHERICHIA COLI AND CHARACTERIZATION OF THE PROTEIN

    Institute of Scientific and Technical Information of China (English)

    Zhang Shu; Ye Qinong

    1998-01-01

    Objective:To determine the biological activity of rhG-CSF and it's characterization. Methods: The prokaryotic expression vector pG01 containing human GCSF cDNA were constructed with DNA recombination technology. Results: We had achieved high level expression of the human G-CSF in E. Coli, where it represented at least 23.6% of the total protein as determined from SDS-PAGE gels. The human G-CSF was expressed as inclusion bodies in E. Coli. The inclusion bodies were solubilized in a solution containing 7M urea,renatured by dialysis, isolated and purified by DEAEsepharose CL-6B ion exchange and Superdex 75 gel filtration chromatography. The purified rhG-CSF was confirmed by coincidence of biological activity and protein demonstrated by SDS-PAGE. It was homogeneous with respect to mol. Wt (18400). The purity of the rhGCSF might be >90 per cent. Conclusion: The purified rhG-CSF in our laboratory had dramatically the biological activity of regulating proliferation and differentiation of the human G-CSF-dependent cell line NSF-1 and the progenitor cells of granulocytes of human bone marrow.

  20. Transcripts of pectin-degrading enzymes and isolation of complete cDNA sequence of a pectate lyase gene induced by coffee white stem borer (Xylotrechus quadripes) in the bark tissue of Coffea canephora (robusta coffee).

    Science.gov (United States)

    Bharathi, Kosaraju; Santosh, P; Sreenath, H L

    2017-05-01

    Of the two commercially cultivated coffee (Coffea) species, C. arabica (arabica) is highly susceptible and C. canephora (robusta) is highly resistant to the insect pest Xylotrechus quadripes (Coleoptera: Cerambycidae), commonly known as coffee white stem borer (CWSB). We constructed a forward-subtracted cDNA library by Suppression Subtractive Hybridization (SSH) from robusta bark tissue for profiling genes induced by CWSB infestation. Among the 265 unigenes of the SSH EST library, 7 unigenes (5 contigs and 2 singletons) matching different pectin-degrading enzymes were discovered. These ESTs matched one pectate lyase, three polygalacturonases, and one pectin acetylesterase gene. Quantitative real-time PCR (qRT-PCR) revealed that CWSB infestation strongly induces the pectate lyase gene at 72 h. Complete cDNA sequence of the pectate lyase gene was obtained through 3' and 5' RACE reactions. It was a 1595 bp long sequence that included full CDS and both UTRs. Against C. canephora genome sequences in Coffee Genome Hub database ( http://coffee-genome.org/ ), it had 22 matches to different pectate lyase genes mapped on 9 of the 11 pseudochromosomes, the top match being Cc07_g00190 Pectate lyase. In NCBI database, it matched pectate lyase sequences of several plants. Apart from C. canephora, the closest pectate lyase matches were from Sesamum indicum and Nicotiana tabacum. The pectinolytic enzymes discovered here are thought to play a role in the production of oligogalacturonides (OGs) which act as Damage-Associated Molecular Pattern (DAMP) signals eliciting innate immunity in plants. The pectate lyase gene, induced by CWSB infestation, along with other endogenous pectinolytic enzymes and CWSB-specific elicitors, may be involved in triggering basal defense responses to protect the CWSB-damaged tissue against pathogens, as well as to contain CWSB in robusta.

  1. A 7872 cDNA microarray and its use in bovine functional genomics.

    Science.gov (United States)

    Everts, Robin E; Band, Mark R; Liu, Z Lewis; Kumar, Charu G; Liu, Lei; Loor, Juan J; Oliveira, Rosane; Lewin, Harris A

    2005-05-15

    The strategy used to create and annotate a 7872 cDNA microarray from cattle placenta and spleen cDNA sequences is described. This microarray contains approximately 6300 unique genes, as determined by BLASTN and TBLASTX similarity search against the human and mouse UniGene and draft human genome sequence databases (build 34). Sequences on the array were annotated with gene ontology (GO) terms, thereby facilitating data analysis and interpretation. A total of 3244 genes were annotated with GO terms. The array is rich in sequences encoding transcription factors, signal transducers and cell cycle regulators. Current research being conducted with this array is described, and an overview of planned improvements in our microarray platform for cattle functional genomics is presented.

  2. Genome-Wide Screening of Genes Showing Altered Expression in Liver Metastases of Human Colorectal Cancers by cDNA Microarray

    Directory of Open Access Journals (Sweden)

    Rempei Yanagawa

    2001-01-01

    Full Text Available In spite of intensive and increasingly successful attempts to determine the multiple steps involved in colorectal carcinogenesis, the mechanisms responsible for metastasis of colorectal tumors to the liver remain to be clarified. To identify genes that are candidates for involvement in the metastatic process, we analyzed genome-wide expression profiles of 10 primary colorectal cancers and their corresponding metastatic lesions by means of a cDNA microarray consisting of 9121 human genes. This analysis identified 40 genes whose expression was commonly upregulated in metastatic lesions, and 7 that were commonly downregulated. The upregulated genes encoded proteins involved in cell adhesion, or remodeling of the actin cytoskeleton. Investigation of the functions of more of the altered genes should improve our understanding of metastasis and may identify diagnostic markers and/or novel molecular targets for prevention or therapy of metastatic lesions.

  3. 水稻EPSP合酶cDNA克隆、序列分析及其拷贝数测定%Isolation of Rice EPSP Synthase cDNA and Its Sequence Analysis and Copy Number Determination

    Institute of Scientific and Technical Information of China (English)

    徐军望; 魏晓丽; 李旭刚; 陈蕾; 冯德江; 朱祯

    2002-01-01

    根据本室分离的水稻EPSP合酶基因的基因组序列设计一对引物,利用RT-PCR方法首次从水稻(Oryza sativa L. subsp. indica)叶片的RNA中扩增获得了水稻编码EPSP合酶的全长为1 585 bp的cDNA片段,它含有一个完整的开放读码框,编码511个氨基酸,包括444个氨基酸组成的成熟肽序列以及N端的67个氨基酸组成的叶绿体转运肽序列.成熟肽氨基酸序列对比表明,除真菌来源的EPSP合酶变异较大外,其他来源的EPSP合酶同源性较高,均在51%以上.而叶绿体转运肽氨基酸序列同源性较低.Southern杂交表明水稻EPSP合酶基因在水稻基因组中以单拷贝形式存在.RT-PCR分析表明,水稻EPSP合酶基因在根、未成熟种子和叶片中均有转录表达,在叶片中表达量最高.%In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first-strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in previous study. A 1 585-bp cDNA fragment was amplified and cloned. The 1 585-bp cDNA contains an open reading frame (ORF) comprising of 1 533 nucleotides (nt) which encodes a 511 residue polypepetides, including 67 amino acids chloroplast transit peptide and 444 amino acids EPSP synthase mature peptide. A comparison between the EPSP synthase of different sources indicates that the mature peptide shows more than 51% identity except for the fungi EPSP synthase and the transit peptide shows considerably less sequence conservation. The copy number of rice epsps gene is estimated to be one copy per haploid rice genome using southern blot. RT-PCR indicated that rice epsps gene is expressed in rice leaves, endosperms and roots and has the highest expression level in leaves.

  4. 人干细胞因子(SCF)5’旁侧调控序列与 其全长cDNA融合克隆的构建及鉴定%Construction and identification of the fusion clone of the SCF 5’flanking sequence and its full-length cDNA

    Institute of Scientific and Technical Information of China (English)

    谭运年; 谭文斌; 彭兴华

    2001-01-01

    Objective:To study the effect of different length DNA sequences of the 5’SCF 1.42 kb flanking sequence in the expression and regulation of full-length cDNA in eukaryotic cells,a fused clone of the 5’SCF 1.42kb flanking sequence and its full-length cDNA were constructed.Methods:A 1.42 kb flanking sequence and a 1.2 kb full-length cDNA were achieved by PCR from human genomic DNA and by RT-PCR from HepG2 mPNA,respectively,and then cloned into pGEM-T cloning vector and identified.Both clones were digested with fitly restricted endonuclease and three DNA fragments,480 bp,980 bp,1.2 kb cDNA were harvested.Finally,these three DNA fragments were subcloned into pUC19 cloning vector in turn.Results:The fused clone of 5’SCF 1.42 kb flanking sequence and its full-length cDNA were successfully constructed.Conclusion:To avoid the interruption of some restricted endonuclease sites,the way to cut larger fragments into smaller fragments is still useful and effective in the process of gene cloning%目的:为研究人干细胞因子(SCF)5’旁侧1.42 kb区域内不同序列对全长cDNA在真核细胞中表达的调控作用,构建了SCF5’旁侧1.42 kb的调控序列与其1.2 kb的全长cDNA融合克隆。方法:将PCR获得的SCF5’旁侧1.42 kb的调控序列与RT-PCR获得的其1.2 kb的全长cDNA克隆入pGEM-T载体,筛选正确插入方向,利用合适的限制性内切酶从中切出三个片段依次亚克隆入pUC19克隆载体中。结果:获得了SCF5’旁侧 1.42 kb的调控序列与其1.2 kb的全长cDNA并成功地构建了它们的融合克隆。结论:T-载体在克隆添加了少量具有3’→5’外切核酸酶的PCR产物中仍然有效;在稍大片段的基因克隆操作中,利用分段亚克隆的方法,避开干扰另外的酶切位点,依次分段亚克隆更为可行。

  5. Nucleotide sequence of a tobacco cDNA encoding plastidic glutamine synthetase and light inducibility, organ specificity and diurnal rhythmicity in the expression of the corresponding genes of tobacco and tomato.

    Science.gov (United States)

    Becker, T W; Caboche, M; Carrayol, E; Hirel, B

    1992-06-01

    A full-length cDNA encoding glutamine synthetase (GS) was cloned from a lambda gt10 library of tobacco leaf RNA, and the nucleotide sequence was determined. An open reading frame accounting for a primary translation product consisting of 432 amino acids has been localized on the cDNA. The calculated molecular mass of the encoded protein is 47.2 kDa. The predicted amino acid sequence of this precursor shows higher homology to GS-2 protein sequences from other species than to a leaf GS-1 polypeptide sequence, indicating that the cDNA isolated encodes the chloroplastic isoform (GS-2) of tobacco GS. The presence of C- and N-terminal extensions which are characteristic of GS-2 proteins supports this conclusion. Genomic Southern blot analysis indicated that GS-2 is encoded by a single gene in the diploid genomes of both tomato and Nicotiana sylvestris, while two GS-2 genes are very likely present in the amphidiploid tobacco genome. Western blot analysis indicated that in etiolated and in green tomato cotyledons GS-2 subunits are represented by polypeptides of similar size, while in green tomato leaves an additional GS-2 polypeptide of higher apparent molecular weight is detectable. In contrast, tobacco GS-2 is composed of subunits of identical size in all organs examined. GS-2 transcripts and GS-2 proteins could be detected at high levels in the leaves of both tobacco or tomato. Lower amounts of GS-2 mRNA were detected in stems, corolla, and roots of tomato, but not in non-green organs of tobacco. The GS-2 transcript abundance exhibited a diurnal fluctuation in tomato leaves but not in tobacco leaves. White or red light stimulated the accumulation of GS-2 transcripts and GS-2 protein in etiolated tomato cotyledons. Far-red light cancelled this stimulation. The red light response of the GS-2 gene was reduced in etiolated seedlings of the phytochrome-deficient aurea mutant of tomato. These results indicate a phytochrome-mediated light stimulation of GS-2 gene expression

  6. 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.

  7. Screening Target Specificity of siRNAs by Rapid Amplification of cDNA Ends (RACE) for Non-Sequenced Species

    OpenAIRE

    Sabirzhanov, Boris; Sabirzhanova, Inna B.; Keifer, Joyce

    2011-01-01

    RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propos...

  8. Generation and analysis of a large-scale expressed sequence Tag database from a full-length enriched cDNA library of developing leaves of Gossypium hirsutum L.

    Directory of Open Access Journals (Sweden)

    Min Lin

    Full Text Available BACKGROUND: Cotton (Gossypium hirsutum L. is one of the world's most economically-important crops. However, its entire genome has not been sequenced, and limited resources are available in GenBank for understanding the molecular mechanisms underlying leaf development and senescence. METHODOLOGY/PRINCIPAL FINDINGS: In this study, 9,874 high-quality ESTs were generated from a normalized, full-length cDNA library derived from pooled RNA isolated from throughout leaf development during the plant blooming stage. After clustering and assembly of these ESTs, 5,191 unique sequences, representative 1,652 contigs and 3,539 singletons, were obtained. The average unique sequence length was 682 bp. Annotation of these unique sequences revealed that 84.4% showed significant homology to sequences in the NCBI non-redundant protein database, and 57.3% had significant hits to known proteins in the Swiss-Prot database. Comparative analysis indicated that our library added 2,400 ESTs and 991 unique sequences to those known for cotton. The unigenes were functionally characterized by gene ontology annotation. We identified 1,339 and 200 unigenes as potential leaf senescence-related genes and transcription factors, respectively. Moreover, nine genes related to leaf senescence and eleven MYB transcription factors were randomly selected for quantitative real-time PCR (qRT-PCR, which revealed that these genes were regulated differentially during senescence. The qRT-PCR for three GhYLSs revealed that these genes express express preferentially in senescent leaves. CONCLUSIONS/SIGNIFICANCE: These EST resources will provide valuable sequence information for gene expression profiling analyses and functional genomics studies to elucidate their roles, as well as for studying the mechanisms of leaf development and senescence in cotton and discovering candidate genes related to important agronomic traits of cotton. These data will also facilitate future whole-genome sequence

  9. Structure, sequence, expression, and chromosomal localization of the human V{sub 1a} vasopressin receptor gene

    Energy Technology Data Exchange (ETDEWEB)

    Thibonnier, M.; Graves, M.K.; Wagner, M.S. [Case Western Reserve Univ. School of Medicine, Cleveland, OH (United States)] [and others

    1996-02-01

    We recently reported the structure and functional expression of a human V{sub 1a} vasopressin receptor (V{sub 1a}R) cDNA isolated from human liver cDNA libraries. To understand further the expression and regulation of the V{sub 1a}R, we now describe the genomic characteristics, tissue expression, chromosomal localization, and regional mapping of the human V{sub 1a}R gene, AVPR1A. Tissue distribution of the human V{sub 1a}R mRNA explored by Northern blot analysis of various human tissues or organs revealed the presence of a 5.5-kb mRNA transcript expressed in the liver and to a lesser degree in the heart, the kidney, and skeletal muscle. Screening of human genomic libraries revealed that the human AVPR1A gene is included entirely within a 6.4-kb rated by a 2.2-kb intron located before the corresponding seventh transmembrane domain of the receptor sequence. The first exon also contains 2 kb of 5{prime}-untranslated region, and the second exon includes 1 kb of 3{prime}-untranslated region. 5{prime}-RACE analysis of human liver mRNA by PCR localized the V{sub 1a}R mRNA transcription start site 1973 bp upstream of the translation the intron sequence were used as primers in polymerase chain reaction (PCR) analysis of human/rodent somatic cell hybrids. AVPR1A was localized by PCR analysis of a somatic cell hybrid panel to chromosome 12. Fluorescence in situ hybridization using a yeast artificial chromosome physically mapped AVPR1A to region 12q14-q15. 34 refs., 4 figs.

  10. Stress responses in alfalfa (Medicago sativa L.) 12. Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants.

    Science.gov (United States)

    Gowri, G; Paiva, N L; Dixon, R A

    1991-09-01

    An expression library containing cDNAs derived from transcripts from fungal elicitor-treated alfalfa cell suspension cultures was screened with an antiserum raised against phenylalanine ammonia-lyase (PAL) from alfalfa. A single immunoreactive clone was isolated which encoded a full-length PAL cDNA (APAL1) consisting of a 2175 bp open reading frame, 96 bp 5'-untranslated leader and 128 bp 3'-non-coding region. The deduced amino acid sequence was 86.5% similar to that of the PAL2 gene of bean, and encoded a polypeptide of Mr 78,865. A second PAL cDNA species was isolated, whose 3'-untranslated region was 86% identical to that of APAL1. Southern blot analysis indicated that PAL is encoded by a small multigene family in alfalfa. PAL transcript levels were rapidly and massively induced, and preceded increased PAL extractable activity, on exposure of alfalfa suspension cells to elicitor from baker's yeast. PAL transcripts were most abundant in roots, stems and petioles during growth and development of alfalfa seedlings. These studies provide the basis for an examination of the developmental and environmental control of a key enzyme of phenylpropanoid synthesis in a plant species which is readily amenable to stable genetic transformation.

  11. Cloning of the VASP (Vasodilator-Stimulated Phosphoprotein) genes in human and mouse: Structure, sequence, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Zimmer, M.; Fischer, L.; Hauser, W. [Medizinische Universitaetsklinik, Wuerzburg (Germany)] [and others

    1996-09-01

    The genes encoding the vasodilator-stimulated phosphoprotein (VASP) in human and mouse were isolated, and major parts were sequenced. In both species the gene is composed of 13 exons with conserved exon-intron positions. The mouse VASP cDNA sequence was deduced from the genomic sequence. The predicted amino acid sequence is 89% identical to the human protein. The high nucleotide sequence homology extends not only over the coding regions but also into the 3{prime}-UTRs, indicating a possible function in mRNA targeting or regulation of translation. Prominent 5{prime} CpG islands including multiple SP1 sites indicate a housekeeping function of VASP. Using cosmid DNA as a probe for fluorescence in situ hybridization, the human VASP gene was assigned to chromosome 19q13.2-q13.3, an extended region with homology to mouse chromosome 7. A sequence overlap of the VASP 5{prime}-region with the telomeric end of a cosmid contig physically links the VASP gene with ERCC1. VASP is located about 92 kb distal to ERCC1 and about 300 kb proximal to the myotonic dystrophy protein kinase gene. 43 refs., 6 figs.

  12. Building the sequence map of the human pan-genome

    DEFF Research Database (Denmark)

    Li, Ruiqiang; Li, Yingrui; Zheng, Hancheng

    2010-01-01

    Here we integrate the de novo assembly of an Asian and an African genome with the NCBI reference human genome, as a step toward constructing the human pan-genome. We identified approximately 5 Mb of novel sequences not present in the reference genome in each of these assemblies. Most novel...... analysis of predicted genes indicated that the novel sequences contain potentially functional coding regions. We estimate that a complete human pan-genome would contain approximately 19-40 Mb of novel sequence not present in the extant reference genome. The extensive amount of novel sequence contributing...... to the genetic variation of the pan-genome indicates the importance of using complete genome sequencing and de novo assembly....

  13. Targeted sequencing of the human X chromosome exome.

    Science.gov (United States)

    Mondal, Kajari; Shetty, Amol Carl; Patel, Viren; Cutler, David J; Zwick, Michael E

    2011-10-01

    We used a RainDance Technologies (RDT) expanded content library to enrich the human X chromosome exome (2.5 Mb) from 26 male samples followed by Illumina sequencing. Our multiplex primer library covered 98.05% of the human X chromosome exome in a single tube with 11,845 different PCR amplicons. Illumina sequencing of 24 male samples showed coverage for 97% of the targeted sequences. Sequence from 2 HapMap samples confirmed missing data rates of 2-3% at sites successfully typed by the HapMap project, with an accuracy of at least ~99.5% as compared to reported HapMap genotypes. Our demonstration that a RDT expanded content library can efficiently enrich and enable the routine sequencing of the human X chromosome exome suggests a wide variety of potential research and clinical applications for this platform.

  14. Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP)

    Energy Technology Data Exchange (ETDEWEB)

    Hoskins, Roger A.; Stapleton, Mark; George, Reed A.; Yu, Charles; Wan, Kenneth H.; Carlson, Joseph W.; Celniker, Susan E.

    2005-04-22

    The production of comprehensive cDNA clone collections is an important goal of the human and model organism genome projects. cDNA sequences are used to determine the structures of transcripts, including splice junctions, polyadenylation sites, and 5' and 3' untranslated regions (UTRs). cDNA collections are also valuable resources for functional studies of genes and proteins. Expressed Sequence Tag (EST)sequencing is the method of choice for recovering cDNAs representing a majority of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a library at random, so it realizes diminishing returns as the project progresses. To drive cDNA collections toward completion new methods are needed to recover cDNAs representing specific genes and alternative transcripts, including transcripts with low expression levels. We describe a simple and effective inverse-PCR-based method for screening plasmid libraries to recover intact cDNAs for specific transcripts. We tested the method by screening libraries used in our Drosophila EST projects for 153 transcription factor genes that were not yet represented by full-length cDNAs. We recovered target-specific clones for 104 of the genes: 46 exactly match, 30 improve and 28partially match current gene annotations. Successful application of the screening method depends on cDNA library complexity and quality of the gene models. The approach should be effective for improving cDNA collections for other model organisms and the human. It also provides a simple and rapid method for isolating cDNAs of interest in any system for which plasmid cDNA libraries and complete or partial gene sequences are available.

  15. Sequence and expression analysis of gaps in human chromosome 20

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Seemann, Stefan; Mang, Yuan;

    2012-01-01

    The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 (chr 20) currently has three unfinished gaps remaining on its q-arm. All three gaps are within gene-dense regions and....../or overlap disease-associated loci, including the DLGAP4 locus. In this study, we sequenced ~99% of all three unfinished gaps on human chr 20, determined their complete genomic sizes and assessed epigenetic profiles using a combination of Sanger sequencing, mate pair paired-end high-throughput sequencing...... and chromatin, methylation and expression analyses. We found histone 3 trimethylated at Lysine 27 to be distributed across all three gaps in immortalized B-lymphocytes. In one gap, five novel CpG islands were predominantly hypermethylated in genomic DNA from peripheral blood lymphocytes and human cerebellum...

  16. Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding

    Directory of Open Access Journals (Sweden)

    Douglas Carl J

    2008-01-01

    Full Text Available Abstract Background The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions. Results As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa × P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones

  17. Main: Sequences [KOME

    Lifescience Database Archive (English)

    Full Text Available Sequences Nucleotide Sequence Nucleotide sequence of full length cDNA (trimmed sequence) kome_ine_full_seq...uence_db.fasta.zip kome_ine_full_sequence_db.zip kome_ine_full_sequence_db ...

  18. Cloning, sequence analysis and detection of vitellogenin cDNA from Colisa fasciata%条纹密鲈卵黄蛋白原基因的克隆与检测

    Institute of Scientific and Technical Information of China (English)

    梁岳; 方展强

    2011-01-01

    Using RT-PCR,the partial length vitellogenin( VTG)cDNA sequence and (3-actin cDNA sequence of Colisa fasciata were cloned. VTG cDNA sequence contains 3 464 bp nucleotides and encodes 1 150 amino acids, p-actin cDNA sequence contains 1 253 bp nucleotides and encodes 375 amino acids. In vivo and in vitro methods were employed to investigate VTG mRNA expression under exposure to estradiol (E2), octylphenol (OP), cadmium ( Cd2 + ) and perfluorooctane sulfonates ( PFOS) and evaluate the estrogenic activity. The results showed that E2 and OP could induce VTG mRNA expression in dose-dependent way by in vivo and in vitro test. Cd2+ could induce VTG mRNA expression only in the low dose by in vivo test,but VTG mRNA expression was not observed in PFOS groups by in vivo and in vitro test. The results indicated that the strength of estrogenic effects was in the order E2 > OP > Cd2 +. Cd2 + estrogenic effects in vivo and in vitro results are inconsistent, suggesting that the mechanism of Cd2 + induced effects of estrogen and E2 may be different. The results also indicated that VTG cDNA of C. Fasciata is very sensitive to environmental hormone and very suitable to be a biomarker for monitoring the environmental hormones.%采用RT-PCR方法克隆并分析了条纹密鲈卵黄蛋白原(VTG)和β-肌动蛋白(β-actin)cDNA部分序列.获得的VTG cDNA序列片段长3464 bp,全部处于编码区,编码1150个氨基酸;β-actin cDNA序列片段长1253 bp,编码375个氨基酸.使用活体与离体的实验方法,检测了VTG mRNA转录情况,并以此评价雌二醇(E2)、辛基酚(OP)、镉(Cd2+)和全氟辛烷磺酸类化合物( PFOS)引起的雌激素效应.结果显示,E2和OP在活体和离体实验中均能剂量依赖性地诱导VTG mRNA表达.Cd2+仅在活体实验低剂量组诱导VTG mRNA表达,PFOS在活体和离体实验的各个浓度组均未见显著的VTG mRNA表达.结果表明,所诱导的雌激素效应强弱的排列顺序为E2>OP> Cd2.Cd2的雌激素效应

  19. An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

    Science.gov (United States)

    Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

    2012-07-01

    cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

  20. cDNA cloning and immunological characterization of the rye grass allergen Lol p I.

    Science.gov (United States)

    Perez, M; Ishioka, G Y; Walker, L E; Chesnut, R W

    1990-09-25

    The complete amino acid sequence of two "isoallergenic" forms of Lol p I, the major rye grass (Lolium perenne) pollen allergen, was deduced from cDNA sequence analysis. cDNA clones isolated from a Lolium perenne pollen library contained an open reading frame coding for a 240-amino acid protein. Comparison of the nucleotide and deduced amino acid sequence of two of these clones revealed four changes at the amino acid level and numerous nucleotide differences. Both clones contained one possible asparagine-linked glycosylation site. Northern blot analysis shows one RNA species of 1.2 kilobases. Based on the complete amino acid sequence of Lol p I, overlapping peptides covering the entire molecule were synthesized. Utilizing these peptides we have identified a determinant within the Lol p I molecule that is recognized by human leukocyte antigen class II-restricted T cells obtained from persons allergic to rye grass pollen.

  1. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Pittius, C.W.; Hennighausen, L.; Lee, E.; Westphal, H.; Nicols, E.; Vitale, J.; Gordon, K. (National Institutes of Health, Bethesda, MD (USA))

    1988-08-01

    Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.

  2. De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora).

    Science.gov (United States)

    Liu, Le; Zhang, Shijie; Lian, Chunlan

    2015-12-04

    Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora.

  3. De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora

    Directory of Open Access Journals (Sweden)

    Le Liu

    2015-12-01

    Full Text Available Japanese red pine (Pinus densiflora is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98% unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%, Amborella trichopoda (9.83%, and Pinus taeda (4.15%. A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite software, of which the tri-nucleotide repeats were most abundant (50.18% and 629 EST-SSR (expressed sequence tag- simple sequence repeats primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s mapping for pine for P. densiflora.

  4. Mutation of miRNA target sequences during human evolution

    DEFF Research Database (Denmark)

    Gardner, Paul P; Vinther, Jeppe

    2008-01-01

    It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes...... containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits.......It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes...

  5. Deep sequencing extends the diversity of human papillomaviruses in human skin.

    OpenAIRE

    Bzhalava, Davit; Mühr, Laila Sara Arroyo; Lagheden, Camilla; Ekström, Johanna; Forslund, Ola; Dillner, Joakim; Hultin, Emilie

    2014-01-01

    Most viruses in human skin are known to be human papillomaviruses (HPVs). Previous sequencing of skin samples has identified 273 different cutaneous HPV types, including 47 previously unknown types. In the present study, we wished to extend prior studies using deeper sequencing. This deeper sequencing without prior PCR of a pool of 142 whole genome amplified skin lesions identified 23 known HPV types, 3 novel putative HPV types and 4 non-HPV viruses. The complete sequence was obtained for one...

  6. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

    DEFF Research Database (Denmark)

    Gododkin, Jan; Cirera, Susanna; Hedegaard, Jakob

    2007-01-01

    Background: Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from ...

  7. A human gut microbial gene catalogue established by metagenomic sequencing

    DEFF Research Database (Denmark)

    dos Santos, Marcelo Bertalan Quintanilha; Sicheritz-Pontén, Thomas; Nielsen, Henrik Bjørn

    2010-01-01

    , from faecal samples of 124 European individuals. The gene set, ,150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes......To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence...

  8. Expressed sequence tag analysis of adult human optic nerve for NEIBank: Identification of cell type and tissue markers

    Directory of Open Access Journals (Sweden)

    Peterson Katherine

    2009-09-01

    Full Text Available Abstract Background The optic nerve is a pure white matter central nervous system (CNS tract with an isolated blood supply, and is widely used in physiological studies of white matter response to various insults. We examined the gene expression profile of human optic nerve (ON and, through the NEIBANK online resource, to provide a resource of sequenced verified cDNA clones. An un-normalized cDNA library was constructed from pooled human ON tissues and was used in expressed sequence tag (EST analysis. Location of an abundant oligodendrocyte marker was examined by immunofluorescence. Quantitative real time polymerase chain reaction (qRT-PCR and Western analysis were used to compare levels of expression for key calcium channel protein genes and protein product in primate and rodent ON. Results Our analyses revealed a profile similar in many respects to other white matter related tissues, but significantly different from previously available ON cDNA libraries. The previous libraries were found to include specific markers for other eye tissues, suggesting contamination. Immune/inflammatory markers were abundant in the new ON library. The oligodendrocyte marker QKI was abundant at the EST level. Immunofluorescence revealed that this protein is a useful oligodendrocyte cell-type marker in rodent and primate ONs. L-type calcium channel EST abundance was found to be particularly low. A qRT-PCR-based comparative mammalian species analysis reveals that L-type calcium channel expression levels are significantly lower in primate than in rodent ON, which may help account for the class-specific difference in responsiveness to calcium channel blocking agents. Several known eye disease genes are abundantly expressed in ON. Many genes associated with normal axonal function, mRNAs associated with axonal transport, inflammation and neuroprotection are observed. Conclusion We conclude that the new cDNA library is a faithful representation of human ON and EST data

  9. Antisense EGFR sequence reverses the growth properties of human liver carcinoma cell line BEL-7404 in vitro

    Institute of Scientific and Technical Information of China (English)

    XUYONGHUA; WANLIJIANG; SUFENGPENG; YINGHUACHEN

    1993-01-01

    A recombinant plasmid containing a full length human epidermal growth factor receptor (EGFR) cDNA sequence in antisense orientation was transferred into cells of a human liver carcinoma cell line BEL-7404. Compared with the control cell clone JX-0 transferred with the vector plasmid and the parent BEL-7404 cells, the antisense EGFR transferred cell clone JX-1 showed a decreased EGFR gene expression and reduced significantly the growth potential either in anchorage-dependent or anchorage-independent growth. Furthermore. JX-1 cells appeared to be distinctly dependent on serum concentration for monolayer growth. The results suggested that antisense EGFR could partly block the EGFR gene ex-pression and reverse the malignant growth properties of human liver carcinoma cells in vitro.

  10. Study on a cDNA sequence of cold inducible zinc finger protein in albinism tea cultivar "Xiaoxueya"%茶树品种“小雪芽”冷诱导锌指蛋白基因cDNA研究

    Institute of Scientific and Technical Information of China (English)

    王开荣; 李娜娜; 陆建良; 郑新强; 梁月荣; 吴颖; 李明

    2012-01-01

    A cDNA sequence of cold inducible zinc finger protein in leaf of albinism tea cultivar " Xiaoxueya" was investigated. The results shows that the cDNA sequence had 698 bp in length, with 83% and 82% identity to zinc finger protein mRNA of Glycine max and Ricinus communis respectively. It had an opening reading frame encoding 230 amino acids. Compared to that of common tea cuhivar " Fudingdabai" , there were three loci of nucleotide deletion and one locus of nucleotide substitution. Its deduced amino acid sequence had 99% identity to that of cuhivar " Fudingdabai" , among which there were 3 loci of amino acid substitution. The expression of cold inducible zinc finger protein in " Xiaoxueya" ,sas significantly lower than in " Fudingdabai". It is considered that the mutation in gene sequence resulted in the sensitivity of cultivar "Xiaoxueya" to low temperature through low expression and mutation of the cold inducible zinc finger protein.%分析了低温诱导型新梢白化茶树品种“小雪芽”叶片低温诱导锌指蛋白基因cDNA序列。该序列长度为698bp,与大豆锌指蛋白mRNA同源性为83%,与蓖麻锌指蛋白mRNA同源性为82%;具有可编码230个氨基酸的开放阅读框。与“福鼎大白茶”冷诱导锌指蛋白eDNA序列相比,该eDNA序列在50—51位上核苷酸AT缺失,第143位的A被置换为G,第654位T缺失。其翻译的蛋白质氨基酸序列与“福鼎大白茶”同源性达到99%,但有3个位点的氨基酸变异。该基因表达丰度明显低于“福鼎大白茶”。研究认为,基因结构差异,引起表达强度和蛋白质氨基酸序列的差异,可能是引起“小雪芽”品种对低温敏感的重要因素。

  11. The finished DNA sequence of human chromosome 12.

    Science.gov (United States)

    Scherer, Steven E; Muzny, Donna M; Buhay, Christian J; Chen, Rui; Cree, Andrew; Ding, Yan; Dugan-Rocha, Shannon; Gill, Rachel; Gunaratne, Preethi; Harris, R Alan; Hawes, Alicia C; Hernandez, Judith; Hodgson, Anne V; Hume, Jennifer; Jackson, Andrew; Khan, Ziad Mohid; Kovar-Smith, Christie; Lewis, Lora R; Lozado, Ryan J; Metzker, Michael L; Milosavljevic, Aleksandar; Miner, George R; Montgomery, Kate T; Morgan, Margaret B; Nazareth, Lynne V; Scott, Graham; Sodergren, Erica; Song, Xing-Zhi; Steffen, David; Lovering, Ruth C; Wheeler, David A; Worley, Kim C; Yuan, Yi; Zhang, Zhengdong; Adams, Charles Q; Ansari-Lari, M Ali; Ayele, Mulu; Brown, Mary J; Chen, Guan; Chen, Zhijian; Clerc-Blankenburg, Kerstin P; Davis, Clay; Delgado, Oliver; Dinh, Huyen H; Draper, Heather; Gonzalez-Garay, Manuel L; Havlak, Paul; Jackson, Laronda R; Jacob, Leni S; Kelly, Susan H; Li, Li; Li, Zhangwan; Liu, Jing; Liu, Wen; Lu, Jing; Maheshwari, Manjula; Nguyen, Bao-Viet; Okwuonu, Geoffrey O; Pasternak, Shiran; Perez, Lesette M; Plopper, Farah J H; Santibanez, Jireh; Shen, Hua; Tabor, Paul E; Verduzco, Daniel; Waldron, Lenee; Wang, Qiaoyan; Williams, Gabrielle A; Zhang, Jingkun; Zhou, Jianling; Allen, Carlana C; Amin, Anita G; Anyalebechi, Vivian; Bailey, Michael; Barbaria, Joseph A; Bimage, Kesha E; Bryant, Nathaniel P; Burch, Paula E; Burkett, Carrie E; Burrell, Kevin L; Calderon, Eliana; Cardenas, Veronica; Carter, Kelvin; Casias, Kristal; Cavazos, Iracema; Cavazos, Sandra R; Ceasar, Heather; Chacko, Joseph; Chan, Sheryl N; Chavez, Dean; Christopoulos, Constantine; Chu, Joseph; Cockrell, Raynard; Cox, Caroline D; Dang, Michelle; Dathorne, Stephanie R; David, Robert; Davis, Candi Mon'Et; Davy-Carroll, Latarsha; Deshazo, Denise R; Donlin, Jeremy E; D'Souza, Lisa; Eaves, Kristy A; Egan, Amy; Emery-Cohen, Alexandra J; Escotto, Michael; Flagg, Nicole; Forbes, Lisa D; Gabisi, Abdul M; Garza, Melissa; Hamilton, Cerissa; Henderson, Nicholas; Hernandez, Omar; Hines, Sandra; Hogues, Marilyn E; Huang, Mei; Idlebird, DeVincent G; Johnson, Rudy; Jolivet, Angela; Jones, Sally; Kagan, Ryan; King, Laquisha M; Leal, Belita; Lebow, Heather; Lee, Sandra; LeVan, Jaclyn M; Lewis, Lakeshia C; London, Pamela; Lorensuhewa, Lorna M; Loulseged, Hermela; Lovett, Demetria A; Lucier, Alice; Lucier, Raymond L; Ma, Jie; Madu, Renita C; Mapua, Patricia; Martindale, Ashley D; Martinez, Evangelina; Massey, Elizabeth; Mawhiney, Samantha; Meador, Michael G; Mendez, Sylvia; Mercado, Christian; Mercado, Iracema C; Merritt, Christina E; Miner, Zachary L; Minja, Emmanuel; Mitchell, Teresa; Mohabbat, Farida; Mohabbat, Khatera; Montgomery, Baize; Moore, Niki; Morris, Sidney; Munidasa, Mala; Ngo, Robin N; Nguyen, Ngoc B; Nickerson, Elizabeth; Nwaokelemeh, Ogechi O; Nwokenkwo, Stanley; Obregon, Melissa; Oguh, Maryann; Oragunye, Njideka; Oviedo, Rodolfo J; Parish, Bridgette J; Parker, David N; Parrish, Julia; Parks, Kenya L; Paul, Heidie A; Payton, Brett A; Perez, Agapito; Perrin, William; Pickens, Adam; Primus, Eltrick L; Pu, Ling-Ling; Puazo, Maria; Quiles, Miyo M; Quiroz, Juana B; Rabata, Dina; Reeves, Kacy; Ruiz, San Juana; Shao, Hongmei; Sisson, Ida; Sonaike, Titilola; Sorelle, Richard P; Sutton, Angelica E; Svatek, Amanda F; Svetz, Leah Anne; Tamerisa, Kavitha S; Taylor, Tineace R; Teague, Brian; Thomas, Nicole; Thorn, Rachel D; Trejos, Zulma Y; Trevino, Brenda K; Ukegbu, Ogechi N; Urban, Jeremy B; Vasquez, Lydia I; Vera, Virginia A; Villasana, Donna M; Wang, Ling; Ward-Moore, Stephanie; Warren, James T; Wei, Xuehong; White, Flower; Williamson, Angela L; Wleczyk, Regina; Wooden, Hailey S; Wooden, Steven H; Yen, Jennifer; Yoon, Lillienne; Yoon, Vivienne; Zorrilla, Sara E; Nelson, David; Kucherlapati, Raju; Weinstock, George; Gibbs, Richard A

    2006-03-16

    Human chromosome 12 contains more than 1,400 coding genes and 487 loci that have been directly implicated in human disease. The q arm of chromosome 12 contains one of the largest blocks of linkage disequilibrium found in the human genome. Here we present the finished sequence of human chromosome 12, which has been finished to high quality and spans approximately 132 megabases, representing approximately 4.5% of the human genome. Alignment of the human chromosome 12 sequence across vertebrates reveals the origin of individual segments in chicken, and a unique history of rearrangement through rodent and primate lineages. The rate of base substitutions in recent evolutionary history shows an overall slowing in hominids compared with primates and rodents.

  12. Repeat Sequences and Base Correlations in Human Y Chromosome Palindromes

    Institute of Scientific and Technical Information of China (English)

    Neng-zhi Jin; Zi-xian Liu; Yan-jiao Qi; Wen-yuan Qiu

    2009-01-01

    On the basis of information theory and statistical methods, we use mutual information, n-tuple entropy and conditional entropy, combined with biological characteristics, to analyze the long range correlation and short range correlation in human Y chromosome palindromes. The magnitude distribution of the long range correlation which can be reflected by the mutual information is P5>P5a>P5b (P5a and P5b are the sequences that replace solely Alu repeats and all interspersed repeats with random uncorrelated sequences in human Y chromosome palindrome 5, respectively); and the magnitude distribution of the short range correlation which can be reflected by the n-tuple entropy and the conditional entropy is P5>P5a>P5b>random uncorrelated sequence. In other words, when the Alu repeats and all interspersed repeats replace with random uncorrelated sequence, the long range and short range correlation decrease gradually. However, the random uncorrelated sequence has no correlation. This research indicates that more repeat sequences result in stronger correlation between bases in human Y chromosome. The analyses may be helpful to understand the special structures of human Y chromosome palindromes profoundly.

  13. Sequence-level mechanisms of human epigenome evolution.

    Science.gov (United States)

    Prendergast, James G D; Chambers, Emily V; Semple, Colin A M

    2014-06-24

    DNA methylation and chromatin states play key roles in development and disease. However, the extent of recent evolutionary divergence in the human epigenome and the influential factors that have shaped it are poorly understood. To determine the links between genome sequence and human epigenome evolution, we examined the divergence of DNA methylation and chromatin states following segmental duplication events in the human lineage. Chromatin and DNA methylation states were found to have been generally well conserved following a duplication event, with the evolution of the epigenome largely uncoupled from the total number of genetic changes in the surrounding DNA sequence. However, the epigenome at tissue-specific, distal regulatory regions was observed to be unusually prone to diverge following duplication, with particular sequence differences, altering known sequence motifs, found to be associated with divergence in patterns of DNA methylation and chromatin. Alu elements were found to have played a particularly prominent role in shaping human epigenome evolution, and we show that human-specific AluY insertion events are strongly linked to the evolution of the DNA methylation landscape and gene expression levels, including at key neurological genes in the human brain. Studying paralogous regions within the same sample enables the study of the links between genome and epigenome evolution while controlling for biological and technical variation. We show DNA methylation and chromatin divergence between duplicated regions are linked to the divergence of particular genetic motifs, with Alu elements having played a disproportionate role in the evolution of the epigenome in the human lineage.

  14. Strategies for sequencing human chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, G.R.

    1996-06-01

    This project funded for four years (02.92 to 01.96) was a renewal of a project funded for 2.5 years (07.89 to 01.92). This report covers the period 07.89 to 07.94. The original project was entitled {open_quotes}Correlation of physical and genetic maps of Human Chromosome 16{close_quotes}. The aim over this period was to construct a cytogenetic-based physical map of chromosome 16, to enable integration of its physical and genetic maps. This was achieved by collaboration and isolation of new markers until each bin on the physical map contained a polymorphic marker on the linkage map. A further aim was to integrate all mapping data for this chromosome and to achieve contig closure over band q24.

  15. Screening for Novel Binding Proteins Interacting with Human Papillomavirus Type 18 E6 Oncogene in the Hela cDNA Library by Yeast Two-Hybrid System

    Institute of Scientific and Technical Information of China (English)

    Shuang LI; Ping LIU; Ling XI; Xuefeng JIANG; Jianfeng ZHOU; Shixuan WANG; Li MENG; Yunping LU; Ding Ma

    2008-01-01

    To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH109 was transformed with pGBKT7-HPV18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPVl8 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate im- muno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transeriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

  16. Characterization of minisatellites in Arabidopsis thaliana with sequence similarity to the human minisatellite core sequence.

    Science.gov (United States)

    Tourmente, S; Deragon, J M; Lafleuriel, J; Tutois, S; Pélissier, T; Cuvillier, C; Espagnol, M C; Picard, G

    1994-08-25

    A strategy based on random PCR amplification was used to isolate new repetitive elements of Arabidopsis thaliana. One of the random PCR product analyzed by this approach contained a tandem repetitive minisatellite sequence composed of 33 bp repeated units. The genomic locus corresponding to this PCR product was isolated by screening a lambda genomic library. New related loci were also isolated from the genomic library by screening with a 14 mer oligonucleotide representing a region conserved among the different repeated units. Alignment of the consensus sequence for each minisatellite locus allowed the definition of an Arabidopsis thaliana core sequence that shows strong sequence similarities with the human core sequence and with the generalized recombination signal Chi of Escherichia coli. The minisatellites were tested for their ability to detect polymorphism, and their chromosomal position was established.

  17. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  18. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  19. Ancient Human Genome Sequence of an Extinct Palaeo-Eskimo

    DEFF Research Database (Denmark)

    Rasmussen, Morten; Li, Yingrui; Lindgreen, Stinus;

    2010-01-01

    We report here the genome sequence of an ancient human. Obtained from approximately 4,000-year-old permafrost-preserved hair, the genome represents a male individual from the first known culture to settle in Greenland. Sequenced to an average depth of 20x, we recover 79% of the diploid genome, an...... for a migration from Siberia into the New World some 5,500 years ago, independent of that giving rise to the modern Native Americans and Inuit....

  20. A human gut microbial gene catalogue established by metagenomic sequencing

    DEFF Research Database (Denmark)

    dos Santos, Marcelo Bertalan Quintanilha; Sicheritz-Pontén, Thomas; Nielsen, Henrik Bjørn;

    2010-01-01

    To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence...... gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively....

  1. Triplex-forming oligonucleotide target sequences in the human genome

    OpenAIRE

    Goñi, J Ramon; de la Cruz, Xavier; Orozco, Modesto

    2004-01-01

    The existence of sequences in the human genome which can be a target for triplex formation, and accordingly are candidates for anti-gene therapies, has been studied by using bioinformatics tools. It was found that the population of triplex-forming oligonucleotide target sequences (TTS) is much more abundant than that expected from simple random models. The population of TTS is large in all the genome, without major differences between chromosomes. A wide analysis along annotated regions of th...

  2. A novel approach to propagate flavivirus infectious cDNA clones in bacteria by introducing tandem repeat sequences upstream of virus genome.

    Science.gov (United States)

    Pu, Szu-Yuan; Wu, Ren-Huang; Tsai, Ming-Han; Yang, Chi-Chen; Chang, Chung-Ming; Yueh, Andrew

    2014-07-01

    Despite tremendous efforts to improve the methodology for constructing flavivirus infectious cDNAs, the manipulation of flavivirus cDNAs remains a difficult task in bacteria. Here, we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7×TRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7×TRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7×TRE-CMVmin, five repeats of the GAL4 upstream activating sequence, or five repeats of BamHI linkers upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably, 7×TRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs, which will facilitate research in virology, viral pathogenesis and vaccine development of flaviviruses and other RNA viruses. © 2014 The Authors.

  3. Pharmacovirological impact of an integrase inhibitor on human immunodeficiency virus type 1 cDNA species in vivo.

    Science.gov (United States)

    Goffinet, Christine; Allespach, Ina; Oberbremer, Lena; Golden, Pamela L; Foster, Scott A; Johns, Brian A; Weatherhead, Jason G; Novick, Steven J; Chiswell, Karen E; Garvey, Edward P; Keppler, Oliver T

    2009-08-01

    Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients.

  4. Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

    Science.gov (United States)

    Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

    1993-01-01

    Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss

  5. The cDNA Cloning and Sequence Analysis of Fragment of CART mRNA in Porcine Hypothalamus%猪下丘脑CART完整CDS区结构域分析

    Institute of Scientific and Technical Information of China (English)

    李鹏飞; 李富禄; 于秀菊; 吕丽华

    2011-01-01

    In order to obtain and analysis the complete CDS and its domain of porcine CART (Cocain and amphetamine- regulated transcript, CART), based on the cDNA sequences of CART in other species published in GenBank, three pairs of specific primers were designed and part mRNA sequence was amplified by RT-PCR. By alignment with other species, the sequences had high similarities, suggesting that the sequence was the porcine CART,and the mRNA expressed in porcine hypothalamus; Analysis of the 3D structure of the CART protein,showed that a CART superfamily (CART Superfamily) existed between amino acids 40 to 116. The superfamily may be as the ligand of iron to achieve a variety of physiological functions in vivo.%为获得猪可卡因苯异丙胺调控转录肽(Cocain and amphetamine-regulated transcript,CART)的完整CDS区并分析其结构城,根据GenBank中公布的其他物种CART的cDNA序列,设计了三对特异性引物,采用RT- PCR技术扩增出猪下丘脑组织CART mRNA的部分序列.经NCBI比对,与其他物种的相似性较高,提示该序列为猪CART的mRNA序列,CART mRNA在猪下丘脑上有表达;对该序列进行3D结构分析,发现猪CART蛋白在第40~116位氨基酸间存在一个CART超家族(CART Superfamily),这个超家族可能通过作为铁离子的配合基,在动物体内实现多种生理功能.

  6. Combining two technologies for full genome sequencing of human.

    Science.gov (United States)

    Skryabin, K G; Prokhortchouk, E B; Mazur, A M; Boulygina, E S; Tsygankova, S V; Nedoluzhko, A V; Rastorguev, S M; Matveev, V B; Chekanov, N N; D A, Goranskaya; Teslyuk, A B; Gruzdeva, N M; Velikhov, V E; Zaridze, D G; Kovalchuk, M V

    2009-10-01

    At present, the new technologies of DNA sequencing are rapidly developing allowing quick and efficient characterisation of organisms at the level of the genome structure. In this study, the whole genome sequencing of a human (Russian man) was performed using two technologies currently present on the market - Sequencing by Oligonucleotide Ligation and Detection (SOLiD™) (Applied Biosystems) and sequencing technologies of molecular clusters using fluorescently labeled precursors (Illumina). The total number of generated data resulted in 108.3 billion base pairs (60.2 billion from Illumina technology and 48.1 billion from SOLiD technology). Statistics performed on reads generated by GAII and SOLiD showed that they covered 75% and 96% of the genome respectively. Short polymorphic regions were detected with comparable accuracy however, the absolute amount of them revealed by SOLiD was several times less than by GAII. Optimal algorithm for using the latest methods of sequencing was established for the analysis of individual human genomes. The study is the first Russian effort towards whole human genome sequencing.

  7. Molecular cloning and expression analysis of cDNA ends of chicken neuropathy target esterase.

    Science.gov (United States)

    Chang, Ping-An; Sun, Quan; Ni, Xiao-Min; Qv, Feng-Qiong; Wu, Yi-Jun; Song, Fang-Zhou

    2008-03-10

    Neuropathy target esterase (NTE) was proposed as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN) in human and some sensitive animals. Adult hens are usually the animal model for experimental studies of OPIDN. However, little is known about the sequence and characteristics of chicken NTE. We report here the cloning of the 5' and 3' cDNA ends of chicken NTE through rapid amplification of cDNA ends (RACE) and their expression profiles in different tissues with northern blotting. The cloned 3' cDNA end of chicken NTE is 801 base pair (bp) in length with an open reading frame (ORF) of 379 bp. It contains a termination codon (TAG) and a 422-nucleotide noncoding sequence with the polyA sequence (GenBank accession no. DQ126678). The chicken NTE 5' cDNA end is 665 bp in length with an ORF of 552 bp. It contains an initiation codon (ATG) and a 113-bp untranslated region (GenBank accession no. DQ126677). The deduced proteins from 5' and 3' cDNA ends have a high degree of homology to humans and mouse NTE at the amino acid level. Chicken NTE is suggested to be a transmembrane protein by the transmembrane helix prediction of the deduced N-terminal sequence. The chicken NTE gene is expressed as a 4.5k b transcript in different tissues, including brain, kidney, liver and testis. Moreover, the mRNA expression of chicken NTE is highest in brain, and the mRNA levels of chicken NTE in testis, kidney and liver are about 75%, 47% and 24% of that in brain, respectively. These results should be helpful in cloning chicken full-length NTE gene.

  8. Tiered Human Integrated Sequence Search Databases for Shotgun Proteomics.

    Science.gov (United States)

    Deutsch, Eric W; Sun, Zhi; Campbell, David S; Binz, Pierre-Alain; Farrah, Terry; Shteynberg, David; Mendoza, Luis; Omenn, Gilbert S; Moritz, Robert L

    2016-11-04

    The results of analysis of shotgun proteomics mass spectrometry data can be greatly affected by the selection of the reference protein sequence database against which the spectra are matched. For many species there are multiple sources from which somewhat different sequence sets can be obtained. This can lead to confusion about which database is best in which circumstances-a problem especially acute in human sample analysis. All sequence databases are genome-based, with sequences for the predicted gene and their protein translation products compiled. Our goal is to create a set of primary sequence databases that comprise the union of sequences from many of the different available sources and make the result easily available to the community. We have compiled a set of four sequence databases of varying sizes, from a small database consisting of only the ∼20,000 primary isoforms plus contaminants to a very large database that includes almost all nonredundant protein sequences from several sources. This set of tiered, increasingly complete human protein sequence databases suitable for mass spectrometry proteomics sequence database searching is called the Tiered Human Integrated Search Proteome set. In order to evaluate the utility of these databases, we have analyzed two different data sets, one from the HeLa cell line and the other from normal human liver tissue, with each of the four tiers of database complexity. The result is that approximately 0.8%, 1.1%, and 1.5% additional peptides can be identified for Tiers 2, 3, and 4, respectively, as compared with the Tier 1 database, at substantially increasing computational cost. This increase in computational cost may be worth bearing if the identification of sequence variants or the discovery of sequences that are not present in the reviewed knowledge base entries is an important goal of the study. We find that it is useful to search a data set against a simpler database, and then check the uniqueness of the

  9. Developmental and Subcellular Organization of Single-Cell C₄ Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing.

    Science.gov (United States)

    Offermann, Sascha; Friso, Giulia; Doroshenk, Kelly A; Sun, Qi; Sharpe, Richard M; Okita, Thomas W; Wimmer, Diana; Edwards, Gerald E; van Wijk, Klaas J

    2015-05-01

    Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

  10. Lysosomal {beta}-mannosidase: cDNA cloning and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Leipprandt, J.R.; Traviss, C.E. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1994-09-01

    Lysosomal {beta}-mannosidase is an exoglycosidase that cleaves the single {beta}-linked mannose residue from the non-reducing end of all N-linked glycoprotein oligosaccharides. Deficiency of this enzyme results in {beta}-mannosidosis, a severe neurodegenerative disease in goats and cattle. The human cases described have a milder, highly variable presentation. Study of the molecular pathology of this disease in ruminants and humans and development of the animal model for gene therapy studies required cloning of the gene for {beta}-mannosidase has been cloned. {beta}-Mannosidase cDNA were obtained from a bovine thyroid cDNA library by screening with mixed oligonucleotides derived from peptide sequences resulting from microsequencing of bovine {beta}-mannosidase peptides. A total of six independent positive clones were identified from 5 x 10{sup 5} plaques, covering about 80% of the C-terminal region. The missing 5{prime} region was obtained using 5{prime} RACE. The full-length construct contains 3852-bp nucleotides, encoding 879 amino acids. The initiation codon is followed by 17 amino acids containing the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrated a 4.2 kb single transcript in various tissues from both normal and affected goats and calves. The mRNA level was decreased in affected {beta}-mannosidosis animals. The gene encoding {beta}-mannosidase was localized on human chromosome 4 by Southern analysis of rodent/human somatic cell hybrids. The mutation in bovine {beta}-mannosidosis has been identified. This is the first report of cloning of the {beta}-mannosidase gene.

  11. Cloning and sequence analysis of squalene synthase gene and cDNA in Glycyrrhiza uralensis%甘草鲨烯合酶基因及cDNA的克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    荣齐仙; 刘春生; 黄璐琦; 张宁; 南博; 呙未

    2011-01-01

    Objective: To clone and sequence the open reading frame and genomic sequence of squalene synthase (SQS) from Glycyrrhiza uralensis. Method: The primers were designed according to cDNA sequence of SQS from G. glabra reported by Hiroaki HAYASHI, SQS cDNA was cloned with total RNA extracted from roots of G. uralensis. Specific fragments were amplified by RT-PCR and then were cloned and sequenced. SQS DNA was cloned with total DNA extracted from roots of G. uralensis. Specific fragments were amplified by PCR and then were cloned and sequenced. Result: GuSQS1 (GenBank accession number: GQ266154) was 1 242 bp in length encoding proteins with 412 amino acid. NCBI Blast x search results showed GuSQS1 had the highest amino acid similarity to the corresponding proteins from G. uralensis The identities of GuSQS1 with the two proteins were 98. 55% and 88. 62%. SQS ( GenBank accession number: GQ180932) gene with 4 484 bp containing 13 exons and 12 introns was then amplified by PCR with genomic DNA extracted from roots of G. uralensis. Conclusion: These findings of cloning and sequencing the open reading frame and genomic sequence of squalene synthase (SQS) from G. uralensis brought some new clues for the further exploration of SmSQS function in sterol and terpenes biosynthesis.%目的:对甘草鲨烯合酶(SQS)基因的cDNA及DNA进行克隆及序列分析.方法:根据已报道的光果甘草SQS1基因的cDNA序列设计引物,采用RT-PCR的方法,提取甘草根的RNA然后反转录成cDNA,以cDNA为模板,扩增出SQS基因的cDNA序列,以甘草总DNA为模板,扩增SQS的DNA序列.结果:序列分析表明,克隆获得的甘草SQS1的cDNA编码区为1242 bp,编码413个氨基酸残基,命名为GuSQS1,登录号为GQ266154,与卢虹玉等报道的甘草的2个SQS(SQS1和SQS2)的氨基酸序列一致性为98.55%,88.62%,对应DNA序列全长为4484 bp,含有13个外显子,12个内含子,登录号为GQ180932.结论:甘草SQS的cDNA及DNA序列的获得为进一步研究

  12. Analysis of full-length cDNA sequence of FAD2 gene in Vernicia fordii seeds%油桐种子FAD2基因全长cDNA序列分析

    Institute of Scientific and Technical Information of China (English)

    谢禄山; 谭晓风; 张琳; 龙洪旭

    2012-01-01

    Linoleic acid produced in seeds of Verniciafordii is the direct material for synthesize eleostearic acid through catalysis of FAD2, the researches on FAD2 gene in seed from V. fordii has practical significance on improving yield of eleostearic acid. Taking 16 FAD2 clones in cDNA library of nearly mature V. fordii 'Duinian tung' seeds as materials, CAP3 splicing, BLAST alignment and DNAMAN analysis were carried on. The results showed that the cloned gene sequence was FAD2 full-length cDNA sequence, its length was 1 537 bp. The sequence contained a complete coding sequence, length of 1 146 bp (106-1 255 bp), encoding 383 amino acids. The relative molecular mass of the enzyme protein was 44 144.4 u, jsoelectric point was 8.57. The N end of amino acid sequence had a signal peptide sequence of 6 residues, 5 transmembrane domains, 3 strong hydrophilic sequences existed at the N end, C end and intermediate part, respectively, and the activity center of enzyme was 3 conserved histidine clusters. In system evolution, FAD2 gene in V. fordii had a nearest phylogenetic relationship with V. montana, nearer relationship with Euphorbiaceae plants such as Ricinus communis, Triadica sebifrea, Hevea brasiliensis, Jatropha curcas, and far relationship with Olea europaea, Arachis hypogaea, Sesamum indicum, further relationship with Camelia oleifera.%油桐种子中FAD2催化形成的亚油酸是合成桐油酸的直接原料,研究油桐种子中的FAD2基因对提高桐油酸的产量具有实际意义.将油桐对年桐近成熟种子cDNA文库中的16个FAD2克隆子进行CAP3拼接,再进行BLAST比对,并进行DNAMAN分析,结果表明所克隆的基因序列为FAD2全长cDNA序列,其长度为1 537 bp,含有1个完整的编码序列,长度为1 146 bp( 106~1 255 bp),编码383个氨基酸.酶蛋白相对分子质量44 144.4 u,等电点为8.57,氨基酸序列N端有6个残基的信号肽序列,有5个跨膜结构域,N端、C端及中间各有一段表现为强

  13. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jacob

    2007-01-01

    of genes between different tissues, in particular brain/spinal cord, and found patterns of correlation between genes that share expression in pairs of libraries. Finally, there was remarkable agreement in expression between specialized tissues according to Gene Ontology categories. CONCLUSION: This EST......BACKGROUND: Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from...... approximately 25% have a high confidence match to UniProt. Approximately 6,000 new porcine gene clusters were identified. Expression analysis based on the non-normalized libraries resulted in the following findings. The distribution of cluster sizes is scaling invariant. Brain and testes are among the tissues...

  14. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Cirera, Susanna; Hedegaard, Jakob;

    2007-01-01

    of genes between different tissues, in particular brain/spinal cord, and found patterns of correlation between genes that share expression in pairs of libraries. Finally, there was remarkable agreement in expression between specialized tissues according to Gene Ontology categories. Conclusion: This EST......Background: Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from...... approximately 25% have a high confidence match to UniProt. Approximately 6,000 new porcine gene clusters were identified. Expression analysis based on the non-normalized libraries resulted in the following findings. The distribution of cluster sizes is scaling invariant. Brain and testes are among the tissues...

  15. cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Molecular cloning of the porcine leukemia inhibitor factor(pLIF) has not been reported. A full-length cDNA encoding pLIF was cloned, expressed and characterized. The full-length porcine LIF cDNA encodes a 202 amino acid protein that has an 84% sequence identity to mouse LIF and 86% sequence identity to human LIF. The deduced amino acid sequence of a pLIF protein contains six conserved consensus N-linked glycosylation sites and six cysteine groups to form potential disulfide bonds. The pLIF was expressed in E coli, as a mature form, and in CHO cells as a secreted form. Both the forms of the recombinant pLIFs can maintain murine embryonic stem cells in an undifferentiated state in a culture. The recombinant pLIFs will be useful in establishing a long-term culture of stable pluripotent porcine embryonic stem cells for further manipulation.

  16. Four genes encode acetylcholinesterases in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. cDNA sequences, genomic structures, mutations and in vivo expression.

    Science.gov (United States)

    Combes, D; Fedon, Y; Grauso, M; Toutant, J P; Arpagaus, M

    2000-07-21

    We report the full coding sequences and the genomic organization of the four genes encoding acetylcholinesterase (AChE) in Caenorhabditis elegans and Caenorhabditis briggsae, in relation to the properties of the encoded enzymes. ace-1 and ace-2, located on chromosome X and I, respectively, encode two AChEs (ACE-1 and ACE-2) that present 35% identity. The C-terminal end of ACE-1 is homologous to the C terminus of T subunits of vertebrate AChEs. ACE-1 oligomerizes into amphiphilic tetramers. ACE-2 has a hydrophobic C terminus of H type. It associates into glycolipid-anchored dimers. In C. elegans and C. briggsae, ace-3 and ace-4 are organized in tandem on chromosome II, with only 356 nt and 369 nt, respectively, between the stop codon of ace-4 (upstream gene) and the ATG of ace-3. ace-3 produces only 5 % of the total AChE activity. It encodes an H subunit that associates into dimers of glycolipid-anchored catalytic subunits, which are highly resistant to the usual AChE inhibitors, and which hydrolyze butyrylthiocholine faster than acetylthiocholine. ACE-4 is closer to ACE-3 (54 % identity) than to ACE-1 or ACE-2. The usual sequence FGESAG surrounding the active serine residue in cholinesterases is changed to FGQSAG in ace-4. ACE-4 was not detected by our current biochemical methods, although the gene is transcribed in vivo. However the level of ace-4 mRNAs is far lower than those of ace-1, ace-2 and ace-3. The ace-2, ace-3 and ace-4 transcripts were found to be trans-spliced by both SL1 and SL2, although these genes are not included in typical operons. The molecular bases of null mutations g72 (ace-2), p1304 and dc2 (ace-3) have been identified. Copyright 2000 Academic Press.

  17. 杜仲HDR基因全长cDNA克隆与序列分析%Cloning and Sequence Analysis of 1-Hydroxy-2-Methyl-2-E-Butenyl-4-Diphosphate Reductase Gene cDNA from Eucommia ulmoides

    Institute of Scientific and Technical Information of China (English)

    刘攀峰; 杜红岩; 乌云塔娜; 杜兰英; 孙志强

    2013-01-01

    以杜仲叶片cDNA为模板,采用反转录RCR及RACE技术分离出HDR基因的cDNA克隆,命名为EuHDR.EuHDR基因cDNA全长1 653 bp,5'端非编码区长82 bp,3'端非编码区长188 bp,编码460个氨基酸,与喜树HDR基因序列相似性最高,达82%;推导EuHDR氨基酸序列中包含转运肽序列(A1-A33)及植物HDR蛋白多个保守的功能位点(A117,A208,A262,A345);EuHDR蛋白二级结构α-螺旋占35.65%,β-折叠占19.78%,螺环结构占44.57%;EuHDR蛋白三级结构为单体形式,呈不规则的三叶草形状;系统进化分析表明EuHDR蛋白与葡萄HDR蛋白的亲缘关系最为接近.%1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) synthesizes IPP and DMAPP in the last step of the plant 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.Homologous HDR gene cDNA was isolated from the leaves of Eucommia ulmoides by the method of reverse transcription polymerase chain reaction (RTPCR) and rapid amplification of cDNA ends (RACE) technique,and named as EuHDR.With the highest gene sequence similarity to Camptotheca acuminata (82%),the full-length cDNA of EuHDR was 1 653 bp including 5'non-coding region of 82 bp and 3' non-coding region of 188 bp and encoded 460 amino acids.The transit peptide sequence (A1-A33) and multiple conserved functional sites(A117,A208,A262,A345)of plant HDR protein were found in the deduced coding sequence of EuHDR.The secondary structure of EuHDR protein was predicted with proportion of α-helix to 35.65%,β-sheet to 19.78% and loop/coil to 44.57%.The calculated protein tertiary structure of EuHDR was formed as monomer,which in space displayed asymmetrical shamrock-like shape.Phylogenetic analysis revealed that the evolutionary relationship of EuHDR protein was the closest to Vitis vinifera HDR protein.

  18. cDNA Clone of Prophenoloxidase for Litopenaeus Stylirostris and Sequence Structure Analysis%细角滨对虾酚氧化酶原cDNA 克隆及序列结构分析

    Institute of Scientific and Technical Information of China (English)

    许尤厚; 胡超群

    2015-01-01

    采用 RT-PCR 原理和长片段扩增技术克隆细角滨对虾酚氧化酶原基因。结果表明,细角滨对虾血淋巴细胞内存在2个 proPO 基因。 proPO gene 1的 cDNA 序列包含有372氨基酸,前190个氨基酸为一个M 家族血蓝蛋白,是一个铜结合位点区域,191-372为一个 C 家族的血蓝蛋白,是一个免疫球蛋白样的区域。proPO gene 2的2个功能位点之间的序列有重叠,proPO gene 2 cDNA 序列的6-935bp 包含了第一个功能位点,928-1464bp 则包含了第二个功能位点。系统进化树比对分析发现2个基因之间的序列差异非常大。细角滨对虾和凡纳滨对虾的 proPO gene 2同处于一个密切相关的群,proPO gene 1则和其他几种对虾的 proPO gene 处于一个群。 proPO gene 2与 proPO gene 1在对虾免疫活动中是否存在不同的功能还有待于进一步的研究。%Prophenoloxidase (proPO) is one of the important factors on humoral immunity of shrimp, so far there are no re-ports for Litopenaeus stylirostris. Depend on techniques of RT-PCR and long fragment amplification cloning, prophenoloxidase gene of L. stylirostris was cloned. The results show that, there are two proPO genes in the lymphocytes of L. stylirostris. ProPO gene 1 cDNA sequence contains 372 amino acids, the first 190 amino acids are a family of M hemocyanin, a copper binding site region, 191-372 is one of the C family of hemocyanin, is an immunoglobulin like region. There are sequence overlap between the 2 functional sites of proPO gene 2, which means that 6-935bp contains the first functional sites, while 928-1464bp contains sec-ond functional sites. The phylogenetic tree alignment analysis showed that sequence structures of two genes is very different. Pro-PO gene 2 of L. stylirostris and L. vannamei was in a closely related group; but proPO gene 1 of L. stylirostris and L. vannamei was in another group with other several shrimp. The function of ProPO gene 2 and proPO gene 1 in shrimp immune

  19. The sequence and analysis of duplication rich human chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Martin, J; Han, C; Gordon, L A; Terry, A; Prabhakar, S; She, X; Xie, G; Hellsten, U; Chan, Y M; Altherr, M; Couronne, O; Aerts, A; Bajorek, E; Black, S; Blumer, H; Branscomb, E; Brown, N; Bruno, W J; Buckingham, J; Callen, D F; Campbell, C S; Campbell, M L; Campbell, E W; Caoile, C; Challacombe, J F; Chasteen, L A; Chertkov, O; Chi, H C; Christensen, M; Clark, L M; Cohn, J D; Denys, M; Detter, J C; Dickson, M; Dimitrijevic-Bussod, M; Escobar, J; Fawcett, J J; Flowers, D; Fotopulos, D; Glavina, T; Gomez, M; Gonzales, E; Goodstein, D; Goodwin, L A; Grady, D L; Grigoriev, I; Groza, M; Hammon, N; Hawkins, T; Haydu, L; Hildebrand, C E; Huang, W; Israni, S; Jett, J; Jewett, P B; Kadner, K; Kimball, H; Kobayashi, A; Krawczyk, M; Leyba, T; Longmire, J L; Lopez, F; Lou, Y; Lowry, S; Ludeman, T; Manohar, C F; Mark, G A; McMurray, K L; Meincke, L J; Morgan, J; Moyzis, R K; Mundt, M O; Munk, A C; Nandkeshwar, R D; Pitluck, S; Pollard, M; Predki, P; Parson-Quintana, B; Ramirez, L; Rash, S; Retterer, J; Ricke, D O; Robinson, D; Rodriguez, A; Salamov, A; Saunders, E H; Scott, D; Shough, T; Stallings, R L; Stalvey, M; Sutherland, R D; Tapia, R; Tesmer, J G; Thayer, N; Thompson, L S; Tice, H; Torney, D C; Tran-Gyamfi, M; Tsai, M; Ulanovsky, L E; Ustaszewska, A; Vo, N; White, P S; Williams, A L; Wills, P L; Wu, J; Wu, K; Yang, J; DeJong, P; Bruce, D; Doggett, N A; Deaven, L; Schmutz, J; Grimwood, J; Richardson, P; Rokhsar, D S; Eichler, E E; Gilna, P; Lucas, S M; Myers, R M; Rubin, E M; Pennacchio, L A

    2005-04-06

    Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,637 aligned transcripts, 19 tRNA genes, 341 pseudogenes, and 3 RNA pseudogenes. These genes include metallothionein, cadherin, and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. While the segmental duplications of chromosome 16 are enriched in the relatively gene poor pericentromere of the p-arm, some are involved in recent gene duplication and conversion events likely to have had an impact on the evolution of primates and human disease susceptibility.

  20. The wild boar (Sus scrofa Lymphocyte function-associated antigen-1 (CD11a/CD18 receptor: cDNA sequencing, structure analysis and comparison with homologues

    Directory of Open Access Journals (Sweden)

    Bergh Philippe

    2007-10-01

    Full Text Available Abstract Background The most predominant beta2-integrin lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2, expressed on all leukocytes, is essential for many adhesive functions of the immune system. Interestingly, RTX toxin-producing bacteria specifically target this leukocyte beta2-integrin which exacerbates lesions and disease development. Results This study reports the sequencing of the wild boar beta2-integrin CD11a and CD18 cDNAs. Predicted CD11a and CD18 subunits share all the main structural characteristics of their mammalian homologues, with a larger interspecies conservation for the CD18 than the CD11a. Besides these strong overall similarities, wild boar and domestic pig LFA-1 differ by 2 (CD18 and 1 or 3 (CD11a substitutions, of which one is located in the crucial I-domain (CD11a, E168D. Conclusion As most wild boars are seropositive to the RTX toxin-producing bacterium Actinobacillus pleuropneumoniae and because they have sustained continuous natural selection, future studies addressing the functional impact of these polymorphisms could bring interesting new information on the physiopathology of Actinobacillus pleuropneumoniae-associated pneumonia in domestic pigs.

  1. [Whole cDNA sequence cloning and expression of chicken L-FABP gene and its relationship with lipid deposition of hybrid chickens].

    Science.gov (United States)

    Yu, Ying; Wang, Dong; Sun, Dong-Xiao; Xu, Gui-Yun; Li, Jun-Ying; Zhang, Yuan

    2011-07-01

    Liver fatty acid-binding protein (L-FABP) is closely related to intracellular transportation and deposition of lipids. A positive differential displayed fragment was found in the liver tissue among Silkie (CC), CAU-brown chicken (CD), and their reciprocal hybrids (CD and DC) at 8 weeks-old using differential display RT-PCR techniques (DDRT-PCR). Through recycling, sequencing, and alignment analysis, the fragment was identified as chicken liver fatty acid-binding protein gene (L-FABP, GenBank accession number AY321365). Reverse Northern dot blot and semi-quantitative RT-PCR revealed that the avian L-FABP gene was over-expressed in the liver tissue of the reciprocal hybrids (CD and DC) compared to their parental lines (CC and DD), which was consistent with the fact that higher abdomen fat weight and wider inter-muscular fat width observed in the reciprocal hybrids. Considering the higher expression of L-FABP may contribute to the increased lipid deposition in the hybrid chickens, the functional study of avian L-FABP is warranted in future.

  2. Rapid construction of directional cDNA library from human nasopharynx%鼻咽上皮组织定向cDNA文库的快速构建

    Institute of Scientific and Technical Information of China (English)

    张必成; 余鹰; 邱元正; 钱骏; 周鸣; 李忠花; 张小慧; 向娟娟; 朱诗国; 李桂源

    2001-01-01

    Objective To construct a directional cDNA library from human adult nasopharynx by SMART (switching mechanism at 5′ end of RNA transcript) technique. Methods The total RNA was separated from human adult nasopharynx epithelial fissue and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo(dT) primer(contained sfi IB site) while the SMART oligonudeotide(contained sfi IA site) was utilized as a template so that the frist-strand cDNA could be extended over the 5′end of mRNA. The double-strand cDNA was amplified by LD-PCR(long-distance PCR) with the above two primers and then digested by sfi I (IA & IB) restriction enzyme.After cDNA size fractionation through CHROMA SPIN column,the double-strand cDNA was ligated into the sfi I-digested λTripIEx2 vector and then the recombinant DNA was packaged in vitro. Results The unamplified human adult nasopharynx cDNA library consists of 1.5×106 independent clones in which the percentage of recombinant clones is about 100%.The titer of the amplified cDNA library is 3.8×109 pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb. Conclusion These results shows that the human adult nasopharynx cDNA library has an excellent quality and lays solid foundation for screening and cloning new tumor suppressor genes of nasopharyngeal carcinoma(NPC) and tissue-specific genes of human nasopharynx.%目的采用SMART (switching mechanism at 5′ end of RNA transcript)技术,快速构建了高质量的成人鼻咽上皮组织定向cDNA文库。方法从成人正常鼻咽上皮组织分离总RNA,利用经修饰的oligo(dT)引物(含sfi IB酶切位点)合成cDNA第一链,同时根据真核生物mRNA 5′端帽子结构特点,利用SMART核苷酸(含sfi IA酶切位点)作为cDNA第二链在mRNA 5′端延伸出去的模板,进而以此序列为引物利用LD-PCR(Long-distance-PCR)合成双链cDNA,双链cDNA经sfi I(IA & IB)酶切和过柱分

  3. Detection of genogroup IV noroviruses in environmental and clinical samples and partial sequencing through rapid amplification of cDNA ends.

    Science.gov (United States)

    La Rosa, G; Pourshaban, M; Iaconelli, M; Muscillo, M

    2008-01-01

    Noroviruses (NoVs) give rise to clinically relevant gastroenteritis in all age groups and are widely distributed in both clinical and environmental settings. NoVs are classified into five genogroups (GI to GV), of which GI, GII and GIV infect humans. While data on the epidemiology of human NoVs GI and GII have been steadily increasing, very little information has been published on the spread of GIV in either the health care system or the environment, resulting in a lack of information about its clinical significance and pathogenesis. In order to investigate the distribution of GIV strains in the environment, we analyzed sewage samples collected from five treatment plants, by using newly designed nested RT-PCR assays. A collection of clinical stool samples, originating from pediatric patients with symptoms of acute gastroenteritis, previously analyzed in our laboratory for the presence of NoV GI or GII, was also analyzed for the presence of GIV norovirus. Results of this work attest to the presence of GIV in both clinical and environmental contexts and underline the importance of routinely screening for this genogroup, along with GI and GII, in order to better understand its distribution, prevalence and role during epidemics, which is probably underestimated.

  4. Origin and diversification of minisatellites derived from human Alu sequences.

    Science.gov (United States)

    Jurka, Jerzy; Gentles, Andrew J

    2006-01-01

    We analyze minisatellites derived from Alu fragments corresponding approximately to the first 44 bases of human Alu consensus sequences from different subfamilies. The origin of Alu-derived minisatellites appears to have been mediated by short flanking repeats, as first proposed by Haber and Louis [Haber, J.E., Louis, E.J., 1998. Minisatellite origins in yeast and humans. Genomics 48, 132-135.]. We also present evidence for base substitutions and deletions introduced to minisatellites by gene conversion with partially similar but unrelated flanking regions. Segments flanked by short direct repeats are relatively common in different regions of Alu and other repetitive sequences. Our analysis shows that they can be effectively used in comparative studies of the overall sequence context which may contribute to instability of DNA segments flanked by short direct repeats.

  5. Construction and characterization of replication-deficient adenoviral vector containing the cDNA for human VEGF165 in an antisense orientation%反义VEGF165腺病毒重组体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    王家宁; 黄永章; 王俊峰; 王卫民; 李瑞明; 张群林

    2001-01-01

    objective To construct the recombinant adenovirus vectorcontaining the cDNA for hVEGF165 in an antisense orientation for future study of tumor treatment by antisense hVEGF165 RNA strategy.Methods The VEGF165 cDNA has been extracted from pUCCAGGS/hVEGF165 with EcoRI and then inserted in an antisense orientation into the E1-deleted expression plasmid pHCMVsp1A shuttle vector, called pAd-ahVEGF165. pAd-ahVEGF165 was cotransfected with the plasmid pJM17 into the transformed human embryonic kidney cell line 293 cells by liposome-mediated method. Homologous recombination of the pAd-ahVEGF165 and pJM17 in 293 cells replaced the E1 region with the expression cassette from pAd-ahVEGF165. Ad-ahVEGF165 was confirmed by PCR.Ad-ahVEGF165 was propagated in 293 cells and then underwent CsCl density purification. subsequently, the preparations were didalyzed in dialysis buffer. The titer of each viral stock was determined by measuring the absorbance at 260nm. Results VEGF165 cDNA was successfully inserted into the shuttle vector pHCMVSPIA. pAd-ahVEGF165 was confirmed by NcoI and XhoI digestion. Ad-ahVEGF165 was characterized by PCR coamplification. The virus titer was 5.6×1011pfu/ml. Conclusions Ad-ahVEGF165 containing the antisense VEGF165 sequence was successfully constructd.This investigation provides the basis for future study of tumor treatment based on antisense VEGF RNA strategy.%目的:构建含人反义血管内皮生长因子165(VEGF165)基因的重组腺病毒载体,为采用反义VEGF165RNA防治肿瘤的研究奠定基础。方法:将人VEGF165 cDNA反向插入到穿梭质粒pHCMVSP1A的CMV启动子之下,即pAd-ahVEGF165。后者与pJM17通过脂质体共转染293细胞,经同源重组获得含人反义VEGF165基因的重组腺病毒Ad-ahVEGF165。通过PCR共扩增法鉴别Ad-ahVEGF165的正确与否。根据260nm的紫外光吸收值计算病毒滴度。结果:VEGF165 cDNA成功地反向插入了pHCMVSP1A载体,以重组病毒基因组DNA

  6. Templated sequence insertion polymorphisms in the human genome

    Science.gov (United States)

    Onozawa, Masahiro; Aplan, Peter

    2016-11-01

    Templated Sequence Insertion Polymorphism (TSIP) is a recently described form of polymorphism recognized in the human genome, in which a sequence that is templated from a distant genomic region is inserted into the genome, seemingly at random. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; Class 1 TSIPs show features of insertions that are mediated via the LINE-1 ORF2 protein, including 1) target-site duplication (TSD), 2) polyadenylation 10-30 nucleotides downstream of a “cryptic” polyadenylation signal, and 3) preference for insertion at a 5’-TTTT/A-3’ sequence. In contrast, class 2 TSIPs show features consistent with repair of a DNA double-strand break via insertion of a DNA “patch” that is derive