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Sample records for human cln2 cdna

  1. Management Strategies for CLN2 Disease.

    Science.gov (United States)

    Williams, Ruth E; Adams, Heather R; Blohm, Martin; Cohen-Pfeffer, Jessica L; de Los Reyes, Emily; Denecke, Jonas; Drago, Kristen; Fairhurst, Charlie; Frazier, Margie; Guelbert, Norberto; Kiss, Szilárd; Kofler, Annamaria; Lawson, John A; Lehwald, Lenora; Leung, Mary-Anne; Mikhaylova, Svetlana; Mink, Jonathan W; Nickel, Miriam; Shediac, Renée; Sims, Katherine; Specchio, Nicola; Topcu, Meral; von Löbbecke, Ina; West, Andrea; Zernikow, Boris; Schulz, Angela

    2017-04-01

    CLN2 disease (neuronal ceroid lipofuscinosis type 2) is a rare, autosomal recessive, pediatric-onset, rapidly progressive neurodegenerative lysosomal storage disorder caused by tripeptidyl peptidase 1 (TPP1) enzyme deficiency, and is characterized by language delay, seizures, rapid cognitive and motor decline, blindness, and early death. No management guidelines exist and there is a paucity of published disease-specific evidence to inform clinical practice, which currently draws upon experience from the field of childhood neurodisability. Twenty-four disease experts were surveyed on CLN2 disease management and a subset met to discuss current practice. Management goals and strategies are consistent among experts globally and are guided by the principles of pediatric palliative care. Goals and interventions evolve as the disease progresses, with a shift in focus from maintenance of function early in the disease to maintenance of quality of life. A multidisciplinary approach is critical for optimal patient care. This work represents an initial step toward the development of consensus-based management guidelines for CLN2 disease. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. A model of tripeptidyl-peptidase I (CLN2, a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases

    Directory of Open Access Journals (Sweden)

    Oyama Hiroshi

    2003-11-01

    Full Text Available Abstract Background Tripeptidyl-peptidase I, also known as CLN2, is a member of the family of sedolisins (serine-carboxyl peptidases. In humans, defects in expression of this enzyme lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. Similar enzymes have been found in the genomic sequences of several species, but neither systematic analyses of their distribution nor modeling of their structures have been previously attempted. Results We have analyzed the presence of orthologs of human CLN2 in the genomic sequences of a number of eukaryotic species. Enzymes with sequences sharing over 80% identity have been found in the genomes of macaque, mouse, rat, dog, and cow. Closely related, although clearly distinct, enzymes are present in fish (fugu and zebra, as well as in frogs (Xenopus tropicalis. A three-dimensional model of human CLN2 was built based mainly on the homology with Pseudomonas sp. 101 sedolisin. Conclusion CLN2 is very highly conserved and widely distributed among higher organisms and may play an important role in their life cycles. The model presented here indicates a very open and accessible active site that is almost completely conserved among all known CLN2 enzymes. This result is somehow surprising for a tripeptidase where the presence of a more constrained binding pocket was anticipated. This structural model should be useful in the search for the physiological substrates of these enzymes and in the design of more specific inhibitors of CLN2.

  3. 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.

  4. 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.

  5. 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.

  6. 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...

  7. 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.

  8. 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.

  9. 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...

  10. 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.

  11. 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.

  12. 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.

  13. 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).

  14. 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).

  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. 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...

  17. 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...

  18. 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...

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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....

  7. 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.

  8. 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.

  9. Genetic heterogeneity in neuronal ceroid lipofuscinosis (NCL): Evidence that the late-infantile subtype (Jansky-Bielschowsky disease; CLN2) is not an allelic form of the juvenile or infantile subtypes

    Energy Technology Data Exchange (ETDEWEB)

    Williams, R.; McKay, T.; Mitchison, H.; Gardiner, M. (Univ. College of London Medical School (United Kingdom)); Vesa, J.; Jaervelae, I.; Hellsten, E.; Peltonen, L.; Thompson, A.; Callen, D.; Sutherland, G.; Luna-Battadano, D.; Stallings, R.

    1993-10-01

    The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in neurons and other cell types. Inheritance is autosomal recessive. Three main childhood subtypes are recognized: Infantile (Haltia-Santavuori disease; MIM 256743), late infantile (Jansky-Bielschowsky disease; MIM 204500), and juvenile (Spielmeyer-Sjoegren-Vogt, or Batten disease; MIM 204200). The gene loci for the juvenile (CLN3) and infantile (CLN1) types have been mapped to human chromosomes 16p and 1p, respectively, by linkage analysis. Linkage analysis of 25 families segregating for late-infantile NCL has excluded these regions as the site of this disease locus (CLN2). The three childhood subtypes of NCL therefore arise from mutations at distinct loci. 17 refs., 2 figs., 3 tabs.

  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. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  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. 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).

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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...

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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...

  14. 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...

  15. 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...

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. 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.

  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. 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.

  6. 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).

  7. 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.

  8. 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.

  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. 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. 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.

  13. 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

  14. Data on characterizing the gene expression patterns of neuronal ceroid lipofuscinosis genes: CLN1, CLN2, CLN3, CLN5 and their association to interneuron and neurotransmission markers: Parvalbumin and Somatostatin

    Directory of Open Access Journals (Sweden)

    Helena M. Minye

    2016-09-01

    Full Text Available The article contains raw and analyzed data related to the research article “Neuronal ceroid lipofuscinosis genes, CLN2, CLN3, CLN5 are spatially and temporally co-expressed in a developing mouse brain” (Fabritius et al., 2014 [1]. The processed data gives an understanding of the development of the cell types that are mostly affected by defective function of CLN proteins, timing of expression of CLN1, CLN2, CLN3 and CLN5 genes in a murine model. The data shows relationship between the expression pattern of these genes during neural development. Immunohistochemistry was used to identify known interneuronal markers for neurotransmission and cell proliferation: parvalbumin, somatostatin subpopulations of interneurons. Non-radioactive in-situ hybridization detected CLN5 mRNA in the hippocampus. Throughout the development strong expression of CLN genes were identified in the germinal epithelium and in ventricle regions, cortex, hippocampus, and cerebellum. This provides supportive evidence that CLN1, CLN2, CLN3 and CLN5 genes may be involved in synaptic pruning.

  15. 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.

  16. [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.

  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. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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 ...

  9. 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.

  10. 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.

  11. 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.

  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. 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. 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

  15. 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 ...

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  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)

    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.

  2. 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.

  3. 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.

  4. Disilicon complexes with two hexacoordinate Si atoms: paddlewheel-shaped isomers with (ClN4 )Si-Si(S4 Cl) and (ClN2 S2 )Si-Si(S2 N2 Cl) skeletons.

    Science.gov (United States)

    Wagler, Jörg; Brendler, Erica; Heine, Thomas; Zhechkov, Lyuben

    2013-10-11

    The reaction of 1-methyl-3-trimethylsilylimidazoline-2-thione with hexachlorodisilane proceeds toward substitution of four of the disilane Cl atoms during the formation of disilicon complexes with two neighboring hexacoordinate Si atoms. The N,S-bidentate methimazolide moieties adopt a buttressing role, thus forming paddlewheel-shaped complexes of the type ClSi(μ-mt)4 SiCl (mt=methimazolyl). Most interestingly, three isomers (i.e., with (ClN4 )SiSi(S4 Cl), (ClN3 S)SiSi(S3 NCl), and (ClN2 S2 )SiSi(S2 N2 Cl) skeletons as so-called (4,0), (3,1), and cis-(2,2) paddlewheels) were detected in solution by using (29) Si NMR spectroscopic analysis. Two of these isomers could be isolated as crystalline solids, thus allowing their molecular structures to be analyzed by using X-ray diffraction studies. In accord with time-dependent NMR spectroscopy, computational analyses proved the cis-(2,2) isomer with a (ClN2 S2 )SiSi(S2 N2 Cl) skeleton to be the most stable. The compounds presented herein are the first examples of crystallographically evidenced disilicon complexes with two SiSi-bonded octahedrally coordinated Si atoms and representatives of the still scarcely explored class of Si coordination compounds with sulfur donor atoms.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  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. 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...

  12. 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

  13. 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.

  14. 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.

  15. 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.

  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. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Genetics Home Reference: CLN2 disease

    Science.gov (United States)

    ... 2016 Sep;119(1-2):160-7. doi: 10.1016/j.ymgme.2016.07.011. Epub 2016 Jul ... 2013 Mar 1;516(1):114-21. doi: 10.1016/j.gene.2012.12.058. Epub 2012 Dec ... Acta. 2013 Nov;1832(11):1801-6. doi: 10.1016/j.bbadis.2013.04.008. Epub 2013 Apr ...

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  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. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. Analysis of cellular responses to aflatoxin B{sub 1} in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Guo Yingying [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Breeden, Linda L. [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Fan, Wenhong [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zhao Lueping [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Eaton, David L. [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zarbl, Helmut [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States) and Fred Hutchinson Cancer Research Center, Seattle, WA (United States)]. E-mail: hzarbl@fhcrc.org

    2006-01-29

    Aflatoxin B1 (AFB{sub 1}) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB{sub 1} is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N{sup 7}-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB{sub 1}, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB{sub 1} that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB{sub 1} treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB{sub 1}-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific

  3. 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...

  4. T cell-based functional cDNA library screening identified SEC14-like 1a carboxy-terminal domain as a negative regulator of human immunodeficiency virus replication.

    Science.gov (United States)

    Urano, Emiko; Ichikawa, Reiko; Morikawa, Yuko; Yoshida, Takeshi; Koyanagi, Yoshio; Komano, Jun

    2010-05-26

    Genome-wide screening of host factors that regulate HIV-1 replication has been attempted using numerous experimental approaches. However, there has been limited success using T cell-based cDNA library screening to identify genes that regulate HIV-1 replication. We have established a genetic screening strategy using the human T cell line MT-4 and a replication-competent HIV-1. With this system, we identified the C-terminal domain (CTD) of SEC14-like 1a (SEC14L1a) as a novel inhibitor of HIV-1 replication. Our T cell-based cDNA screening system provides an alternative tool for identifying novel regulators of HIV-1 replication.

  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. Characterization of Leukemia-Inducing Genes Using a Proto-Oncogene/Homeobox Gene Retroviral Human cDNA Library in a Mouse In Vivo Model.

    Directory of Open Access Journals (Sweden)

    Su Hwa Jang

    Full Text Available The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30% had MYC as the only transgene, and seven mice (70% had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.

  7. Characterization of Leukemia-Inducing Genes Using a Proto-Oncogene/Homeobox Gene Retroviral Human cDNA Library in a Mouse In Vivo Model.

    Science.gov (United States)

    Jang, Su Hwa; Lee, Sohyun; Chung, Hee Yong

    2015-01-01

    The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.

  8. 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...

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. High-Resolution Analysis of Gene Copy Number Alterations in Human Prostate Cancer Using CGH on cDNA Microarrays: Impact of Copy Number on Gene Expression

    Directory of Open Access Journals (Sweden)

    Maija Wolf

    2004-05-01

    Full Text Available Identification of target genes for genetic rearrangements in prostate cancer and the impact of copy number changes on gene expression are currently not well understood. Here, we applied high-resolution comparative genomic hybridization (CGH on cDNA microarrays for analysis of prostate cancer cell lines. CGH microarrays identified most of the alterations detected by classical chromosomal CGH, as well as a number of previously unreported alterations. Specific recurrent regions of gain (28 and loss (18 were found, their boundaries defined with sub-megabasepair accuracy. The most common changes included copy number decreases at 13% and gains at iq and 5p. Refined mapping identified several sites, such as at 13q (33-44, 49-51, 74-76 Mbp from the p-telomere, which matched with minimal regions of loss seen in extensive loss of heterozygosity mapping studies of large numbers of tumors. Previously unreported recurrent changes were found at 2p, 2q, 3p, 17q (losses, at 3q, 5p, 6p (gains. Integration of genomic and transcriptomic data revealed the role of individual candidate target genes for genomic alterations as well as a highly significant (P < .0001 overall association between copy number levels and the percentage of differentially expressed genes. Across the genome, the overall impact of copy number on gene expression levels was, to a large extent, attributable to low-level gains and losses of copy number, corresponding to common deletions and gains of often large chromosomal regions.

  15. [Screening differential expression of docetaxel-resistance related genes of human lung adenocarcinoma cell line SPC-A1 by cDNA microarray].

    Science.gov (United States)

    Sun, Hai; Geng, Jian; Chen, Longbang

    2007-10-20

    Docetaxel is one of effective chemotherapeutics in the last few years, however, it is interfered by drug resistance in its further application. The aim of this study is to screen differentially expressed genes of docetaxel resistant cell line SPC-A1/Docetaxel and its parent cell line SPC-A1 with gene chip technique. The cDNA retro-transcribed from equal quantity mRNA derived from SPC-A1/Docetaxel and SPC-A1 cell lines. The mixed probes were hybridized with Affymetrix GeneChip HG-U133A2.0. The acquired image was analyzed by Affymetrix GeneChip Operating Software Version 1.0. Then, part of these results were verified by RT-PCR. A total of 934 differentially expressed genes were screened out, in which up-and down-regulated genes were 428 and 506 respectively. These genes involved in ABC transporter, apoptosis regulator, tubulin, signal transducer, enzyme and so on. These differentially expressed genes may be related to the mechanisms of docetaxel resistance in SPC-A1/Docetaxel cell line.

  16. Possible Mechanism of TP53 Accumulation in Nasopharyngeal Carcinoma by Atlas Human Cancer cDNA Expression Array%用 cDNA Array 探讨鼻咽癌组织 TP53积聚的可能原因

    Institute of Scientific and Technical Information of China (English)

    李虹; 韩为农; 张玲; 谢鹭; 姚开泰

    2001-01-01

    目的:用 cDNA Array 比较鼻咽癌组织及正常组织基因表达谱,寻找鼻咽癌组织 TP53积聚的可能原因。方法:( 1) Atlas Human Cancer cDNA Expression Array 7742 1滤膜杂交。( 2) AtlasImage 1.01a分析滤膜杂交结果。( 3) RT PCR反应验证滤膜杂交结果。( 4)免疫组化证实基因在蛋白质水平的表达改变。结果:( 1) 588个肿瘤相关基因中,共有 134个基因表达上调, 88个基因表达下调。( 2)膜上有 TP53调节基因 32种。其中 13种显示差异表达,有 11个表达上调, 2个表达下调。( 3) TP53上游的蛋白激酶基因 ATM和 JNK2表达呈上调趋势。( 4)泛素降解系统的两个基因 ubiquitin conjugating enzyme E2( M74524) ubiquitin conjugating enzyme E2 ( L22005) mRNA在鼻咽癌和正常鼻咽组织内的表达没有明显改变。结论:( 1)在鼻咽癌组织内, TP53的功能失控。( 2) ATM和 JNK极有可能是 TP53积聚的重要原因。

  17. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: Chromosomal assignment of the gene in the human, mouse, and rat genomes

    Energy Technology Data Exchange (ETDEWEB)

    Pausova, Z.; Bourdon, J.; Clayton, D.; Janicic, N.; Goltzman, D.; Hendy, G.N. (McGill Univ. and Royal Victoria Hospital, Montreal Quebec (Canada)); Mattei, M.G. (INSERM, Marseille (France)); Seldin, M.F. (Duke Univ. Medical Center, Durham, NC (United States)); Riviere, M.; Szpirer, J. (Universite Libre de Bruxelles, Rhode-St-Genese (Belgium)) (and others)

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acelpeptide hydrolase gene (APEH). These results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes. 34 refs., 7 figs. 1 tab.

  18. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: chromosomal assignment of the gene in the human, mouse, and rat genomes.

    Science.gov (United States)

    Pausova, Z; Bourdon, J; Clayton, D; Mattei, M G; Seldin, M F; Janicic, N; Rivière, M; Szpirer, J; Levan, G; Szpirer, C

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acylpeptide hydrolase gene (APEH). Our results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes.

  19. 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.

  20. 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.

  1. Construction and Identification ofa cDNA Library of Human Rheumatoid Arthritis Synovial Tissue%人类风湿性关节炎滑膜组织cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    闫永毅; 任蕾; 高飞; 卢秀敏; 刘彦虹

    2012-01-01

    Objective:To construct a cDNA library of human synovial tissue of RA and indentify the quality of the library. Methods: Total RNA was extracted and mRNA was purified. mRNA was reversed to first-strand cDNA which was amplified to double-strand cD-NA by long distance PCR. PCR products were digested by proteinase K and Sfi I, and were fractionated by CHROMA SPIN-400 column. The cDN A of length longer than 0.4kb were collected and ligated toλ TriplEx2 vector. The λ phage packaging reaction for the ligated DNA was performed to produce an unamplified library. Thereafter, the unamplified library was titered and the percentage of recombinant clones were detected. In the end, fourty plaques were randomly selected and amplified by PCR using universal primers from vector in order to test the qualify of the obtained library. Results: The titers of unamplifed and amplified libraries were 6.89x106 pfu/mL and 2.63x 109 pfu/mL respectively. The rate of recombinant was 93%. The insert size range from 300 to 1800 bp. Conclusions: A high quality cD-NA library from human synovial tissue of RA was constructed successfully, and it lays solid foundation not only for screening and cloning new special genes associated with the occurrence of RA, but also for gene therapy of it.%目的:构建人类风湿关节炎(RA)滑膜组织cDNA文库.筛查与RA相关的特异基因,为探讨RA的发病机制及基因治疗奠定基础.方法:提取人类风湿关节炎滑膜组织RNA并使用Trizol法纯化mRNA;运用mRNA5'末端的模板转换方法以powerscriptTM逆转录酶进行转录,使用COS Ⅲ/3'PCR引物合成第1链cDNAs;长距离聚合酶链反应(LD-PCR)合成双链cDNA; PCR产物经蛋白酶K水解并纯化后,经SfiI酶切、柱层析洗脱,重组于TripIEx2载体并包装后,测定滴度、重组率、扩增文库,随机挑取40个噬菌斑,用载体克隆位点两端的通用引物进行PCR扩增,以检测所构建cDNA文库的质量.结果:未扩增文库的滴度为6.89× 106pfu/m

  2. Cloning and analysis of the mouse Fanconi anemia group a cDNA and an overlapping penta zinc finger cDNA

    NARCIS (Netherlands)

    Wong, JCY; Alon, N; Norga, K; Kruyt, FAE; Youssoufian, H; Buchwald, M

    2000-01-01

    Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a mode I system, we cloned and characterized the mouse homolog of the human FANCA cDNA, The mouse cDNA

  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. Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

    Institute of Scientific and Technical Information of China (English)

    史耕先; 靳玉兰; 王壮志; 崔巍; 刘音; 王讯; 朱立平

    2001-01-01

    The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa's staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa's staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase. The transduction did not affect the expression of Fas molecule on cells.

  5. 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

  6. Cloning of a cDNA encoding a putative human very low density lipoprotein/Apolipoprotein E receptor and assignment of the gene to chromosome 9pter-p23[sup 6

    Energy Technology Data Exchange (ETDEWEB)

    Gafvels, M.E.; Strauss, J.F. III (Univ. of Pennyslvania, Philadelphia, PA (United States)); Caird, M.; Patterson, D. (Eleanor Roosevelt Institute, Denver, CO (United States)); Britt, D.; Jackson, C.L. (Brown Univ., Providence, RI (United States))

    1993-11-01

    The authors report the cloning of a 3656-bp cDNA encoding a putative human very low density lipoprotein (VLDL)/apolipoprotein E (ApoE) receptor. The gene encoding this protein was mapped to chromosome 9pter-p23. Northern analysis of human RNA identified cognate mRNAs of 6.0 and 3.8 kb with most abundant expression in heart and skeletal muscle, followed by kidney, placenta, pancreas, and brain. The pattern of expression generally paralleled that of lipoprotein lipase mRNA but differed from that of the low density lipoprotein (LDL) receptor and the low density lipoprotein receptor-related protein/[alpha][sub 2]-macroglobulin receptor (LRP), which are members of the same gene family. VLDL/ApoE receptor message was not detected in liver, whereas mRNAs for both LDL receptor and LRP were found in hepatic tissue. In mouse 3T3-L1 cells, VLDL/ApoE receptor mRNA was induced during the transformation of the cells into adipocytes. Expression was also detected in human choriocarcinoma cells, suggesting that at least part of the expression observed in placenta may be in trophoblasts, cells which would be exposed to maternal blood. Expression in brain may be related to high levels of ApoE expression in that organ, an observation of potential relevance to the recently hypothesized role for ApoE in late onset Alzheimer disease. The results suggest that the putative VLDL/ApoE receptor could play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs including striated and cardiac muscle and adipose tissue and in the transport of maternal lipids across the placenta. The findings presented here, together with recent observations from other laboratories, bring up the possibility that a single gene, the VLDL/ApoE receptor, may play a role in the pathogenesis of certain forms of atherosclerosis, Alzheimer disease, and obesity.

  7. Expression of sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase (ST6GalNac I) cDNA.

    Science.gov (United States)

    Julien, S; Krzewinski-Recchi, M A; Harduin-Lepers, A; Gouyer, V; Huet, G; Le Bourhis, X; Delannoy, P

    2001-01-01

    Sialyl-Tn antigen (STn) is a cancer associated carbohydrate antigen over-expressed in several cancers including breast cancer, and currently associated with more aggressive diseases and poor prognosis. However, the commonly used breast cancer cell lines (MDA-MB-231, T47-D and MCF7) do not express STn antigen. The key step in the biosynthesis of STn is the transfer of a sialic acid residue in alpha2,6-linkage to GalNAc alpha-O-Ser/Thr. This reaction is mainly catalyzed by a CMP-Neu5Ac GalNAc alpha2,6-sialyltransferase: ST6GalNAc I. In order to generate STn-positive breast cancer cells, we have cloned a cDNA encoding the full-length human ST6GalNAc I from HT-29-MTX cells. The stable transfection of MDA-MB-231 with an expression vector encoding ST6GalNAc I induces the expression of STn antigen at the cell surface. The expression of STn short cuts the initial O-glycosylation pattern of these cell lines, by competing with the Core-1 beta1,3-galactosyltransferase, the first enzyme involved in the elongation of O-glycan chains. Moreover, we show that STn expression is associated with morphological changes, decreased growth and increased migration of MDA-MB-231 cells.

  8. 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

  9. 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

  10. 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.

  11. GenBank blastx search result: AK110394 [KOME

    Lifescience Database Archive (English)

    Full Text Available similar to human ceroid-lipofuscinosis, neuronal 2, late infantile(Jansky-Bielschowsky disease) (CLN2), mRNA, RefSeq: NM_000391.2.|PRI PRI 5e-22 +2 ... ...AK110394 002-165-E11 AB169876.1 Macaca fascicularis brain cDNA, clone: QflA-12618,

  12. Cloning and expression of cDNA for a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase from the CEM T-cell line.

    Science.gov (United States)

    Giordanengo, V; Bannwarth, S; Laffont, C; Van Miegem, V; Harduin-Lepers, A; Delannoy, P; Lefebvre, J C

    1997-07-15

    Complementary DNA encoding a human Gal(beta1-3)GalNAc alpha2,3-sialyltransferase type II (hST3Gal II) was cloned from a CEM T-cell cDNA library using a 23-base oligonucleotide probe. The sequence of this probe was established on the basis of a slightly divergent sialylmotif L that was obtained by polymerase chain reaction with degenerate oligonucleotide primers based on the conserved sialylmotif L of mammalian Gal(beta1-3)GalNAc alpha2,3-sialyltransferases. It was thus confirmed that a short oligonucleotide probe may be sensitive and highly specific. The nucleotide and amino acid sequences of hST3Gal II show, respectively, 56.3% and 49.3% similarity to hST3Gal I [Kitagawa, H. & Paulson, J. C. (1994) J. Biol. Chem. 269, 17872-17878] and 88.1% and 93.7% similarity to murine ST3Gal II [Lee, Y. C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T. & Tsuji, S. (1994) J. Biol. Chem. 269, 10028-10033]. hST3Gal II mRNA was highly expressed in heart, liver, skeletal muscle and various lymphoid tissues but not in brain and kidney. A soluble form of hST3Gal II expressed in COS-7 cells was tested in vitro for substrate specificity and kinetic properties. Asialofetuin and asialo-bovine submaxillary mucin appeared better substrates for hST3Gal II than for its murine counterpart as previously reported [Kojima, N., Lee, Y.-C., Hamamoto, T., Kurosawa, N. & Tsuji, S. (1994) Biochemistry 33, 5772-5776]. In previous studies, we have shown hyposialylation of O-glycans attached to two major lymphocyte CD43 and CD45 cell surface molecules in human-immunodeficiency-virus-1(HIV-1)-infected T-cell lines. Since comparable levels of hST3Gal I and hST3Gal II mRNA and enzymatic activity were observed in parental and HIV-1-infected CEM T-cell lysates, the sialylation defect associated with HIV infection of this cell line is probably due to a mechanism different from a simple altered catalytic activity of these sialyltransferases.

  13. 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.

  14. Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene

    DEFF Research Database (Denmark)

    Andresen, B S; Bross, P; Vianey-Saban, C

    1996-01-01

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE...

  15. 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 ...

  16. 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.

  17. 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.

  18. 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 ...

  19. Cloning and prokaryotic expression of HGLP cDNA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A novel cDNA, HGLP, which encodes a G- protein coupled receptor (GPCR) like protein, has been isolated and cloned. The coding region of the human HGLP predicts a 7-transmembrane region protein with motifs of rhodopsin-like GPCR superfamily. Northern blot analysis reveals a 3-kb transcript in various human tissues examined. The N- and C-terminal coding regions of HGLP, which are deduced as non-transmembrane regions, have been amplified by PCR and cloned into pET30a+ vector. Then the recombinant proteins are highly expressed in E. coli.

  20. cDNA Cloning of a Short Isoform of Human Liver NADP(H)-Dependent Retinol Dehydrogenase/Reductase and Analysis of Its Characteristics%一种人肝辅酶Ⅱ依赖性视黄醇脱氢酶剪接体cDNA的克隆及特征分析

    Institute of Scientific and Technical Information of China (English)

    杜晶; 黄东阳; 刘戈飞; 王桂玲; 徐晓琳; 王博; 朱莉

    2004-01-01

    In this report we found a new short PCR product when we amplified a 635 bp of NRDR fragment by RT-PCR.With 3′-Race and 5′-Race,we obtained two full-length Cdna sequences from human liver tissue,one 1 261 bp NRDR Cdna,another 1 003 bp NRDR isoform (NRDRiso,GenBank accession number:AY071856).The NRDR gene comprises eight exons and seven introns.The NRDRiso Cdna is produced by alternative splicing of NRDR Cdna,with the removal of 4,5,6 exons composed of consecutive 258 bp.The open reading frames of the NRDRiso Cdna predict a single polypeptide of 174 amino acids with the calculated minimum molecular mass of 18.6 kDa.%在用RT-PCR法局部扩增人与小鼠肝脏635 bp的NRDR DNA时,在人肝中发现了另一短序列PCR产物,克隆后测序显示其整个序列与NRDR cDNA编码区的前后序列完全一致.采用3'-Race和5'-Race方法,从人肝组织细胞中扩增得到两个全长cDNA,除1 261 bp的NRDR cDNA外,另一个为全长1 003 bp、编码区长为525 bp的NRDRiso(GenBank 登录号:AY071856).数据库分析表明,NRDRiso编码区是由人NRDR 8个外显子中的第1、2、3、7、8外显子选择性剪接而成.缺失的NRDR第4、5、6外显子共258 bp,编码86个氨基酸.因此,与人NRDR的260个氨基酸残基相比,NRDRiso由174个氨基酸残基组成,分子量为18.6 kDa,并且NRDRiso的组织表达与NRDR明显不同.

  1. 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.

  2. Construction and characterization of a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B%类泛素FAT10高表达肝癌细胞株Hep3B酵母双杂交cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    余新; 刘天; 德洪波; 李国惠; 邵江华

    2011-01-01

    AIM: To construct a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B.METHODS: Total RNA was prepared from Hep3B cells and used to purify poly (A) mRNA.Double-stranded cDNA was synthesized from the purified mRNA, ligated to EcoR Ⅰ adaptor,digested with EcoR Ⅰ/Xho Ⅰ enzymes, and then cloned into the pGADT7 vector.The recombinant vector was transformed into E.coli DH10B to obtain a primary cDNA library.The primary library was amplified and used to determine the size of cDNA inserts through enzyme digestion.RESULTS: The primary cDNA library contained 1.03 × 106independent clones.The titer of the cDNA library was estimated to be 2.50 × 106 cfu/mL, and that of the amplified library was 3.60 × 109 cfu/mL.The size of the inserts varied from 0.5 to 3.5 kb, with an average value of about 2.0 kb.CONCLUSION: A yeast two-hybrid cDNA library has been successfully generated from FAT10-over-expressing Hep3B ceils and can be used for future screening of proteins interacting with FAT10.%目的:构建类泛素FAT10高表达肝癌细胞株Hep3B的酵母双杂交用cDNA文库.方法:从肝癌细胞Hep3B中提取总RNA,分离mRNA.利用反转录酶M-MLV与Oligo(dT)AnchorPrimer合成1 Strand cDNA,用E.coli DNAPolymerase与E.coli DNA Ligase将RNA链置换成DNA链,合成2 Strand cDNA.将双链cDNA与EcoR Ⅰ Adaptor连接,然后用EcoR Ⅰ /Xho Ⅰ进行酶切.使用Spin Column除去短链cDNA与pGADT7载体连接,转化入E.coli DH10B,建成原始文库.然后对其进行扩增并随机挑取单菌落,酶切鉴定重组子插入片段大小.结果:提取的总RNA降解少且分子完整;RNA纯度高,相对分子质量为400-5000 bp;成功合成双链cDNA,均符合建库要求:库容量达到1.03×10克隆,原始文库滴度为2.50×10 cfu/L,扩增后的文库滴度为3.60×10 cfu/L.插入片段大小分布为0.5-3.5 kb,平均长度约为2.0 kb.结论:所构建文库的各项指标均达到要求,为筛选FAT10作用蛋白奠定了重要基础.

  3. Cloning of cDNA and genomic DNA encoding human type XVIII collagen and localization of the [alpha]1 (XVIII) collagen gene to mouse chromosome 10 and human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Oh, S.P.; Warman, M.L.; Timmons, S.; Olsen, B.R.; Knoll, J.H.M. (Harvard Medical School, Boston, MA (United States)); Seldin, M.F. (Duke Univ. Medical Center, Durham, NC (United States)); Cheng, Sou-De (Children' s Hospital/Harvard Medical School, Boston, MA (United States))

    1994-02-01

    Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which the authors have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, they report the isolation of human cDNAs and genomic DNAs encoding the [alpha]1 (XVIII) collagen chain. Utilizing a genomic clone as probe, they have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, they have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2. 16 refs., 5 figs.

  4. Full-length Cloning of Human Melanoma Antigen-encoding Gene D1 Using Rapid Amplification of cDNA Ends%采用RACE技术获得全长人新基因MAGE-D1

    Institute of Scientific and Technical Information of China (English)

    邢桂春; 张成岗; 魏汉东; 贺福初

    2001-01-01

    cDNA 末端快速扩增(RACE)技术是快速获得新基因5'和3'端未知序列的有效手段.鉴于新报导的人肿瘤基因MAGE家族成员之一MAGE-D1的cDNA序列不完全,从而拟用RACE技术获得MAGE-D1的全长cDNA序列.结果显示,MAGE-D1的cDNA全长为2 800个碱基,编码778个氨基酸.同时对RACE技术应用时的一些关键问题进行了讨论.

  5. 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...

  6. 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.

  7. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  8. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    李权; 闻宏; 敖世洲

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corresponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  9. Identification and cloning of the cDNA of a Rb-associated protein RAP140a

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corre-sponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was named RAP140a and predicted to encode a 1 233 amino acids hydrophilic protein. RAP140a was mapped to chromosome 3p13-p14.1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

  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. 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.

  12. 人牙周膜成纤维细胞与牙龈成纤维细胞差异表达基因扣除文库的构建%Subtractive cDNA library construction of genes differentially xpressed in human periodontalligament fibroblast in comparison with gingival fibroblasts

    Institute of Scientific and Technical Information of China (English)

    郭希民; 吴补领; 肖明振; 蒲勤; 柴玉波; 朱峰; 赵忠良

    2000-01-01

    AIM: To construct a subtractive cDNA library of genes differentially expressed in cultured primary human PDLF in comparison with GF. METHODS: mRNAs were isolated fiom primary cultures of human PDLF and GF. Subtractive eDNA library of PDLF was constructed with a recently developed gene cloning technique that is based on PCR and subtractive hybridization. Part of randomly selected clones were identified by restriction analysis and reverse Southern dotblot. RESULTS: A subtbtetive cDNA library of PDLF was constructed with a capieity of 4×l02 clones. CONCLUSION:The gene cloning method we used in this study is both simple and effective in cloning differentially expressed gene.%目的:建立人牙周膜成纤维细胞(periodontal ligament fibroblast, PDLF) 与牙龈成纤维细胞(gin-gival fibroblast, GF)差异表达基因的扣除文库。方法:体外培养人PDLC和GF,分别提取mRNA,采用基于PCR和消减杂交的基因克隆技术构建人PDLC与GF差异表达基因的扣除文库。结果:成功构建了人PDLF与GF细胞差异表达基因的扣除文库,文库容量为4×l02。结论:基于PCR和消减杂交的基因克隆技术是构建细胞间差异表达基因扣除文库的较为简洁而有效的方法。

  13. 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...

  14. 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.

  15. Analysis of gene expression profile of aspermia using cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    杨波; 高晓康; 王禾; 刘贺亮; 陈宝琦; 秦荣良; 康福霞; 邵国兴; 邵晨

    2003-01-01

    Objective: To identify the differential gene expression profiles between the normal and aspermia human testes utilizing cDNA microarray. Methods: cDNA probes were prepared by labeling mRNA of aspermia testes tissues with Cy5-dUTP and mRNA of normal testes tissues with Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with 4096 cDNA arrays (4096 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 2.0 or less-than 0.5. A randomly chosen gene RAP1A was studied by in situ hybridization to evaluate the accuracy of the results. Results: 623 differential expressed genes related to aspermia were found. There were 303 up-expressed genes and 320 down-expressed genes. A distinct up-expressed gene RAP1A was confirmed by in situ hybridization. Conclusions: Screening the differential gene expression profiles between the normal and aspermia human testis by cDNA microarray can be used in the study of aspermia-related genes and the further research due to its properties, RAP1A may play some roles in the development and progression of aspermia.

  16. 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.

  17. Isolation of RNA and DNA from leukocytes and cDNA synthesis.

    NARCIS (Netherlands)

    Jansen, J.H.; Reijden, B.A. van der

    2006-01-01

    In this chapter, methods to isolate RNA and DNA from human leukocytes for the subsequent use in molecular diagnostic tests are described. In addition, protocols for cDNA synthesis are given, both for the use in conventional reverse transcription (RT)-polymerase chain reaction (PCR), and for the use

  18. 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.

  19. 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.

  20. Optimization of yeast surface-displayed cDNA library screening for low abundance targets.

    Science.gov (United States)

    Kim, Juh-Yung; Kim, Hyung Kyu; Jang, Hye Jeong; Kim, Eun-Kyung; Kim, Moon Kyu

    2015-04-01

    The yeast surface-displayed cDNA library has been used to identify unknown antigens. However, when unknown target antigens show moderate-to-low abundance, some modifications are needed in the screening process. In this study, a directional random-primed cDNA library was used to increase the number of candidates for the unknown antigen. To avoid the loss of target yeast clones that express proteins at a low frequency in the cDNA library, a comprehensive monitoring system based on magnetic-activated cell sorting, fluorescence-activated cell sorting, and immunofluorescence was established, and a small number of target yeast cells was successfully enriched. These results showed that our optimized method has potential application for identifying rare unknown antigens of the human monoclonal antibody.

  1. Construction and expression of eukaryotic expression plasmid with mouse angiostatin cDNA in human gastric cancer cells%鼠血管抑素Angiostating真核表达载体的构建及在人胃癌细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    吴静; 樊代明; 时永全; 苗继延

    2001-01-01

    AIM To construct angiostatin cDNA eukaryotic expression plasmid, transfect human gastric tumor cells SCG7901, and identify the expression of angiostatin. METHODS Angiostatin cDNA (plasminogen k1-k3 with signal peptid es and HA tag) was subclon ed into eukaryotic expression plasmid pcDNA3.1(+). Gene transfer into gastric tumor cell s SGC7901 was carried out by liposome (TfxTM), according to the transfection protocol in the manual. Then through G418 screening, stable resistant clones were obtained. Wester n blot was used to confirm the expression of angiostatin in selected clones. RESULTS The constructed plasmid pcDNA3.1(+)-angiostatin were confirmed with the right gene sequence by enzymati c digestion and gene sequencing. Western blot showed that angiostatin protein was expressed and secreted by SGC7901 cells. CONCLUSION Gene of angiostatin in plasmid ,p cDNA3.1(+)-angiostatin was expressed by transfected cells, which had potential value in angiogenesis ge ne theraopy of gastric cancer.%目的构建鼠源性血管抑素angiostatin cDNA真核表达载体,转染人胃癌细胞株SCG7901,检测蛋白表达. 方法采用定向克隆构建鼠源性血管抑素angiostatin cDNA真核表达载体pcDNA3.1(+)-angiostatin,酶切鉴定和测序;采用脂质体基因转染人胃癌细胞株SCG7901,G418抗性筛选;Western blot检测血管抑素的表达. 结果酶切鉴定和测序证明成功构建了基因序列正确的血管抑素真核表达载体,经G418筛选和Western blot检测得到高表达血管抑素的细胞株. 结论真核表达载体pcDNA3.1(+)-angiostatin转导的人胃癌细胞能够表达血管抑素蛋白,在胃癌的血管抑制基因治疗中有潜在临床应用价值.

  2. 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)

  3. Recombinant human tripeptidyl peptidase-1 infusion to the monkey CNS: Safety, pharmacokinetics, and distribution

    Energy Technology Data Exchange (ETDEWEB)

    Vuillemenot, Brian R., E-mail: bvuillemenot@bmrn.com [BioMarin Pharmaceutical Inc., Novato, CA (United States); Kennedy, Derek [BioMarin Pharmaceutical Inc., Novato, CA (United States); Reed, Randall P.; Boyd, Robert B. [Northern Biomedical Research, Inc., Muskegon, MI (United States); Butt, Mark T. [Tox Path Specialists, LLC, Hagerstown, MD (United States); Musson, Donald G.; Keve, Steve; Cahayag, Rhea; Tsuruda, Laurie S.; O' Neill, Charles A. [BioMarin Pharmaceutical Inc., Novato, CA (United States)

    2014-05-15

    CLN2 disease is caused by deficiency in tripeptidyl peptidase-1 (TPP1), leading to neurodegeneration and death. The safety, pharmacokinetics (PK), and CNS distribution of recombinant human TPP1 (rhTPP1) were characterized following a single intracerebroventricular (ICV) or intrathecal-lumbar (IT-L) infusion to cynomolgus monkeys. Animals received 0, 5, 14, or 20 mg rhTPP1, ICV, or 14 mg IT-L, in artificial cerebrospinal fluid (aCSF) vehicle. Plasma and CSF were collected for PK analysis. Necropsies occurred at 3, 7, and 14 days post-infusion. CNS tissues were sampled for rhTPP1 distribution. TPP1 infusion was well tolerated and without effect on clinical observations or ECG. A mild increase in CSF white blood cells (WBCs) was detected transiently after ICV infusion. Isolated histological changes related to catheter placement and infusion were observed in ICV treated animals, including vehicle controls. The CSF and plasma exposure profiles were equivalent between animals that received an ICV or IT-L infusion. TPP1 levels peaked at the end of infusion, at which point the enzyme was present in plasma at 0.3% to 0.5% of CSF levels. TPP1 was detected in brain tissues with half-lives of 3–14 days. CNS distribution between ICV and IT-L administration was similar, although ICV resulted in distribution to deep brain structures including the thalamus, midbrain, and striatum. Direct CNS infusion of rhTPP1 was well tolerated with no drug related safety findings. The favorable nonclinical profile of ICV rhTPP1 supports the treatment of CLN2 by direct administration to the CNS. - Highlights: • TPP1 enzyme replacement therapy to the CNS is in development for CLN2 disease. • Toxicology, pharmacokinetics, and CNS distribution were assessed in monkeys. • TPP1 infusion directly to the brain did not result in any safety concerns. • A positive pharmacokinetic and distribution profile resulted from TPP1 infusion. • This study demonstrates the feasibility of ICV administered

  4. Construction of cDNA Library from Populus euphratica

    Institute of Scientific and Technical Information of China (English)

    Yu Guangjun; Wang Yiqin; Shen Xin

    2003-01-01

    In order to isolate and clone salt-tolerance involved genes of Populus euphratica, we constructed a cDNA library from salt-treated leaves of P. euphratica. In the experiment, double strand cDNA were synthesized by a beads-based method. The syntheses of the first strand and the second strand cDNA, adapter ligation and restriction reaction for releasing cDNA were all conducted on the beads. The double strand cDNA were released from magnetic beads by digestion with NotI, and cDNA fragments smaller than 500 bp and residual adapters were removed through cDNA size fractionation columns. Finally, double strand cDNA were directionally cloned intoλExcell vector. The results show that the primary titer of the cDNA library is 7.46×106 pfu per mL and the packaging efficiency reaches 1.47×107 recombinants per μg DNA. λDNA extracted from two clones of plaque were digested by EcoR I and NotI, both of the clones contained inserts larger than 900 bp. These results show that the cDNA library of salt-treated P. euphratica leaves has been successfully constructed.

  5. SEREX技术筛选及鉴定食管癌肿瘤抗原%Human Esophageal Carcinoma Antigens Screened by Serologic Analysis of Recombinant cDNA Expression Libraries (SEREX)

    Institute of Scientific and Technical Information of China (English)

    遇珑; 胡海; 冉宇靓; 彭良平; 李江伟; 杨治华

    2007-01-01

    背景与目的:正常细胞向癌细胞转化过程中,突变的基因或各种异常表达的蛋白可以成为肿瘤抗原诱导机体的免疫反应,因此肿瘤患者的血清中存在着与肿瘤相关的自身抗体.重组cDNA表达文库血清学分析法(serological analysis of recombinant cDNA expression libraries,SEREX)是利用肿瘤患者血清中的自身抗体筛选、鉴定肿瘤抗原的技术.本研究拟采用SEREX的方法寻找食管癌自身抗体的相关肿瘤抗原,鉴定与食管癌发生、发展相关的基因和免疫治疗分子靶点,并为食管癌的诊断提供候选血清标志物.方法:用食管癌组织建立库容量达1.6×106 pfu的cDNA表达文库,SEREX筛选获得21个不同cDNA序列的阳性克隆,进一步使用SADA法分析其中4个抗原在10例食管癌及10例正常人血清中的反应.结果:在Homosapiens desmin(DES)等21个阳性克隆中,4个克隆与已知EST序列明显无同源性,另外17个克隆与已知基因高度同源.Ribosomal protein S4等4个抗原与食管癌患者和正常人血清反应阳性率分别为40%和0%、60%和10%、70%和20%、30%和20%.结论:Ribosomal protein S4等4个抗原普遍参与了食管癌患者的体液免疫反应,与食管癌患者血清的反应阳性率明显高于正常人的血清.本研究发现的21个食管癌抗原可作为食管癌治疗的潜在分子靶点和食管癌诊断新的候选血清学标志物.

  6. Cloning of a cDNA for steroid sulfatase: frequent occurrence of gene deletions in patients with recessive X chromosome-linked ichthyosis

    Energy Technology Data Exchange (ETDEWEB)

    Bonifas, J.M.; Morley, B.J.; Oakey, R.E.; Kan, Y.W.; Epstein, E.J. Jr.

    1987-12-01

    A human steroid sulfatase cDNA 2.4 kilobases long was isolated from a human placental lambda gt11 cDNA expression library. The library was screened with monospecific rabbit antibodies elicited by injection of steroid sulfatase protein purified from human placentas. Hybridization of the cDNA with EcoRI-digested genomic DNA indicated that patients from 14 of 15 apparently unrelated families have gross deletions of the gene for steroid sulfatase. One patient had genomic DNA fragments that were identical to those from normal individuals, indicating the absence of any major deletions as the cause of his lack of steroid sulfatase enzyme activity.

  7. 与人巨细胞病毒UL128两种不同结构蛋白相互作用蛋白的筛选与分析%Screening of protein interacting with the transcript of UL128 gene showed two protein patterns by yeast two-hybrid from human fetus brain cDNA library

    Institute of Scientific and Technical Information of China (English)

    任高伟; 崔鑫; 马艳萍; 齐莹; 阮强; 孙峥嵘

    2010-01-01

    Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.%目的 利用酵母双杂交系统从人胎脑cDNA文库中筛选与两种不同转录结构的人巨细胞病毒(HCMV)UL128编码蛋白相互作用的蛋白,比较两者相互作用蛋白之间的异同点.方法 通过3'RACE和5'RACE技术扩增出两种HCMV UL128片段,其大小分别为519 bp和642 bp,并将其成功构建到酵母诱饵表达载体pGBKT7中.将以上两种酵母表达载体分别转化到酵母菌AH109中,再将文库DNA转化到已含有酵母表达载体的AH109中,筛选与两种片段大小不同的UL128编码蛋白相互作用的人胎脑蛋白,并对筛选得到的阳性克隆进行测序和生物信息学分析.结果 筛出EFEMP2与UL128-519 bp编码蛋白相互作用,THY-1

  8. Discovery and analysis of pancreatic adenocarcinoma genes using cDNA microarrays

    Institute of Scientific and Technical Information of China (English)

    Gang Jin; Xian-Gui Hu; Kang Ying; Yan Tang; Rui Liu; Yi-Jie Zhang; Zai-Ping Jing; Yi Xie; Yu-Min Mao

    2005-01-01

    AIM: To study the pathogenetic processes and the role of gene expression by microarray analyses in expediting our understanding of the molecular pathophysiology of pancreatic adenocarcinoma, and to identify the novel cancer-associated genes.METHODS: Nine histologically defined pancreatic head adenocarcinoma specimens associated with clinical data were studied. Total RNA and mRNA were isolated and labeled by reverse transcription reaction with Cy5 and Cy3 for cDNA probe. The cDNA microarrays that represent a set of 4 096 human genes were hybridized with labeled cDNA probe and screened for molecular profiling analyses.RESULTS: Using this methodology, 184 genes were screened out for differences in gene expression level after nine couples of hybridizations. Of the 184 genes,87 were upregulated and 97 downregulated, including 11 novel human genes. In pancreatic adenocarcinoma tissue, several invasion and metastasis related genes showed their high expression levels, suggesting that poor prognosis of pancreatic adenocarcinoma might have a solid molecular biological basis.CONCLUSION: The application of cDNA microarray technique for analysis of gene expression patterns is a powerful strategy to identify novel cancer-associated genes, and to rapidly explore their role in clinical pancreatic adenocarcinoma. Microarray profiles provide us new insights into the carcinogenesis and invasive process of pancreatic adenocarcinoma. Our results suggest that a highly organized and structured process of tumor invasion exists in the pancreas.

  9. 基质金属蛋白酶组织抑制剂1基因重组腺病毒载体的构建%Construction of Recombinant Adenovirus Vector Carrying the Human Tissue Inhibitor of Metalloproteinase 1 cDNA

    Institute of Scientific and Technical Information of China (English)

    周毅; 屠冠军

    2012-01-01

    Objective To construct recombinant adenovirus vector carrying the human tissue inhibitor of metalloproteinase 1 (T1MP1) cD-NA and explore the feasibility of reversing or delaying intervertebral disc degeneration by gene therapy. Methods The hTIMPl cDNA was amplified from plasmid containing human TIMP1 ORF sequence by PCR. hTIMPl cDNA was subcloned into the adenovirus shuttle plasmid pDC316 of Ad5MaxTM adenovirus vector system,and the products were co-transfected into HEK293 cells with the helper plasmid pBH- Glox_El ,3Cre by lipofectin transfection method. The recombinant adenovirus Ad5-TIMP1 was generated by homologous recombination of the two plasmids in HEK 293 cells. After identification by PCR and enzyme digestion, Ad5-TTMP1 was amplified in HEK 293 adenoviral packaging cells, purified by column chromatography, and virus activity was measured by TCID50 assay. Results Recombinant adenoviral vector carrying hTIMPl(Ad5-hTIMPl) was successfully constructed with virus activity of l×l010 IU/ml,which was confirmed by PCR,restriction enzyme digestion and DNA sequencing. Conclusion The Recombinant adenoviral vector carrying hTIMPl (Ad5-hTlMPl) was successfully constructed, which provided a foundation for further research on reversing or delaying intervertebral disc degeneration by gene therapy.%目的 为探讨基因治疗逆转或延缓椎间盘退变的可行性,构建及制备人基质金属蛋白酶组织抑制剂1 (tissue inhibitor of metalloproteinase 1,TIMP1) cDNA重组腺病毒载体.方法 以含TIMP1 cDNA序列的质粒为模板,通过PCR方法扩增TIMP1cDNA片段,将TIMP1 cDNA全长定向克隆到Ad5MaxTM腺病毒系统的穿梭质粒pDC316上,构建pDC316-TIMP1穿梭质粒;使用Ad5MaxTM腺病毒包装系统,脂质体介导的穿梭质粒及骨架质粒pBHGlox-E1,3Cre共转染HEK293细胞,同源重组构建含TIMP1 cDNA的重组腺病毒Ad5-TIMP1,通过PCR方法鉴定重组腺病毒Ad5-TIM P1的正确性.通过阴离子柱层析

  10. Infectious Maize rayado fino virus from cloned cDNA

    Science.gov (United States)

    Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

  11. 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.

  12. Comparative efficacy and safety of multiple routes of direct CNS administration of adeno-associated virus gene transfer vector serotype rh.10 expressing the human arylsulfatase A cDNA to nonhuman primates.

    Science.gov (United States)

    Rosenberg, Jonathan B; Sondhi, Dolan; Rubin, David G; Monette, Sébastien; Chen, Alvin; Cram, Sara; De, Bishnu P; Kaminsky, Stephen M; Sevin, Caroline; Aubourg, Patrick; Crystal, Ronald G

    2014-09-01

    Metachromatic leukodystrophy (MLD), a fatal disorder caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA), is associated with an accumulation of sulfatides, causing widespread demyelination in both central and peripheral nervous systems. On the basis of prior studies demonstrating that adeno-associated virus AAVrh.10 can mediate widespread distribution in the CNS of a secreted lysosomal transgene, and as a prelude to human trials, we comparatively assessed the optimal CNS delivery route of an AAVrh.10 vector encoding human ARSA in a large animal model for broadest distribution of ARSA enzyme. Five routes were tested (each total dose, 1.5 × 10(12) genome copies of AAVrh.10hARSA-FLAG): (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen, thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of ARSA activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered saline-administered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter generated the broadest distribution of ARSA, with 80% of the brain displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls. No significant toxicity was observed with any delivery route as measured by safety parameters, although some inflammatory changes were seen by histopathology. We conclude that AAVrh.10-mediated delivery of ARSA via CNS

  13. Significance of RNA reference in tumour-related gene expression analyses by cDNA array.

    Science.gov (United States)

    Laytragoon-Lewin, Nongnit; Lagerlund, Magnus; Lundgren, Jan; Nordlander, Britt; Elmberger, Göran; Södergren, Towe; Lagerros, Christofer; Rutqvist, Lars Erik; Lewin, Freddi

    2005-01-01

    The cDNA array technique is an efficient approach for studying the expression of a large number of genes in a single experiment. The cDNA array analysis indicates the relative level of corresponding gene expression from a specimen and a reference. Our investigation was performed to address the significance of reference RNA on the outcome of the cancer-related gene expression profile obtained from cDNA array analysis. Human head and neck squamous cell carcinoma (HNSCC) biopsies and 5 sources of RNA reference were used for this purpose. In these biopsies, each individual patient expressed a unique set of genes both in normal and tumour tissue. It is important to note that 5 striking patterns of tumour-related gene expression were obtained according to the 5 references used. Significant differences in 60%, 16%, 15% and 15% of the genes expressed were shown when autologous normal matched tissue biopsy references were compared to pooled cell lines, allogenic normal mixed cell types, tumours or allogenic normal matched cell type references, respectively. Thus, theoretically and our study suggested that patient autologous normal cells matching with the tumour type should be the most suitable reference in cDNA array for the identification of individual tumour gene profiles with clinical purpose.

  14. 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...

  15. TRAV gene usage in pig T-cell receptor alpha cDNA.

    Science.gov (United States)

    Yamamoto, Ryuji; Uenishi, Hirohide; Hatsuse, Hiromi; Sato, Eimei; Awata, Takashi; Yasue, Hiroshi; Takagaki, Yohtaroh

    2005-05-01

    Pig (Sus scrofa) TRA clones were isolated from cDNA libraries of total RNA from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes of a 5-month-old Clawn strain pig. Among 103 complete TRA cDNA clones from both sources, 33 different TRAV genes were identified. By comparing their sequence identities against one another, these pig TRAV genes were grouped into 20 subgroups, including 13 subgroups, each containing only a single member. All of these pig subgroups gave corresponding human and mouse functional counterparts, suggesting their functional commonality. An exception was the Va01 gene segment, which lacked a functional human counterpart. The present report provides groundwork for studies on pig TRA expression.

  16. 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.

  17. 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...

  18. [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.

  19. 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.

  20. 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.

  1. 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...

  2. 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...

  3. Expression of human AR cDNA driven by its own promoter results in mild promotion, but not suppression, of growth in human prostate cancer PC-3 cells%自身启动子驱动的人雄激素受体cDNA的表达能够适度促进而不是抑制人前列腺癌细胞PC-3的生长

    Institute of Scientific and Technical Information of China (English)

    Saleh Altuwaijri; Cheng-Chia Wu; Yuan-Jie Niu; Atsushi Mizokami; Hong-Chiang Chang; Chawnshang Chang

    2007-01-01

    Aim: To examine the physiological role of the androgen receptor (AR) in the PC-3 cell line by transfecting full-length functional AR cDNA driven by its natural human AR promoter. Methods: We generated an AR-expressing PC-3(AR)9 stable clone that expresses AR under the control of the natural human AR promoter and compared its proliferation to that of the PC-3(AR)2 (stable clone that expresses AR under the control of the cytomegalovirus (CMV) promoter,established by Heisler et al.) after androgen treatment. Results: We found that dihydrotestosterone (DHT) from 0.001 nmol/L to 10 nmol/L induces cell cycle arrest or inhibits proliferation of PC-3(AR)2 compared with its vector control, PC-3(pIRES). In contrast, PC-3(AR)9 cell growth slightly increased or did not change when treated with physiological concentrations of 1 nmol/L DHT. Conclusion: These data suggest that intracellular control of AR expression levels through the natural AR promoter might be needed for determining AR function in androgen-independent prostate cancer (AIPC) PC-3 cells. Unlike previous publications that showed DHT mediated suppression of PC-3 growth after transfection of viral promoter-driven AR overexpression, we report here that DHT-mediated PC-3 proliferation is slightly induced or does not change compared with its baseline after reintroducing AR expression driven by its own natural promoter, as shown in PC-3(AR)9 prostate cancer cells.

  4. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    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 appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  5. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    1994-01-04

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties. GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  6. Differential cDNA cloning by enzymatic degrading subtraction (EDS).

    OpenAIRE

    1994-01-01

    We describe a new method, called enzymatic degrading subtraction (EDS), for the construction of subtractive libraries from PCR amplified cDNA. The novel features of this method are that i) the tester DNA is blocked by thionucleotide incorporation; ii) the rate of hybridization is accelerated by phenol-emulsion reassociation; and iii) the driver cDNA and hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. We demonstrate th...

  7. Constructing and detecting a cDNA library for mites.

    Science.gov (United States)

    Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

    2015-10-01

    RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

  8. 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...

  9. 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.``

  10. 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.

  11. 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.

  12. Identification of alternative transcripts using rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Yeku, Oladapo; Scotto-Lavino, Elizabeth; Frohman, Michael A

    2009-01-01

    Many organisms, including humans, have many more proteins than are actually coded for by their genes. This discrepancy is partially explained by the existence of alternative transcripts produced by the same gene. Multiple isoforms of the same gene sometimes perform completely different functions, and as such, knowing the sequence of one of the transcripts is not enough. Rapid Amplification of cDNA Ends (RACE) provides an inexpensive and powerful tool to quickly identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5' and 3' cDNA ends using the "New Race" technique.

  13. 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.

  14. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

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

    Institute of Scientific and Technical Information of China (English)

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

    2001-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  17. 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.

  18. Screening cDNA Libraries Using Partial Probes to Isolate Full-Length cDNAs from Vascular Cells.

    Science.gov (United States)

    Csortos, C; Lazar, V; Garcia, J G

    1999-01-01

    The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the utility and power of library screening, we will relate our own library screening efforts utilized to isolate the nonmuscle high molecular weight myosin light chain kinase isoform from a human umbilical vein endothelial cell cDNA library (3). This unique nonmuscle myosin light chain kinase isoform phosphorylates myosin light chains, thereby playing an essential role in agonist-mediated endothelial cell contraction, paracellular gap formation and increased vascular permeability. We are hopeful that this step-by-step approach will help the reader to understand the discussed methods.

  19. A new approach for cloning hLIF cDNA from genomic DNA isolated from the oral mucous membrane.

    Science.gov (United States)

    Cui, Y H; Zhu, G Q; Chen, Q J; Wang, Y F; Yang, M M; Song, Y X; Wang, J G; Cao, B Y

    2011-11-25

    Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.

  20. 人胰腺细胞cDNA 文库中丙型肝炎病毒NS4B 结合蛋白基因的筛选%Screening of proteins binding to hepatitis C virus NS4B protein from human pancreas cDNA library

    Institute of Scientific and Technical Information of China (English)

    温少芳; 张锦前; 孙荣华; 李卓; 高萍; 王琦; 刘顺爱; 成军

    2011-01-01

    Objective To screen proteins from human pancreas cDNA library , which interact with the protein coded hy hepatitis C virus ( HCV) NS4B. Methods The reconstructed plasmid pGBKT7-NS4B was transformed into yeast strain AH109 and identified. The transformed AH109 ( pGBKT7-NS4B) mated with Y187 ( plasmids of pancreatic cDNA library ) . The diploid yeast cells were plated on nutrient deficiency medium containing X-α-gal for selecting. The plasmids in diploid yeast cells were extracted and electrotransformed into E. c.oli DH5a. The plasmids in DH5α were extracted, sequenced and blasted.Results Seven proteins interacting with HCV NS4B were found . such as CDK5RAP3 , colipase , pancreatic stone protein ,lactoprotein . elastase2A , chymotrypsin and bile salt-stimulated esterase . Conclusions Some of the eight pancreatic proteins may be related with metaholisms of glucose and lipid .%目的 从人胰腺细胞cDNA 文库中,采用酵母双杂交方法 筛选HCV NS4B 包膜蛋白的相互作用蛋白.方法 人胰腺细胞cDNA 文库先进行扩增,随后纯化及鉴定,然后将鉴定好的人胰腺细胞cDNA 文库质粒转化入酵母菌株Y187.构建pGBKT7-NS4B 作为诱饵质粒,随之转化酵母菌株AH109,用色氨酸缺陷型培养基(SD/-Trp)筛选阳性菌落.配合两种重组酵母菌株AH109 与Y187,用四缺培养基及X-α-gal 筛选蓝色酵母菌落,提取相应质粒,电转化感受态菌DH5α后再提取质粒,测序仪测序,结果 使用PUBMED 进行序列比对.结果 成功构建pGBKT7-HCV NS4B 重组质粒,并从人胰腺cDNA 文库中筛选出7 种与HCV NS4B 蛋白相结合的蛋白基因,包括CDK5RAP3、辅脂肪酶、胰石蛋白、凝乳蛋白、弹性蛋白酶2A、糜蛋白酶和胆盐刺激酯酶.结论 HCV NS4B 蛋白可能通过与所筛选出前述蛋白中参与代谢过程的相关蛋白结合,影响糖、脂类代谢过程.

  1. Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunobiology*

    Directory of Open Access Journals (Sweden)

    Coussens PM

    2003-03-01

    Full Text Available Recent developments in expressed sequence tag (EST and cDNA microarray technology have had a dramatic impact on the ability of scientists to study the responses of thousands of genes to external stimuli, such as infection, nutrient flux, and stress. To date however, these studies have largely been limited to human and rodent systems. Despite the tremendous potential benefit of EST and cDNA microarray technology to studies of complex problems in domestic animal species, a lack of integrated resources has precluded application of these technologies to domestic species. To address this problem, the Center for Animal Functional Genomics (CAFG at Michigan State University has developed a normalized bovine total leukocyte (BOTL cDNA library, generated EST clones from this library, and printed cDNA microarrays suitable for studying bovine immunobiology. Our data revealed that the normalization procedure successfully reduced highly abundant cDNA species while enhancing the relative percentage of clones representing rare transcripts. To date, a total of 932 EST sequences have been generated from this library (BOTL and the sequence information plus BLAST results made available through a web-accessible database http://gowhite.ans.msu.edu. Cluster analysis of the data indicates that a total of 842 unique cDNAs are present in this collection, reflecting a low redundancy rate of 9.7%. For creation of first generation cDNA microarrays, inserts from 720 unique clones in this library were amplified and microarrays were produced by spotting each insert or amplicon 3 times on glass slides in a 48-patch arrangement with 64 total spots (including blanks and positive controls per patch. To test our BOTL microarray, we compared gene expression patterns of concanavalin A stimulated and unstimulated peripheral blood mononuclear cells (PBMCs. In total, hybridization signals on over 90 amplicons showed upregulation (>3× in response to Con A stimulation, relative to

  2. Impairment of HIV-1 cDNA synthesis by DBR1 knockdown.

    Science.gov (United States)

    Galvis, Alvaro E; Fisher, Hugh E; Nitta, Takayuki; Fan, Hung; Camerini, David

    2014-06-01

    Previous studies showed that short hairpin RNA (shRNA) knockdown of the RNA lariat debranching enzyme (DBR1) led to a decrease in the production of HIV-1 cDNA. To further characterize this effect, DBR1 shRNA was introduced into GHOST-R5X4 cells, followed by infection at a multiplicity near unity with HIV-1 or an HIV-1-derived vector. DNA and RNA were isolated from whole cells and from cytoplasmic and nuclear fractions at different times postinfection. Inhibition of DBR1 had little or no effect on the formation of minus-strand strong-stop cDNA but caused a significant reduction in the formation of intermediate and full-length cDNA. Moreover, minus-strand strong-stop DNA rapidly accumulated in the cytoplasm in the first 2 h of infection but shifted to the nuclear fraction by 6 h postinfection. Regardless of DBR1 inhibition, greater than 95% of intermediate-length and full-length HIV-1 cDNA was found in the nuclear fraction at all time points. Thus, under these experimental conditions, HIV-1 cDNA synthesis was initiated in the cytoplasm and completed in the nucleus or perinuclear region of the infected cell. When nuclear import of the HIV-1 reverse transcription complex was blocked by expressing a truncated form of the mRNA cleavage and polyadenylation factor CPSF6, the completion of HIV-1 vector cDNA synthesis was detected in the cytoplasm, where it was not inhibited by DBR1 knockdown. Refinement of the cell fractionation procedure indicated that the completion of reverse transcription occurred both within nuclei and in the perinuclear region. Taken together the results indicate that in infections at a multiplicity near 1, HIV-1 reverse transcription is completed in the nucleus or perinuclear region of the infected cell, where it is dependent on DBR1. When nuclear transport is inhibited, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. Thus, there are at least two mechanisms of HIV-1 reverse transcription that require different

  3. Preparation of cDNA libraries from vascular cells.

    Science.gov (United States)

    Lieb, M E; Taubman, M B

    1999-01-01

    The vast majority of past and present efforts in the molecular cloning of expressed sequences involve isolation of clones from cDNA libraries constructed in bacteriophage lambda (1,2). As discussed in Chapter 6 , screening these cDNA libraries using labeled probes remains the most straightforward method to isolate full length cDNAs for which some partial sequence information is known. Although the availability of high quality reagents and kits over the past decade has made the process of library construction increasingly straightforward, generation of high-quality libraries is a task that still requires a fair amount of dedicated effort. Because alternative PCR-based cloning strategies have become increasingly popular alternatives to cDNA library screening, it is useful to consider the advantages and disadvantages of each strategy before embarking on a project to construct a cDNA library (Table 1). In our opinion, it is worthwhile to construct a cDNA library when the transcript of interest is not exceedingly rare (i.e., can readily be detected by Northern blot analysis of total RNA), when multiple cDNAs will need to be cloned over a period of time, and in situations where occasional mutations can not be tolerated (for example, if the cDNA is to be expressed in mammalian cells to examine function). In situations where the transcript of interest is expressed at exceedingly low levels, or when only a single cDNA needs to be cloned, a PCR-based strategy should be considered. When the tissue source is precious (such as a unique clinical specimen), successful construction of a phage library provides a resource that can be amplified and used for multiple cloning projects over many years, but runs the risk of consuming the available RNA if the library construction fails. Table 1 Comparison of Relative Advantages of cDNA Cloning from Lambda Phage Libraries by Plaque Hybridization Compared to Newer PCR- Based Strategies Lambda phage cDNA library PCR-based strategy Freedom

  4. Analysis of gene expression profile of pancreatic carcinoma using CDNA microarray

    Institute of Scientific and Technical Information of China (English)

    ZhiJun Tan; Xian-Gui Hu; Gui-Song Cao; Yan Tang

    2003-01-01

    AIM: To identify new diagnostic markers and drug targets,the gene expression profiles of pancreatic cancer were compared with that of adjacent normal tissues utilizing cDNA microarray analysis.METHODS: cDNA probes were prepared by labeling mRNA from samples of six pancreatic carcinoma tissues with Cy5dUTP and mRNA from adjacent normal tissues with Cy3dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 12 800cDNA arrays (12 648 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3 000scanner (General Scanning, Inc.). The values of CyS-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 0.69.RESETS: Among 6 samples investigated, 301 genes, which accounted for 2.38% of genes on the microarry slides,exhibited differentially expression at least in 5. There were 166 over-expressed genes including 136 having been registered in Genebank, and 135 under-expressed genes including 79 in Genebank in cancerous tissues.CONCLUSION: Microarray analysis may provide invaluable information on disease pathology, progression, resistance to treatment, and response to cellular microenvironments of pancreatic carcinoma and ultimately may lead to improving early diagnosis and discovering innovative therapeutic approaches for cancer.

  5. Rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Yeku, Oladapo; Frohman, Michael A

    2011-01-01

    Rapid Amplification of cDNA ends (RACE) provides an inexpensive and powerful tool to quickly obtain full-length cDNA when the sequence is only partially known. Starting with an mRNA mixture, gene-specific primers generated from the known regions of the gene and non-specific anchors, full-length sequences can be identified in as little as 3 days. RACE can also be used to identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5(') and 3(') cDNA ends using the "new RACE" technique.

  6. Procedure for normalization of cDNA libraries

    Science.gov (United States)

    Bonaldo, Maria DeFatima; Soares, Marcelo Bento

    1997-01-01

    This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

  7. 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.

  8. 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.

  9. Analysis of common bean (Phaseolus vulgaris L., genotype BAT93 calmodulin cDNA using computational tools

    Directory of Open Access Journals (Sweden)

    Kassim Amelia

    2015-01-01

    Full Text Available Background: Common bean (Phaseolus vulgaris L. is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it. Objective: The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM gene cDNA and its deduced protein (amino acids sequence. Materials and Methods: Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D structure of PvCaM protein were predicted using the Phyre automatic fold recognition server. Results: Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM. Conclusions: This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is

  10. 转染胸苷磷酸化酶基因对5'-脱氧氟尿苷抑制结肠癌细胞的影响%Effect of thymidine phosphorylase cDNA transfection on the inhibition of human colon carcinoma cell line by 5'-deoxy-5-fluorouridine

    Institute of Scientific and Technical Information of China (English)

    高庆; 张继民; 刘剑; 王绮雯; 叶钿均; 刘影

    2013-01-01

    目的 探讨转染胸苷磷酸化酶(TP)cDNA对5’-脱氧氟尿苷(5’-DFUR)抑制人结肠癌细胞LOVO作用的影响.方法 将TP cDNA序列克隆以慢病毒表达载体包装后转染人结肠癌细胞LOVO(LOVO-TP组),另设空白对照组和LOVO-载体组.以流式细胞仪检测3组细胞转染效率,RTPCR法检测3组细胞TP mRNA表达;Western blot法检测转染前后TP蛋白水平;MTT法检测转染前后LOVO对5’-DFUR药物敏感性的变化;高效液相色谱法(HPLC)检测3组细胞转化不同浓度5'-DFUR生成5-FU量的情况.结果 转染TP基因并传代5代后,LOVO细胞的转染效率在95%左右.转染TP基因后,LOVO-TP组的TP mRNA表达水平为空白对照组的(282.5±86.8)倍(P<0.01),而LOVO-载体组与对照组相比差异无统计学意义(P>0.05);Western blot检测显示,LOVO-TP组的TP蛋白表达明显高于对照组和LOVO-载体组.5’-DFUR对LOVO细胞半数有效剂量(IC50)对照组为(1607.3±56.8) μmol/L,明显高于LOVO-TP组的(1087.7±89.1) μmol/L(P<0.01);而LOVO-染载体组为(1699.5±38.7)tμmol/L,与对照组相比差异无统计学意义(P>0.05).培养基中分别加入0、500、1000和2000 μmol/L的5'-DFUR后,对照组培养基中分别检出0、2.10、3.13和7.19 μmol/L的氟尿嘧啶(5-FU);而在LOVO-TP组的细胞培养基中则分别检出0、22.16、30.94和40.02 μmol/L的5-FU;而LOVO-载体组的培养基中则几乎无5-FU检出.结论 转染TP cDNA能够明显提高LOVO细胞的TP mRNA及TP蛋白表达水平,使细胞外5’-DFUR转化为5-FU增多,明显提高5’-DFUR对LOVO的细胞毒性作用.%Objective To investigate the inhibiting impact of 5'-deoxy-5-fluorouridine(5'-DFUR)on human colon carcinoma cell line LOVO after transfection of thymidine phosphorylase (TP) cDNA.Methods TP cDNA was transfected into human colon carcinoma cell line LOVO with lentiviral vector pLenti6.3_MCS_IRES2-EGFP,and the transfection efficiency was analyzed by flow cytometry.TP mRNA and protein

  11. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

    A Bioinformatic Strategy to Rapidly Characterize cDNA LibrariesG. Charles Ostermeier1, David J. Dix2 and Stephen A. Krawetz1.1Departments of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, & Institute for Scientific Computing, Wayne State Univer...

  12. NORMAL NASAL GENE EXPRESSION LEVELS USING CDNA ARRAY TECHNOLOGY

    Science.gov (United States)

    Normal Nasal Gene Expression Levels Using cDNA Array Technology. The nasal epithelium is a target site for chemically-induced toxicity and carcinogenicity. To detect and analyze genetic events which contribute to nasal tumor development, we first defined the gene expressi...

  13. Rescue of mumps virus from cDNA.

    Science.gov (United States)

    Clarke, D K; Sidhu, M S; Johnson, J E; Udem, S A

    2000-05-01

    A complete DNA copy of the genome of a Jeryl Lynn strain of mumps virus (15,384 nucleotides) was assembled from cDNA fragments such that an exact antigenome RNA could be generated following transcription by T7 RNA polymerase and cleavage by hepatitis delta virus ribozyme. The plasmid containing the genome sequence, together with support plasmids which express mumps virus NP, P, and L proteins under control of the T7 RNA polymerase promoter, were transfected into A549 cells previously infected with recombinant vaccinia virus (MVA-T7) that expressed T7 RNA polymerase. Rescue of infectious virus from the genome cDNA was demonstrated by amplification of mumps virus from transfected-cell cultures and by subsequent consensus sequencing of reverse transcription-PCR products generated from infected-cell RNA to verify the presence of specific nucleotide tags introduced into the genome cDNA clone. The only coding change (position 8502, A to G) in the cDNA clone relative to the consensus sequence of the Jeryl Lynn plaque isolate from which it was derived, resulting in a lysine-to-arginine substitution at amino acid 22 of the L protein, did not prevent rescue of mumps virus, even though an amino acid alignment for the L proteins of paramyxoviruses indicates that lysine is highly conserved at that position. This system may provide the basis of a safe and effective virus vector for the in vivo expression of immunologically and biologically active proteins, peptides, and RNAs.

  14. Isolation and characterization of a cDNA clone of UDP-galactose: flavonoid 3-O-galactosyltransferase (UF3GaT) expressed in Vigna mungo seedlings.

    Science.gov (United States)

    Mato, M; Ozeki, Y; Itoh, Y; Higeta, D; Yoshitama, K; Teramoto, S; Aida, R; Ishikura, N; Shibata, M

    1998-11-01

    Four cDNA clones were isolated from Vigna mungo seedlings by the screening with cDNA encoding UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) of Antirrhinum majus as a probe; the product of the gene corresponding to one cDNA was more highly expressed in the first simple leaves than in stems. Nucleotide sequence analysis revealed 1,691 bp (including 326 bp non-reading) containing an open reading frame of 455 amino acids. The deduced amino acid sequence showed 42% and 23% identity with those of A. majus UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) and Petunia hybrida UDP-rhamnose:anthocyanidin 3-O-glucoside rhamnosyltransferase (RT), respectively. One region of the cDNA (amino acids 325 to 387) showed similarity to ceramide UDP-galactosyltransferases of mice, rats and humans. A crude extract from Escherichia coli, in which the protein was expressed from the cDNA, showed high UF3GaT activity but low UF3GT activity, and was similar in K(m), optimal pH and substrate specificity to UF3GaT from V. mungo. We conclude that we have obtained UDP-galactose:flavonoid 3-O-galactosyltransferase (UF3GaT) cDNA from V. mungo.

  15. Cloning of Splicing Variants of αl, 3-galactosyltransferase cDNA of Chinese Banna Minipig Inbred Line and Its Expression in Human Cells%中国近交系版纳微型猪α1,3-半乳糖基转移酶基因剪接变异体的克隆及其在人类细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    刘美; 朱圣明; 郑鸿; 王宇; 王竹; 杨娅君; 伍艳霞; 曾养志; 王艳萍

    2012-01-01

    目的 克隆中国近交系版纳微型猪(Chinese Banna Minipig Inbred Line,BMI)的α1,3-半乳糖基转移酶(α1,3-GT)基因并分离其剪接变异体,构建其真核表达载体并观察BMI α1,3-GT基因在人肺腺癌A549细胞中的表达和功能.方法 提取BMI肝组织总RNA,RT-PCR扩增全长的α1,3-GT cDNA,并克隆到pMD18-T载体,挑选15个阳性克隆进行测序,获得GT1、GT2两种基因剪接变异体.将GT1、GT2克隆到pEGFP-N1上构建其真核表达载体,分别命名为pN-GT1、pN-GT2.将pN-GT1、pN-GT2分别转染人肺腺癌A549细胞,RT-PCR检测转染细胞中α1,3-GT mRNA的表达,倒置荧光显微镜和流式细胞术观察转染细胞上α-半乳糖基(α-Gal)的表达,流式细胞术检测人血清中IgM抗体和补体C3与转染细胞上α-Gal的结合.结果 在BMI中发现了碱基长度为1116bp和1080 bp的两个α1,3-GT基因剪接变异体,后者缺失了外显子5.pN-GT1或pN-GT2转染的A549细胞中,均检测到了α1,3-GT mRNA和α-Gal的表达,转染细胞膜上也检测到IgM和C3的沉积,且两种转染细胞中α-Gal的表达及IgM和C3的沉积没有差异(P>0.05).结论 成功克隆了BMI的α1,3-GT基因,并发现两种α1,3-GT基因剪接变异体.两种基因剪接变异体转染的细胞中,均检测到α1,3-GT mRNA的表达且α1,3-GT能催化合成具有生物学效应的α-Gal,这为BMI用于异种移植中涉及α1,3-GT基因操作的研究提供了基因背景资料.%Objevtive To study the transfection and expression of the splicing variants of al, 3galactosyltransferase cDNA of Chinese Banna Minipig Inbred Line (BMI) in human A549 cells. Methods Full length of αl,3-GT gene cDNA was amplified by RT-PCR from total RNA of BMI liver tissue and cloned into T-A cloning vector. Two different splicing variants of BMI αl.3-GT cDNA were confirmed by sequencing 15 positive clones and inserted respectively into pEGFP-N1 to construct eukaryotic expression vectors pN-GTl and pN-GT2. The vectors were

  16. RNA transcripts of full-length cDNA clones of rabbit hepatitis E virus are infectious in rabbits.

    Science.gov (United States)

    Cossaboom, Caitlin M; Huang, Yao-Wei; Yugo, Danielle M; Kenney, Scott P; Piñeyro, Pablo; Matzinger, Shannon R; Heffron, C Lynn; Pierson, F William; Meng, Xiang-Jin

    2014-11-07

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is a single-stranded positive-sense RNA virus belonging to the family Hepeviridae. At least four genotypes of the family infect humans: genotypes 1 and 2 are transmitted to humans through contaminated water, while genotypes 3 and 4 are zoonotic and have animal reservoirs. A novel strain of HEV recently identified in rabbits is a distant member of genotype 3, and thus poses a potential risk of zoonotic transmission to humans. The objective of this study was to construct and characterize an infectious cDNA clone of the rabbit HEV. Two full-length cDNA clones of rabbit HEV, pT7g-rabHEV and pT7-rabHEV, were constructed and their infectivity was tested by in vitro transfection of Huh7 human liver cells and by direct intrahepatic inoculation of rabbits with capped RNA transcripts. Results showed that positive signal for rabbit HEV protein was detected by an immunofluorescence assay with a HEV-specific antibody in Huh7 human liver cells transfected with capped RNA transcripts from the two full-length cDNA clones. Rabbits intrahepatically inoculated with capped RNA transcripts from each of the two clones developed active HEV infection as evidenced by seroconversion to anti-HEV antibodies, and detection of rabbit HEV RNA in sera and feces of inoculated animals. The availability of a rabbit HEV infectious cDNA clone now affords us the ability to delineate the mechanism of HEV replication and cross-species infection in a small animal model.

  17. Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene

    Energy Technology Data Exchange (ETDEWEB)

    Tebbs, R.S.; Tucker, J.D.; Hwang, M. [Lawrence Livermore National Lab., CA (United States)] [and others

    1995-07-03

    The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to displatin and {gamma}-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is {approx} 2.5 kb and detects an {approx} 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels. 30 refs., 5 figs., 2 tabs.

  18. The preparation of an infectious full-length cDNA clone of Saffold virus.

    Science.gov (United States)

    Himeda, Toshiki; Hosomi, Takushi; Asif, Naeem; Shimizu, Hiroyuki; Okuwa, Takako; Muraki, Yasushi; Ohara, Yoshiro

    2011-03-09

    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-3 genome. To obtain the infectious RNA using T7 promoter, a variant of T7 RNA polymerase, which fails to recognize the PTH signal, was useful. This study will provide a valuable technical insight into the reverse genetics of SAFV.

  19. The preparation of an infectious full-length cDNA clone of Saffold virus

    Directory of Open Access Journals (Sweden)

    Okuwa Takako

    2011-03-01

    Full Text Available 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-3 genome. To obtain the infectious RNA using T7 promoter, a variant of T7 RNA polymerase, which fails to recognize the PTH signal, was useful. This study will provide a valuable technical insight into the reverse genetics of SAFV.

  20. 微量RNA的cDNA PCR文库的构建%The Construction of cDNA PCR Library from a Small Amount of RNA

    Institute of Scientific and Technical Information of China (English)

    李晶泉; 袁晓东; 汤敏谦

    2001-01-01

    By the method of PCR (Polymerase Chain Reaction),we have constructed the cDNA PCR library from mRNA.The cDNA PCR library can amplify the original cDNA up to hundreds of times.With the total RNA of human K562 cultured cell,the cDNA of β-Actin has been obtained by the methods of cDNA PCR library and reverse transcription respectively.As contrast,the amount of β-Actin′s cDNA from the cDNA PCR library is much higher than from reverse transcription.75pg total RNA of human K562 Cultured cell is employed to construct 50μl cDNA PCR library,and the cDNA of β-Actin can even be detected by using 1μl of the library as template to perform the PCR.Therefore cDNA PCR library can greatly enlarge the amount of information.%使用PCR(polymerase chain reaction)技术,调制了mRNA的cDNA PCR文库,实验证明,cDNA PCR文库能使原cDNA的量放大数百倍。同时,使用人体K562培养细胞的总RNA,对cDNA PCR文库法和反转录中的β-Actin的cDNA量进行了比较,cDNA PCR文库法中的β-Actin的cDNA量大大高于反转录中的β-Actin的cDNA量。使用75pg的人体K562培养细胞的总RNA,调制成50μl的cDNA PCR文库,使用1μl的cDNA PCR文库进行PCR反应时,可对文库中的β-Actin的cDNA进行PCR检测。因此,cDNA PCR文库显示了良好的信息放大性能。

  1. 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

  2. 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

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

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

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

  4. 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.

  5. ANALYSIS OF GENES ASSOCIATED WITH LYMPHATIC METASTASIS IN PANCREATIC CARCINOMA USING cDNA MICROARRAY

    Institute of Scientific and Technical Information of China (English)

    谭志军; 胡先贵; 曹贵松; 唐岩

    2003-01-01

    Objective: To identify new markers for prediction of lymph node metastasis. Methods: cDNA probes were prepared by labeling mRNA from samples of four pancreatic carcinoma tissues with Cy5-dUTP and mRNA from adjacent normal tissues with Cy3-dUTP respectively through reverse transcription. The mixed probes of each sample were then hybridized with 4,096 cDNA arrays (4,000 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Genes that differentially expresses in each cancerous tissue were sought out according to the standard that the absolute value of natural logarithm of the ratio of Cy5 to Cy3 is greater than 0.69, i. e., more than 2 times change of gene expression, and the signal value of either Cy3 and Cy5 need to be greater than 600. Then, the genes differently expressed in cancer with and without lymphatic metastasis were screened out for further analysis. Results: Among 2 samples with lymphatic metastasis and 2 samples without metastasis, 56 genes, which accounted for 1.40% of genes on the microarray slides, exhibited differentially expression in cancerous tissues with lymphatic metastasis. There were 32 over-expressed genes including 11 having been registered in Genebank, and 24 under-expressed genes including 3 in Genebank. Conclusion: Microarray analysis may provide invaluable information to identify specific gene expression profile of lymphatic metastasis in pancreatic cancer.

  6. Induction of a mutant phenotype in human repair proficient cells after overexpression of a mutated human DNA repair gene.

    NARCIS (Netherlands)

    P.B.G.M. Belt; M.F. van Oostenrijk; H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude)

    1991-01-01

    textabstractAntisense and mutated cDNA of the human excision repair gene ERCC-1 were overexpressed in repair efficient HeLa cells by means of an Epstein-Barr-virus derived CDNA expression vector. Whereas antisense RNA did not influence the survival of the transfected cells, a mutated cDNA generating

  7. 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.

  8. cDNA table - RPD | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us RPD cDNA table Data detail Data name cDNA table DOI 10.18908/lsdba.nbdc00191-004 Description...age About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA table - RPD | LSDB Archive ...

  9. 基因芯片技术筛选人不同发育阶段表皮干细胞差异表达基因的研究%Screening of differential expression genes of human skin epidermal stem cells at different development stages by cDNA microarray technique

    Institute of Scientific and Technical Information of China (English)

    蓝蔚; 刘德伍; 李国辉; 毛远桂; 陈桦; 易先锋; 王联群; 彭燕; 钟清玲

    2011-01-01

    ,with 10 cases in each group. Epidermis were separated using trypsin digestion and EDTA, and human epidermal stem cells were isolated and purified with type Ⅳ collagen attachment method. The monoclonal antibody of integrin β1 and keratin 19 were used for detection and identification of epidermal stem cells by immunohistochemical staining. Total RNA was extracted from above cells by Trizol one-step method, and were detected by formaldehyde denaturing agarose gel electrophoresis. Probes were prepared and hybridized into cDNA microarray for scanning fluorescent signals and analysis of images, with two-fold differential expression value for screening. Significantly up/down-regulated genes were selected for verification by real time RT-PCR. Results By comparing expression profile between A and C groups, a total of 1808 genes with differential expression were detected, including 1089 up-regulated genes and 719 down-regulated genes, and they were classified into 128 categories. Among them, 1462 genes were known (found in GeneBank), 346 genes were unknown. A total of 4534 genes with differential expression were detected between C and F groups, in which 1783 genes were up-regulated and 2751 genes were down-regulated, and they were classified into 216 categories. Among them, 3577 genes were known (found in GeneBank), and 957 genes were unknown. There were 1104 genes with differential expression consistently detected in F, C and A groups,which were classified into 32 categories according to gene function. Among them, 94 genes were consistently up-regulated and 75 genes consistently down-regulated. Test results of real time RT-PCR were in accordance with above-mentioned results. Conclusions Gene expression profiles of epidermal stem cells cultured in vitro, harvested from fetuses, children, and adult, exhibit obvious difference. This may be closely related to different stages of proliferation and differentiation of human epidermal stem cell and self-repair ability of wound at

  10. Analysis of differences of gene expressions in keloid and normal skin with the aid of cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    Chen Wei; Fu Xiaobing; Sun Xiaoqing; Sun Tongzhu; Zhao Zhili; Yang Yinhui; Sheng Zhiyong

    2003-01-01

    Background: Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of the disease. Keloid is a intricate lesion which is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skins with the aid of cDNA microarray in order to explore the molecular mechanism underlying keloid formation. Methods: The PCR products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs was isolated from freshly excised human keloids and normal skin, and then was purified to mRNA by Oligotex. Both the mRNA from keloids and normal skin was reversely transcribed to cDNAs with the incorporations of fluorescent dUTP, for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. Results: Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250were up-regulated (2.98%) and 152 down-regulated (1.81%). Analyses of collagen, fibronectin, proteoglycan,growth factors and apoptosis related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with published biochemical and clinical observations of keloids. Conclusions: DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help understand the molecular mechanism of keloid formation.

  11. Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

    Institute of Scientific and Technical Information of China (English)

    周兆斓; 朱祯; 刘春明; 张海涛; 肖桂芳; 李向辉

    1996-01-01

    Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.

  12. PG25, a pineal-specific cDNA, cloned by differential display PCR (DDPCR) and rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Wang, X; Brownstein, M J; Young, W S

    1997-05-16

    Synthesis of melatonin in the mammalian pineal gland is regulated by the rhythmic expression of acetyl-CoA: serotonin N-acetyltransferase (SNAT) and other unknown factors. To screen for pineal-specific mRNAs potentially involved in melatonin synthesis and/or regulation, differential display PCR (DDPCR) was employed. We used 80 primer pairs and examined 40 bands of interest. One of the pineal specific clones (relative to brain and eye), PG25, was studied further. Hybridization histochemical and Northern analyses confirmed its tissue specificity. The size of the corresponding mRNA is 2.4 kb. A cDNA (2 kb) containing the coding region was obtained using a long-template PCR-based RACE technique. A data base search indicates that PG25 is highly homologous to a recently identified human lung endothelial cell-specific gene, ESM-1. Interestingly, not only the amino acid sequences but also the cDNA sequences, including the long 3' untranslated regions, are highly similar. This suggests that the conserved 3' untranslated region may carry information to regulate its own expression. Northern analysis revealed that PG25 is also expressed in the rat lung, but at a much lower (10%) level compared to the pineal. Finally, our work shows the feasibility of a fast, integrated PCR-based cloning method for obtaining long, potentially full-length cDNAs with restricted expression in anatomically complex regions of the brain. This protocol combining several existing methodologies is suitable for use with limited tissue sources and uses minimal amounts of isotopes.

  13. Expression of HNP1cDNA in CHO-dhfr- cells

    Institute of Scientific and Technical Information of China (English)

    LIU Juan; SUN Yong-tao; DU De-wei; WANG Lin-xu; ZHAI Song; WANG Shao-yang; WANG Ding-cheng

    2004-01-01

    To prepare secretary recombinant human neutrophil peptide1 (HNP1)and test its antimicrobialactivity. Methods: The eukaryotic expression vector pcDNA3. 1/V5-His-TOPO-HNP1 was cotransfected with plasmidpDCH1P11 carrying dhfr gene into dhfr- negative CHO (CHO-dhfr-) cells and recombinant protein was verified by ELISA;G418 selective medium was used to screen the stably expressing cell clones followed by serial passages in 5 × 10-8 mol/L and5 × 10-7 mol/L methotrexate (MTX) for gene amplification. Finally 4 cell clones with high expression level were obtainedand confirmed by ELISA, RT-PCR and IFA. The bacteriastatic activity of concentrated supernatants was tested in vitro asthat was almost 200-fold increase than that in G418 selective medium. 303 bp segments were amplified from 4 tably tranfec-tant cloneswhich matched the length of HNP1 cDNA by RT-PCR. Strong fluorescence was visible in cell plasma in the sta-blly transfectant cells by IFA. K-B disc agar diffusion test showed obvious bacteriastatic diffusion on MH plate of E. Coli.Conclusion: HNP1cDNA can be strongly expressed in CHO-dhfr- cells, which supernatants exhibited high inhibitive effectagainst bacteria.

  14. cDNA cloning, characterization, and expression analysis of the Rac1 gene from Scophthalmus maximus.

    Science.gov (United States)

    Jia, Airong; Zhang, Xiao-Hua

    2009-09-01

    Rac1 is a small GTP-binding protein that belongs to the Rho small GTPases, which are important signaling molecules that regulate the dynamics of the actin cytoskeleton and mediate changes in cell morphology and motility. The EST sequence of Rac1 from turbot (Scophthalmus maximus L.) was obtained from a subtractive cDNA library previously. In this study, the full-length cDNA sequence of turbot Rac1 was obtained, which was 2420 nucleotides (nt) encoding a protein of 192 amino acids, with a putative molecular weight of 21.3 kDa. At the amino-acid level, turbot Rac1 was highly conserved to previously characterized GTPases of Rac sub-family, and was nearly identical to human Rac1 (95.3% identity). Quantitative real-time PCR demonstrated that the Rac1 was constitutively expressed in all tissues examined, but at different levels. Upon challenge with Vibrio harveyi, the expression level of Rac1 fluctuated in the liver at different time points. In the head kidney, its expression level decreased to the lowest at 4 h, and then increased to the background level at 24 h. The remarkable degree of evolutionary conservation observed in turbot Rac1 primary structure together with its changing in expression level upon challenge suggested a functionally important role for this Rho family member in the immune response.

  15. cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

    Institute of Scientific and Technical Information of China (English)

    HUANG; Shengbing; SONG; Wei; LIN; Qishui

    2005-01-01

    A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5'-terminal and 3'-terminal were obtained by RACE technique. The full-length cDNA that encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-Qop. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.

  16. Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

    Science.gov (United States)

    Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

    1992-01-01

    We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

  17. Molecular Cloning of Myostatin Partial cDNA of Beijing Duck and Its Expression in Breast Muscle

    Institute of Scientific and Technical Information of China (English)

    WANG Yong-sheng; HOU Shui-sheng; HUANG Wei; KANG Jun-mei

    2006-01-01

    In this experiment, 500 bp cDNA of myostatin gene was cloned from a Beijing duck's breast. The duck myostatin gene was found to have 98, 96, 95, 88, and 87% sequence similarity at the cDNA level with domestic goose, chicken, domestic pigeon, human, and pig, respectively. The predicted amino acid sequence has an overall similarity with a comparable region of turkey 99%, domestic goose 98%, and chicken 99%. Conserved domains of deduced amino acids showed that it belonged to the TGF-beta family. Myostatin expression in breast muscle was higher at 28, 35, and 42 days than at 7, 14, and 21 days. The pattern of myostatin expression was closely parallel to the trend of breast muscle growth, suggesting that myostatin might play an important role in breast muscle development. It was possible to postulate that myostatin may be a major determinant of muscle mass in breast muscle, as shown in other species.

  18. [cDNA library construction from panicle meristem of finger millet].

    Science.gov (United States)

    Radchuk, V; Pirko, Ia V; Isaenkov, S V; Emets, A I; Blium, Ia B

    2014-01-01

    The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

  19. Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.

    Science.gov (United States)

    Ares, Manuel

    2014-01-01

    This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.

  20. Isolation of novel non-HLA gene fragments from the hemochromatosis region (6p21. 3) by cDNA hybridization selection

    Energy Technology Data Exchange (ETDEWEB)

    Goei, V.L.; Capossela, A.; Gruen, J.R.; Parimoo, S.; Chu, T.W. (Yale Univ. School of Medicine, New Haven, CT (United States))

    1994-02-01

    It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC. 39 refs., 4 figs., 3 tabs.

  1. Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Hai-Tao Wang; Jian-Ping Kong; Fang Ding; Xiu-Qin Wang; Ming-Rong Wang; Lian-Xin Liu; Min Wu; Zhi-Hua Liu

    2003-01-01

    AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1.METHODS: The authors first constructed pcDNA3.1/mychis expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes,classification was performed according to their function and cellular component.RESULTS: Human EMP-1 gene can be stably expressed in ECg706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion.CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved incell signaling, cell communication and adhesion regulators.

  2. Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

    DEFF Research Database (Denmark)

    Jensen, T G; Andresen, B S; Bross, P

    1992-01-01

    An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter...... and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild......-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild...

  3. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends.

    Science.gov (United States)

    Schaefer, B C

    1995-05-20

    Rapid amplification of cDNA ends (RACE) is a polymerase chain reaction (PCR)-based technique which was developed to facilitate the cloning of full-length cDNA 5'- and 3'-ends after a partial cDNA sequence has been obtained by other methods. While RACE can yield complete sequences of cDNA ends in only a few days, the RACE procedure frequently results in the exclusive amplification of truncated cDNA ends, undermining efforts to generate full-length clones. Many investigators have suggested modifications to the RACE protocol to improve the effectiveness of the technique. Based on first-hand experience with RACE, a critical review of numerous published variations of the key steps in the RACE method is presented. Also included is a detailed, effective protocol based on RNA ligase-mediated RACE/reverse ligation-mediated PCR, as well as a demonstration of its utility.

  4. Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Watanabe, Kousuke; Emoto, Noriko; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2011-07-01

    Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.

  5. 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.

  6. Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells

    Institute of Scientific and Technical Information of China (English)

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

    2001-01-01

    AIM: To clone the cDNA of UGT1 A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGTI A9. METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E. CoIl DH5α. The inserted fragment, verified by DNA sequencing, wes subcloned into the Hind III/Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9- UGT1A9, and selected by G418 (400 mg. L-1) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples for UGT1 A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC. RESULTS: The sequence of the cDNA segment cloned,which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9- UGT1 A9, contains the entire coding region, along with 18bp of the 5' and 55 bp of the 3′ untranslated region of the UGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1 A9, and the enzyme activity towards propranolol in S9 protein was found to be 101 ± 24pmol.min-1 .mg-1 protein (n = 3), but was not detectable in parental CHL cells. CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL ceils. The CHL-UGT1A9 cell lines established efficiently expressed the protein of UGT1A9 for the further enzyme study of drug glucuronidation.

  7. Analysis of Gene Expression Profile in Lung Adenosquamous Carcinoma Using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    YANG Fei; YANG Jiong; JIANG Man; YE Bo; ZHANG Yu-xia; CHEN Hong-lei; XIA Dong; LIU Ming-qiu

    2004-01-01

    Gene expression profile of the lung adenosquamous carcinoma was characterized by using cDNA microarray chip containing 4 096 human genes. Among target genes, 508 differentially expressed genes were identified in adenosquamous carcinoma of the lung, 232 genes were overexpressed and 276 genes were underexpressed. Among them, 92 genes are cell signals transduction genes, 34 genes are proto-oncogenes and tumor suppressor genes or cell cycle related genes or cell apoptosis related genes, 29 genes are cell skeleton genes, 28 genes are DNA synthesis, repair and recombination genes, 12 genes are DNA binding and transcription genes. These genes may be associated with the occurence and development of adenosquamous carinome of the lung.

  8. A drosophila full-length cDNA resource

    Energy Technology Data Exchange (ETDEWEB)

    Stapleton, Mark; Carlson, Joseph; Brokstein, Peter; Yu, Charles; Champe, Mark; George, Reed; Guarin, Hannibal; Kronmiller, Brent; Pacleb, Joanne; Park, Soo; Rubin, Gerald M.; Celniker, Susan E.

    2003-05-09

    Background: A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages. Results: We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40 percent of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing. Conclusions: We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.

  9. Cloning a Chymotrypsin-Like 1 (CTRL-1 Protease cDNA from the Jellyfish Nemopilema nomurai

    Directory of Open Access Journals (Sweden)

    Yunwi Heo

    2016-07-01

    Full Text Available An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita or Hydrozoan (Hydra vulgaris. The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT and 3′ acceptor splice sequences (AG are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

  10. Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

    Directory of Open Access Journals (Sweden)

    Børresen-Dale Anne-Lise

    2002-10-01

    Full Text Available Abstract Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation. Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays.

  11. Broiler chickens (Ross strain) lack insulin-responsive glucose transporter GLUT4 and have GLUT8 cDNA.

    Science.gov (United States)

    Seki, Yoshinori; Sato, Kan; Kono, Tatsuyoshi; Abe, Hiroyuki; Akiba, Yukio

    2003-08-01

    Identification of insulin-responsive glucose transporter proteins, GLUT4 and GLUT8, was attempted in chickens that characteristically are hyperglycemic and insulin resistant. Northern blot analysis using rat GLUT4 cDNA probe and RT-PCR using primers designed against the conserved regions in mammalian GLUT4 cDNA were not successful in identifying GLUT4 homologue(s) in various chicken tissues. Furthermore, GLUT4 homologues could not be detected in chicken tissues by genomic Southern blot analyses using a rat GLUT4 cDNA probe. These data, therefore, suggest that the GLUT4 homologous gene is deficient in chicken tissues. However, GLUT8, another insulin-responsive glucose transporter in the blastocyst, was identified with the aid of RACE (rapid amplification of cDNA ends) reactions in the chicken testis. Chicken GLUT8 was composed of 1449 bp with a coding region for a 482 amino acid protein. The deduced amino acid sequence was 58.8, 56.3, and 56.8% identical with human, rat, and mouse GLUT8, respectively. By RT-PCR, GLUT8 mRNA expressions were detected in chicken brain, kidney, adrenal, spleen, lung, testis, and pancreas; and barely detectable in skeletal muscle, liver, adipose tissue, and heart. Here we firstly report that GLUT8 was identified in chickens, while GLUT4, a major insulin-responsive transporter in mammals, is deficient in these animals. We propose the hypothesis that the hyperglycemia and insulin resistance observable in chickens is associated with their possible deficiency of GLUT4.

  12. Rainbow trout cytochrome P-450c17 (17 alpha-hydroxylase/17,20-lyase). cDNA cloning, enzymatic properties and temporal pattern of ovarian P-450c17 mRNA expression during oogenesis.

    Science.gov (United States)

    Sakai, N; Tanaka, M; Adachi, S; Miller, W L; Nagahama, Y

    1992-04-13

    A cDNA clone encoding cytochrome P-450c17 (17 alpha-hydroxylase/17,20-lyase) was isolated from a rainbow trout ovarian follicle cDNA library. The cDNA contained an open reading frame of 1,542 nucleotides encoding a protein of 514 amino acid residues. The amino acid sequence of trout P-450c17 shows a much greater homology with chicken P-450c17 than with that of human, bovine and rat. The trout P-450c17 expressed in non-steroidogenic mammalian COS-1 cells showed both 17 alpha-hydroxylase and 17,20-lyase activities. The cDNA only hybridized to a single species of mRNA (2.4 kb) isolated from rainbow trout ovaries; the 2.4 kb transcripts were abundant in trout ovaries during the later stages of oogenesis.

  13. Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy

    Indian Academy of Sciences (India)

    T Venugopal; S Mathavan; T J Pandian

    2002-06-01

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in Escherichia coli. These sequences show 96–98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.

  14. Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy.

    Science.gov (United States)

    Venugopal, T; Mathavan, S; Pandian, T J

    2002-06-01

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in Escherichia coli. These sequences show 96-98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.

  15. Isolation and Expression of a cDNA Encoding Methylmalonic Aciduria Type A Protein from Euglena gracilis Z

    Directory of Open Access Journals (Sweden)

    Fumio Watanabe

    2013-02-01

    Full Text Available In animals, cobalamin (Cbl is a cofactor for methionine synthase and methylmalonyl-CoA mutase (MCM, which utilizes methylcobalamin and 5′-deoxyadenosylcobalamin (AdoCbl, respectively. The cblA complementation class of inborn errors of Cbl metabolism in humans is one of three known disorders that affect AdoCbl synthesis. The gene responsible for cblA has been identified in humans (MMAA as well as its homolog (meaB in Methylobacterium extorquens. Recently, it has been reported that human MMAA plays an important role in the protection and reactivation of MCM in vitro. However, the physiological function of MMAA is largely unknown. In the present study, we isolated the cDNA encoding MMAA from Euglena gracilis Z, a photosynthetic flagellate. The deduced amino acid sequence of the cDNA shows 79%, 79%, 79% and 80% similarity to human, mouse, Danio rerio MMAAs and M. extorquens MeaB, respectively. The level of the MCM transcript was higher in Cbl-deficient cultures of E. gracilis than in those supplemented with Cbl. In contrast, no significant differences were observed in the levels of the MMAA transcript under the same two conditions. No significant difference in MCM activity was observed between Escherichia coli that expressed either MCM together with MMAA or expressed MCM alone.

  16. Isolation and characterization of goat ovarian aromatase cDNA: assessment of the activity using an intact cell system and placental expression.

    Science.gov (United States)

    Bobes, Raúl José; Miranda, Carolina; Pérez-Martinez, Mario; Luu-The, Van; Romano, Marta C

    2004-08-01

    Goat ovarian follicles produce estrone and estradiol from androgens. The synthesis of C18 estrogens from C19 androgens requires cytochrome P450 aromatase, but little information about this key enzyme is available in the goat. We report here for the first time the cDNA sequence of the goat ovarian aromatase, the activity of the enzyme in a cell system, and its expression in the term goat placenta. A cDNA library from goat ovarian poly(A)+ RNA was constructed. Human aromatase cDNA was selected as probe to screen the library; several clones were isolated, but none was complete. The longest clone was 3.1 kb long, but it lacked the sequence coding for a few amino acids in the NH(2)-terminal. To obtain the missing sequence, we performed reverse amplification of the cDNA end (RACE). Sequence analysis indicated that goat aromatase possessed a very long 3'-untranslated region ( approximately 1790 bp), and a polyadenylation signal (AATAAA) located at position 3320 downstream from the ATG start codon. The coding region of goat cDNA was inserted in an expression vector and transfected into HEK-293 cells that were cultured in presence of [14C]-androstenedione, steroids extracted and further separated by TLC. The transfected cells efficiently transformed [14C]-androstenedione into estrone. This activity was inhibited by 4-hydroxyandrostenedione. We also investigated the presence of mRNA for P450 aromatase in the goat placenta, using reverse transcription-polymerase chain reaction (RT-PCR) and primers derived from the cDNA ovarian sequence and confirmed the expression of the mRNA in term placenta.

  17. Preparation of microbial community cDNA for metatranscriptomic analysis in marine plankton.

    Science.gov (United States)

    Stewart, Frank J

    2013-01-01

    High-throughput sequencing and analysis of microbial community cDNA (metatranscriptomics) are providing valuable insight into in situ microbial activity and metabolism in the oceans. A critical first step in metatranscriptomic studies is the preparation of high-quality cDNA. At the minimum, preparing cDNA for sequencing involves steps of biomass collection, RNA preservation, total RNA extraction, and cDNA synthesis. Each of these steps may present unique challenges for marine microbial samples, particularly for deep-sea samples whose transcriptional profiles may change between water collection and RNA preservation. Because bacterioplankton community RNA yields may be relatively low (microbiology research.

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

    OpenAIRE

    1997-01-01

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

  19. Construction and characterization of infectious cDNA clones of a chicken strain of hepatitis E virus (HEV), avian HEV.

    Science.gov (United States)

    Huang, F F; Pierson, F W; Toth, T E; Meng, X J

    2005-09-01

    Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important human pathogen. Increasing evidence indicates that hepatitis E is a zoonosis. Avian HEV was recently discovered in chickens with hepatitis-splenomegaly syndrome in the USA. Like swine HEV from pigs, avian HEV is also genetically and antigenically related to human HEV. The objective of this study was to construct and characterize an infectious cDNA clone of avian HEV for future studies of HEV replication and pathogenesis. Three full-length cDNA clones of avian HEV, pT7-aHEV-5, pT7G-aHEV-10 and pT7G-aHEV-6, were constructed and their infectivity was tested by in vitro transfection of leghorn male hepatoma (LMH) chicken liver cells and by direct intrahepatic inoculation of specific-pathogen-free (SPF) chickens with capped RNA transcripts from the three clones. The results showed that the capped RNA transcripts from each of the three clones were replication competent when transfected into LMH cells as demonstrated by detection of viral antigens with avian HEV-specific antibodies. SPF chickens intrahepatically inoculated with the capped RNA transcripts from each of the three clones developed active avian HEV infections as evidenced by seroconversion to avian HEV antibodies, viraemia and faecal virus shedding. The infectivity was further confirmed by successful infection of naïve chickens with the viruses recovered from chickens inoculated with the RNA transcripts. The results indicated that all three cDNA clones of avian HEV are infectious both in vitro and in vivo. The availability of these infectious clones for a chicken strain of HEV now affords an opportunity to study the mechanisms of HEV cross-species infection and tissue tropism by constructing chimeric viruses among human, swine and avian HEVs.

  20. Molecular cloning of a cysteine proteinase cDNA from adult Ancylostoma ceylanicum by the method of rapid amplification of cDNA ends using polymerase chain reaction.

    Science.gov (United States)

    Mieszczanek, J; Kofta, W; Wedrychowicz, H

    2000-12-01

    The hookworm Ancylostoma ceylanicum is a parasite of great importance in human and veterinary medicine. The most promising vaccination trials against hookworm infections are based on antigens belonging to the proteinase family. The aim of the present research was to isolate a cysteine proteinase gene from A. ceylanicum. This was achieved by rapid amplification of cDNA ends using polymerase chain reaction (RACE-PCR). A set of consensus oligonucleotide primers was designed to anneal to the conserved coding regions of cysteine proteinase. The PCR products were cloned and sequenced. The novel sequence displayed a high degree of homology with genes of cysteine proteinases known from other hookworm species. In the coding region the nucleotide identity with accp-1, the cysteine proteinase gene of A. caninum, reaches 84.3%. Analysis of the expression of acey-1. the cysteine proteinase gene of A. ceylanicum, suggests that it is produced exclusively in the gland cells of either adult worms or blood-feeding stages of A. ceylanicum.

  1. Application of an improved cDNA competition technique to identify prostate cancer-associated gene.

    Science.gov (United States)

    Rinaldy, A R; Steiner, M S

    1999-11-01

    A technique to improve cDNA library screening was developed by using mixed probes derived from two closely related cDNA populations of high-metastatic MAT-LyLu and low-metastatic AT-1 Dunning R3227 rat prostate cancer sublines. The technique required the generation of a cDNA library from each subline followed by polymerase chain reaction (PCR) amplification of the cDNA insert population. The PCR products derived from the first library were radiolabeled and mixed with an excess amount of PCR products from the second library. The mixture and an excess amount of both the lambda and pBluescript DNA were used as a probe to screen the first cDNA library. This mixed probe (designated the competition probe) differentially cross-hybridized with the plaque lift of the screened first cDNA library. Weak radioactive signals indicated the cross-hybridization of cDNA sequences common to the competition probe mixture and the first cDNA library, whereas strong signals implied unhybridized unique or abundant cDNA sequences in the first cDNA library. The reproducibility of this technique was confirmed by showing that the full-length cDNA clones were associated with the phenotype of the screened first cell line. The isolated clones were characterized as rat nucleolar protein, rat mitochondrial genes coding for 16S and 12S rRNAs, and rat tRNAs specific for valine and phenyl-alanine. This result is consistent with the fact that the first cell line, MAT-LyLu, is metabolically more active than are AT-1 cells because of higher gene dosage or amplification of nucleolar and mitochondrial RNA and its associated genes. Another clone which had a strong signal represented a novel gene associated with the MAT-LyLu cancer phenotype.

  2. Cloning of a cDNA encoding the smallest neurofilament protein from the rat

    NARCIS (Netherlands)

    J-P. Julien (Jean-Pierre); K. Ramachadran; F.G. Grosveld (Frank)

    1985-01-01

    textabstractWe have cloned a cDNA coding for the smallest rat neurofilament protein. The cDNA is 861 nucleotides long coding for 287 amino acids from the internal alpha-helical region and the carboxy-terminal tail domain of the neurofilament protein. Comparison of the porcine, mouse and rat neurofil

  3. 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.

  4. 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.

  5. IDENTIFICATION OF DIFFERENTIAL GENES IN OVARIAN CANCER USING REPRESENTATIONAL DIFFERENCE ANALYSIS OF cDNA

    Institute of Scientific and Technical Information of China (English)

    Hong Chen; Min Wang; Xin-yan Wang; Shan Gao; Jun Wang; Xiao-ming Guan

    2005-01-01

    Objective To identify differential genes between normal ovarian epithelium tissue and ovarian epithelial cancer using representational difference analysis of cDNA (cDNA-RDA). Methods cDNA-RDA was performed to identify the differentially expressed sequences between cDNAs from cancer tissue and cDNAs from normal ovarian tissue in the same patient who was in the early stage of ovarian serous cystadeno carcinoma. These differentially expressed fragments were cloned and analyzed, then sequenced and compared with known genes.Results Three differentially expressed cDNA fragments were isolated using cDNA from normal ovarian tissue as tester and cDNA from cancer tissue as driver amplicon by cDNA-RDA. DP Ⅲ-1 and DP Ⅲ-2 cDNA clone showed significant ho mology to the cDNA of alpha actin gene; DPⅢ-3 cDNA clone showed significant homology to the cDNA of transgelin gene. Conclusion cDNA-RDA can be used to sensitively identify the differentially expressed genes in ovarian serous cystadenocarcinoma. Ovarian serous cystadenocarcinoma involves alteration of multiple genes.

  6. Construction and characterization of a normalized whole-life-cycle cDNA library of rice

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A cDNA library with genomic complete coverage is a powerful tool for functional genomic studies. For studying the functions of rice genes on a large scale, a normalized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63, an elite restorer line for a number of rice hybrids that are widely cultivated in China. This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages. For normalization, the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA labeled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library. This library consists of 62000 clones with an average insert length about 1.4 kb. Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes. Sequencing of 10750 cDNA clones of this library reveals 6399 unique ESTs (expressed sequence tags), indicating that the non-redundancy of the library is about 59.5%. This library has been used to make cDNA microarrays for functional genomic studies.

  7. Peptidomics combined with cDNA library unravel the diversity of centipede venom

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo

    2015-01-01

    of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins...

  8. cDNA library information - Dicty_cDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available (C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA library construction method How to const...dir) 2) Full-length cDNA libraries (oligocapped method)(fl) 3) Gamete-specific subtraction library (sub) cDN

  9. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains

    Energy Technology Data Exchange (ETDEWEB)

    Wun, T.C.; Kretzmer, K.K.; Girard, T.J.; Miletich, J.P.; Broze, G.J. Jr.

    1988-05-05

    Human plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inactivates factor X/sub a/ directly, and in a X/sub a/-dependent fashion also inhibits the VII/sub a/-tissue factor complex of the extrinsic coagulation pathway. Rabbit polyclonal anti-LACI antiserum was used to screen human placental and fetal liver lambdagt11 cDNA libraries for the expression of LACI antigens. Immunologically positive clones were further tested for their ability to bind /sup 125/I-factor X/sub a/. Seven clones were obtained which are immunologically and functionally active. The longest cDNA insert (lambdaP9) of these isolates is 1.4 kilobases (kb) while other clones are 1.0 kb in length. Nucleotide sequence analysis shows that lambdaP9 consists of 1431 bases that include a 5'-noncoding sequence of 132 nucleotides, an open reading frame of 912 nucleotides, and a 3'-noncoding region of 387 nucleotides. The predicted sequence of mature LACI contains 18 cysteines and three potential N-linked glycosylation sites. The amino acid sequence analysis of purified LACI's NH/sub 2/ terminus and two of its proteolytic fragments match exactly those deduced from the cDNA sequence, indicating that the cDNA codes for LACI. The translated amino acid sequence of LACI shows several discernible domains, including a highly negatively charged NH/sub 2/ terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. Northern blot analysis shows that the following liver-derived cell lines, Chang liver, HepG2 hepatoma, and SK hepatoma all, contain two major species of mRNA which hybridize with LACI cDNA.

  10. Methods to determine the transcriptomes of trypanosomes in mixtures with mammalian cells: the effects of parasite purification and selective cDNA amplification.

    Directory of Open Access Journals (Sweden)

    Julius Mulindwa

    2014-04-01

    Full Text Available Patterns of gene expression in cultured Trypanosoma brucei bloodstream and procyclic forms have been extensively characterized, and some comparisons have been made with trypanosomes grown to high parasitaemias in laboratory rodents. We do not know, however, to what extent these transcriptomes resemble those in infected Tsetse flies - or in humans or cattle, where parasitaemias are substantially lower. For clinical and field samples it is difficult to characterize parasite gene expression because of the large excess of host cell RNA. We have here examined two potential solutions to this problem for bloodstream form trypanosomes, assaying transcriptomes by high throughput cDNA sequencing (RNASeq. We first purified the parasites from blood of infected rats. We found that a red blood cell lysis procedure affected the transcriptome substantially more than purification using a DEAE cellulose column, but that too introduced significant distortions and variability. As an alternative, we specifically amplified parasite sequences from a mixture containing a 1000-fold excess of human RNA. We first purified polyadenylated RNA, then made trypanosome-specific cDNA by priming with a spliced leader primer. Finally, the cDNA was amplified using nested primers. The amplification procedure was able to produce samples in which 20% of sequence reads mapped to the trypanosome genome. Synthesis of the second cDNA strand with a spliced leader primer, followed by amplification, is sufficiently reproducible to allow comparison of different samples so long as they are all treated in the same way. However, SL priming distorted the abundances of the cDNA products and definitely cannot be used, by itself, to measure absolute mRNA levels. The amplification method might be suitable for clinical samples with low parasitaemias, and could also be adapted for other Kinetoplastids and to samples from infected vectors.

  11. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.

    OpenAIRE

    Jiang, H P; Serrero, G

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

  12. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein

    OpenAIRE

    Jiang, Hui-Ping; Serrero, Ginette

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

  13. Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Using cDNA representational difference analysis (cDNA RDA) method, we have successfully isolated a gene fragment whose expression was specifically induced by external GA3 application. Screening a G2 pea cDNA library using this fragment as a probe, we obtained a 2036 bp full-length cDNA. It contains a 1746 bp open reading frame and encodes a protein of 581 amino acids with a theoretical molecular weight of 64 ku. It shares high-level sequence identity with AAIR genes from other plant species. This cDNA was cloned into expression vector and recombinant E. coli DH5α cells with remarkable AAIR enzyme activity were obtained.

  14. Cloning and molecular characterization of cDNA encoding a mouse male-enhanced antigen-2 (Mea-2): a putative family of the Golgi autoantigen.

    Science.gov (United States)

    Kondo, M; Sutou, S

    1997-01-01

    The male-enhanced antigen-2 (Mea-2) gene was originally identified with a monoclonal histocompatibility Y (H-Y) antibody (mAb4VII). There is no report of the full length cDNA encode for Mea-2 product until this report. In this study, we isolated the full length mouse Mea-2 cDNA by screening a testis cDNA library with a PCR-amplified Mea-2 product, and direct PCR amplification of its upstream sequences from the cDNA library. The primary structure of the Mea-2 peptide, deduced from this nucleotide sequence, shows that it encode a 150 kDa protein, of 1325 amino acid residues, which contained five putative N-glycosylation sites and four leucine zipper motifs. A data bank search indicated that it has high homology with a human Golgi autoantigen (golgin-160) both in its nucleotides (78%) and amino acids sequence (83%). This suggests that Mea-2 gene product may encode a golgi structural protein. In situ hybridization analysis suggested that the Mea-2 gene is expressed in spermatids during spermatogenesis as already shown by Mea-1, suggesting that Mea-2 gene product as well as Mea-1 have also some role for spermatogenesis.

  15. Optimization and comparison of different methods for RNA isolation for cDNA library construction from the reindeer lichen Cladonia rangiferina

    Directory of Open Access Journals (Sweden)

    Lim Kean-Jin

    2009-10-01

    Full Text Available Abstract Background The reindeer lichen is the product of a mutualistic relationship between a fungus and an algae. Lichen demonstrate a remarkable capacity to tolerate dehydration. This tolerance is driven by a variety of biochemical processes and the accumulation of specific secondary metabolites that may be of relevance to the pharmaceutical, biotechnology and agriculture industries. These protective metabolites hinder in vitro enzymatic reactions required in cDNA synthesis. Along with the low concentrations of RNA present within lichen tissues, the process of creating a cDNA library is technically challenging. Findings An evaluation of existing commercial and published protocols for RNA extraction from plant or fungal tissues has been performed and experimental conditions have been optimised to balance the need for the highest quality total ribonucleotides and the constraints of budget, time and human resources. Conclusion We present a protocol that balances inexpensive RNA extraction methods with commercial RNA clean-up kits to yield sufficient RNA for cDNA library construction. Evaluation of the protocol and the construction of, and sampling from, a cDNA library is used to demonstrate the suitability of the RNA extraction method for expressed sequence tag production.

  16. Optimization and comparison of different methods for RNA isolation for cDNA library construction from the reindeer lichen Cladonia rangiferina.

    Science.gov (United States)

    Junttila, Sini; Lim, Kean-Jin; Rudd, Stephen

    2009-10-05

    The reindeer lichen is the product of a mutualistic relationship between a fungus and an algae. Lichen demonstrate a remarkable capacity to tolerate dehydration. This tolerance is driven by a variety of biochemical processes and the accumulation of specific secondary metabolites that may be of relevance to the pharmaceutical, biotechnology and agriculture industries. These protective metabolites hinder in vitro enzymatic reactions required in cDNA synthesis. Along with the low concentrations of RNA present within lichen tissues, the process of creating a cDNA library is technically challenging. An evaluation of existing commercial and published protocols for RNA extraction from plant or fungal tissues has been performed and experimental conditions have been optimised to balance the need for the highest quality total ribonucleotides and the constraints of budget, time and human resources. We present a protocol that balances inexpensive RNA extraction methods with commercial RNA clean-up kits to yield sufficient RNA for cDNA library construction. Evaluation of the protocol and the construction of, and sampling from, a cDNA library is used to demonstrate the suitability of the RNA extraction method for expressed sequence tag production.

  17. Characterization, cDNA cloning and expression pattern of relaxin gene during embryogenesis of Danio rerio.

    Science.gov (United States)

    Fiengo, Marcella; Donizetti, Aldo; del Gaudio, Rosanna; Minucci, Sergio; Aniello, Francesco

    2012-06-01

    We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone.

  18. 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.

  19. CDNA library from the Latex of Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    Wilaiwan Chotigeat

    2010-12-01

    Full Text Available Latex from Hevea brasiliensis contains 30-50% (w/w of natural rubber (cis-1,4-polyisoprene, the important rawmaterial for many rubber industries. We have constructed a cDNA library from the latex of H. brasiliensis to investigate theexpressed genes and molecular events in the latex. We analyzed 412 expressed sequence tags (ESTs. More than 90% of theEST clones showed homology to previously described sequences in public databases. Functional classification of the ESTsshowed that the largest category were proteins of unknown function (30.1%, 11.4% of ESTs encoded for rubber synthesisrelatedproteins (RS and 8.5% for defense or stress related proteins (DS. Those with no significant homology to knownsequences (NSH accounted for 8.7%, primary metabolism (PM and gene expression and RNA metabolism were 7.8% and6.6%, respectively. Other categories included, protein synthesis-related proteins (6.6%, chromatin and DNA metabolism(CDM 3.9%, energy metabolism (EM 3.4%, cellular transport (CT 3.2%, cell structure (CS 3.2%, signal transduction (ST2.2%, secondary metabolism (SM 1.7%, protein fate (PF 2.2%, and reproductive proteins (RP 0.7%.

  20. The gene for human glutaredoxin (GLRX) is localized to human chromosome 5q14

    Energy Technology Data Exchange (ETDEWEB)

    Padilla, C.A.; Holmgren, A. [Karolinska Inst., Stockholm (Sweden); Bajalica, S.; Lagercrantz, J. [Karolinska Hospital, Stockholm (Sweden)

    1996-03-05

    Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescence in situ hybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human-hamster and a human-mouse hybrid panel and using a human glutaredoxin cDNA as a probe. 13 refs., 2 figs.

  1. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  2. RICD: A rice indica cDNA database resource for rice functional genomics

    Directory of Open Access Journals (Sweden)

    Zhang Qifa

    2008-11-01

    Full Text Available Abstract Background The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones. Results Rice Indica cDNA Database (RICD is an online MySQL-PHP driven database with a user-friendly web interface. It allows investigators to query the cDNA clones by keyword, genome position, nucleotide or protein sequence, and putative function. It also provides a series of information, including sequences, protein domain annotations, similarity search results, SNPs and InDels information, and hyperlinks to gene annotation in both The Rice Annotation Project Database (RAP-DB and The TIGR Rice Genome Annotation Resource, expression atlas in RiceGE and variation report in Gramene of each cDNA. Conclusion The online rice indica cDNA database provides cDNA resource with comprehensive information to researchers for functional analysis of indica subspecies and for comparative genomics. The RICD database is available through our website http://www.ncgr.ac.cn/ricd.

  3. [Construction and analysis of subtractive cDNA library associated with multidrug resistance of acute leukemia].

    Science.gov (United States)

    Ji, Lei; Zhang, Wang-Gang; Liu, Jie; Liu, Xin-Ping; Yao, Li-Bo

    2004-08-01

    The study was aimed to construct subtractive cDNA library associated with multidrug resistance (MDR) of acute leukemia for screening genes related to MDR in leukemia. The improved PCR-based subtractive hybridization was performed to clone differential genes between HL-60/VCR and HL-60 cell line. The mRNA of HL-60/VCR and HL-60 cell line were isolated. Then the mRNA of HL-60/VCR group was reversely transcribed into cDNA by Cap-Finder method, and the mRNA of HL-60 was reversely transcribed into cDNA by ordinary method to be marked by biotin for the hybridization next with HL-60/VCR cDNA. After hybridizing, filtrating through the sephacryl S-400 column, absorbing by the magnetic beads, and amplifying by PCR method, the fragments were cloned by T-A method and the cDNA library was constructed. Then the quality of cDNA library was identified by dot-blotting hybridization method. The results showed that after constriction, the library demonstrated its good quality. There was a high proportion of large fragments in this library. From small amount of samples a large amount of candidate fragments could be screened rapidly at once by dot-blotting hybridization. It is concluded that a differentially-expressed subtractive cDNA library in MDR of leukemia with high quality and larger fragments can be efficiently constructed by improving subtractive hybridization and selective PCR method.

  4. Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon

    Institute of Scientific and Technical Information of China (English)

    罗田; 徐洵

    2002-01-01

    --mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyATtract System 1000 Kit. By using mRNA as template, double- strand cDNA with EcoR I/Xho I ends was synthesized by using a ZAP Express cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP Express vector predigested with EcoR I/Xho I, and the recombinant DNA was in vitro packaged into larnbda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XLl - Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2 × 105pfu in capacity and its recombination ratio is higher than 99%. The size of the inserted cDNAs was determined by EcoR I/Xho I digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library . The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA was amplified from the library by PCR reveals that this library contains full-length cDNAs of Penaeus mod on hepatopancreas and is available for screening and expression of shrimp genes.

  5. Comparative expression analysis of isolated human adipocytes and the human adipose cell lines LiSa-2 and PAZ6

    NARCIS (Netherlands)

    Beek, van E.A.; Bakker, A.H.; Kruyt, P.M.; Vink, C.; Saris, W.H.; Keijer, J.

    2008-01-01

    Objective: To obtain insight in the extent to which the human cell lines LiSa-2 and PAZ6 resemble isolated primary human adipocytes. Design: A combination of cDNA subtraction (representative difference analysis; RDA) and cDNA microarray analysis was used to select adipose specific genes to compare i

  6. Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

    Science.gov (United States)

    Vanhooren, J C; Fransen, M; de Béthune, B; Baumgart, E; Baes, M; Torrekens, S; Van Leuven, F; Mannaerts, G P; Van Veldhoven, P P

    1996-07-15

    The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

  7. cDNA cloning of the murine PEX gene implicated in X-linked hypophosphatemia and evidence for expression in bone

    Energy Technology Data Exchange (ETDEWEB)

    Du, L.; Desbarats, M.; Viel, J. [McGill Univ., Montreal, Quebec (Canada)] [and others

    1996-08-15

    The recently identified human PEX g ene apparently encodes for a neutral endopeptidase that is mutated in patients with X-linked hypophosphatemia. The 3{prime} and 5{prime} ends of the coding region of PEX have not been cloned, nor has the tissue expression of the gene been identified. Here we report the isolation and characterization of the complete open reading frame of the mouse Pex gene and the demonstration of its expression in bone. Mouse Pex cDNA is predicted to encode a protein of 749 amino acids with 95% identity to the available human PEX sequence and significant homology to members of the membrane-bound metalloendopeptidase family. Northern blot analysis revealed a 6.6-kb transcript in bone and in cultured osteoblasts from normal mice that was not detectable in samples from the Hyp mouse, the murine homolog of human X-linked hypophosphatemia. Pex transcripts were, however, detectable in Hyp bone by RT-PCR amplification. Of particular interest, a cDNA clone from rat incisor shows 93% sequence identity to the 5{prime} end of Pex cDNA, suggesting that Pex may be expressed in another calcified tissue, the tooth. The association of impaired mineralization of bone and teeth and disturbed renal phosphate reabsorption with altered expression of Pex suggests that the Pex gene product may play a critical role in these processes. 47 refs., 2 figs., 1 tab.

  8. Molecular cloning and characterization of cDNA encoding a putative stress-induced heat-shock protein from Camelus dromedarius.

    Science.gov (United States)

    Elrobh, Mohamed S; Alanazi, Mohammad S; Khan, Wajahatullah; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Bazzi, Mohammad D

    2011-01-01

    Heat shock proteins are ubiquitous, induced under a number of environmental and metabolic stresses, with highly conserved DNA sequences among mammalian species. Camelus dromedaries (the Arabian camel) domesticated under semi-desert environments, is well adapted to tolerate and survive against severe drought and high temperatures for extended periods. This is the first report of molecular cloning and characterization of full length cDNA of encoding a putative stress-induced heat shock HSPA6 protein (also called HSP70B') from Arabian camel. A full-length cDNA (2417 bp) was obtained by rapid amplification of cDNA ends (RACE) and cloned in pET-b expression vector. The sequence analysis of HSPA6 gene showed 1932 bp-long open reading frame encoding 643 amino acids. The complete cDNA sequence of the Arabian camel HSPA6 gene was submitted to NCBI GeneBank (accession number HQ214118.1). The BLAST analysis indicated that C. dromedaries HSPA6 gene nucleotides shared high similarity (77-91%) with heat shock gene nucleotide of other mammals. The deduced 643 amino acid sequences (accession number ADO12067.1) showed that the predicted protein has an estimated molecular weight of 70.5 kDa with a predicted isoelectric point (pI) of 6.0. The comparative analyses of camel HSPA6 protein sequences with other mammalian heat shock proteins (HSPs) showed high identity (80-94%). Predicted camel HSPA6 protein structure using Protein 3D structural analysis high similarities with human and mouse HSPs. Taken together, this study indicates that the cDNA sequences of HSPA6 gene and its amino acid and protein structure from the Arabian camel are highly conserved and have similarities with other mammalian species.

  9. 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)

    ZHANG; Kai-tai

    2001-01-01

    [1]Tom S, Andrew PR. Human Molecular Genetics [M]. John Wiley & Sons, Inc. United States of America 1996; 335.[2]Zhao Yong-liang, Jin Cui-zhen, Wu De-chang et al. Neoplastic transformation and cytogenetic changes of rat tracheal epithelial cells induced by a-particles irradiation [J]. Chin Med Sci J 1997; 12:202.[3]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[4]Frederick A, Roger B. Current Protocols in Molecular Biology [M]. John Wiley & Sons, Inc. United States of America 1998; 2.1.1.[5]Roux KH. Optimization and troubleshooting in PCR [J]. PCR Methods Appl 1995; 4:5158.[6]Sambrook, J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual [M]. 2nd Ed. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989; 54.[7]Zhang Y, Frohman MA. Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs [J]. Methods Mol Biol 1997; 69:61.[8]Frohman MA. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE [J]. Methods Enzymol 1993; 218:340.[9]Iqbal S, Robinson J, Deere D, et al. Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water [J]. Lett Appl Microbiol 1997; 24:498.[10]Schunck B, Kraft W, Truyen U. A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces [J]. J Virol Methods 1995; 55:427.

  10. Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

    Science.gov (United States)

    Fu, Glenn K; Wang, Jonathan T; Yang, Junming; Au-Young, Janice; Stuve, Laura L

    2004-07-01

    The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

  11. cDNA Cloning of a Short Isoform of Human Neuroblastoma NADP(H)-Dependent Retinol Dehydrogenase/Reductase and Analysis of Its Characteristics%一种人神经母细胞瘤辅酶Ⅱ-依赖性视黄醇脱氢酶cDNA的克隆及特征分析

    Institute of Scientific and Technical Information of China (English)

    李一凡; 刘戈飞; 宋旭红; 杜昆; 黄东阳

    2005-01-01

    背景与目的:检测神经母细胞瘤中新的辅酶Ⅱ-依赖性视黄醇脱氢/还原酶[NADP(H)-dependent retinol dehydrogenase/reductase,NRDR]选择性剪接亚型.材料与方法:我们用NRDR特异引物,从人神经母细胞瘤细胞系SK-N-SH和SK-SY-5Y cDNA中分别经PCR扩增出635 bp和429 bp DNA片段,测序证实635 bp片段是NRDR,而429 bp片段是选择性剪接新亚型.再用快速cDNA末端扩增(Rapid amplification of cDNA ends,RACE)方法得到429 bp片段cDNA全长序列.用绿色荧光蛋白与新亚型的融合蛋白做亚细胞定位.结果:得到新亚型全长并命名为human NRDR A2(humNRDRA2)(AY616182).已知NRDR有8个外显子,而新亚型humNRDRA2由选择性剪接造成了第4和第6外显子丢失,从第5外显子开始读码框架前移1 bp,导致随后的编码区框码漂移(frameshift),同时蛋白翻译终止信号提前出现,产生仅有188个氨基酸的蛋白,其中第137氨基酸以后的序列相对于NRDR完全发生改变,同时C-末端的过氧化物酶体定位信号-SRL丢失,却在160~176氨基酸处出现了一个细胞核定位信号.我们在ESTs库中未找到与其相同的剪接形式,提示它可能是神经母细胞瘤特有的一种NRDR剪接亚型.用GFP-NRDRA2融合蛋白做该蛋白的亚细胞定位,发现发绿色荧光的细胞均已悬浮,但悬浮状态不佳,而作为对照的NRDR另一亚型则能定位成功,提示NRDRA2蛋白可能具有一定的细胞毒性.结论:人神经母细胞瘤NRDRA2选择性剪接亚型羧基端框移突变并有核定位序列;该蛋白可能是一种细胞毒性蛋白.

  12. 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.

  13. Characterization and simulation of cDNA microarray spots using a novel mathematical model

    OpenAIRE

    2007-01-01

    Abstract Background The quality of cDNA microarray data is crucial for expanding its application to other research areas, such as the study of gene regulatory networks. Despite the fact that a number of algorithms have been suggested to increase the accuracy of microarray gene expression data, it is necessary to obtain reliable microarray images by improving wet-lab experiments. As the first step of a cDNA microarray experiment, spotting cDNA probes is critical to determining the quality of s...

  14. A CLINICAL STUDY ON PROSTATE CANCER DIAGNOSIS WITH cDNA MACROARRAY

    Institute of Scientific and Technical Information of China (English)

    ZHONG Wei-de; XIE Ke-ji; WEI Hong-ai; LIU Liang-shi; LIU Wen-hua; JIANG Fu-neng; ZENG Guang-qiao; DAI Qi-shan; HE Hui-chan; BI Xue-cheng; PENG Zhi-qiang

    2005-01-01

    Objective: To diagnose prostatic cancer (CaP) with cDNA macroarray. Methods: Total RNA was isolated from patients with prostate cancer and from normal people, and poly(A) RNA was further purified. Then differentially expressed genes were analysed in CaP and normal prostate by cDNA macroarray system. Results: There were differential expressions of nine prostate-associated specific genes in CaP as compared with normal prostate, among which, 7 were significantly up-regulated and 2 were down-regulated. Conclusion: As a diagnostic approach at molecular level, the cDNA macroarray is supposed to elevate the detection rate of CaP.

  15. 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.

  16. The full-ORF clone resource of the German cDNA Consortium

    Directory of Open Access Journals (Sweden)

    Michel Guenter

    2007-10-01

    Full Text Available Abstract Background With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Results Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen. A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available

  17. 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 ...

  18. Identifying and mapping cDNA fragments related to rice photoperiod sensitive genic male sterility

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The differentially expressed cDNA fragments have been obtained by differential screening with cDNA-RAPD technique in photoperiod sensitive genic male sterile (PGMS) rice.Some of them have been reassessed with Northern blot hybridization,from which a PGMS-related positive fragment,RPG43,has been identified.Further analysis on RPG43 with Southern blot and RAPD indicates that the fragment is a single-copy sequence and its mRNA has been processed after transcription.Sequence analysis reveals that RPG43 is 744 bp in length and contains a 60 bp region (from 126th to 185th bp) showing 72% homology to a human DNA sequence,pac pDJ- 356d6,on chromosome 11.So it is a new sequence found in plant and its GenBank access number is AF126027.In addition,RPG43 has been mapped to a position 3.8 cM away from RFLP marker R1553 on chromosome 5 of rice.

  19. Genome-wide expression profiling of the response to terbinafine in Candida albicans using a cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    ZENG Yue-bin; QIAN Yuan-shu; MA Lian; GU Hong-ni

    2007-01-01

    Background Candida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression.Methods Candida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations (MICs, 4 mg/L) for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).Results A total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette (ABC) or major facilitator superfamily (MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1),genes involved in stress response and detoxification (CDR1, AGP2, HOL3), and gene involved in the ergosterol biosynthesis pathway (ERG12). The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.Conclusions The up-regulation of the gene encoding the multidrug resistance efflux pump

  20. Construction and analysis of a subtracted cDNA library of Betula platyphylla female inflorescence

    Institute of Scientific and Technical Information of China (English)

    WEIJi-cheng; YANGChuan-ping; WANGChao; JIANGJing

    2005-01-01

    Female inflorescence of Betula platyphylla was sampled at an interval of each two days to analyze the background of gene expression in floral phase. On the basis of SMART strategy, the driver cDNA was obtained from total RNA of the last sample and the tester cDNA was from that of the others by RT-PCR which were subsequently used to construct a subtracted cDNA library. The result of the ESTs (expression sequence tags) blastX showed that the genes in the subtracted cDNA library could be mainly clustered into 5 groups related to metabolism, transportation and signal transduction, cell cycle, stress response, and regulation. The relationship between gene expression and development was also discussed.

  1. Quantitative Transcript Analysis in Plants: Improved First-strand cDNA Synthesis

    Institute of Scientific and Technical Information of China (English)

    Nai-Zhong XIAO; Lei BA; Preben Bach HOLM; Xing-Zhi WANG; Steve BOWRA

    2005-01-01

    The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional analysis and quantification. Using a plant-derived α-tubulin as a model system, the effect of oligo sequence and DTT on the quality and quantity of first-strand cDNA synthesis was assessed via a combination of semi-quantitative PCR and real-time PCR. The results indicated that anchored oligo dT significantly improved the quantity and quality of α-tubulin cDNA compared to the conventional oligo dT. Similarly, omitting DTT from the first-strand cDNA synthesis also enhanced the levels of transcript. This is the first time that a comparative analysis has been undertaken for a plant system and it shows conclusively that small changes to current protocols can have very significant impact on transcript analysis.

  2. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Dufva, I.H.; Dufva, Hans Martin

    2006-01-01

    oligonucleotides (pentadecamers) consistently, yielded at least 2 fold as much cDNA as did random hexamers using either-poly(A) RNA or an amplified version of messenger RNA (aRNA) as a template. The cDNA generated using pentadecamers did not differ in size distribution or the amount of incorporated label compared...... with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage...... that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality....

  3. Identifications of SUMO-1 cDNA and Its Expression Patterns in Pacific White Shrimp Litopeanaeus vannamei

    Directory of Open Access Journals (Sweden)

    Yanisa Laoong-u-thai, Baoping Zhao, Amornrat Phongdara, Harry Ako, Jinzeng Yang

    2009-01-01

    Full Text Available Small ubiquitin-like modifiers (SUMO work in a similar way as ubiquitin to alter the biological properties of a target protein by conjugation. A shrimp SUMO cDNA named LvSUMO-1 was identified in Litopenaeus vannamei. LvSUMO-1 cDNA contains a coding sequence of 282 nucleotides with untranslated regions of 37 bp at 5'-end and 347 bp at 3'-end, respectively. The deduced 93 amino acids exhibit 83% identity with the Western Honeybee SUMO-1, and more than 65% homologies with human and mouse SUMO-1. LvSUMO-1 mRNA is expressed in most L. vannamei tissues with the highest level in hepatopancrease. The mRNA expression of LvSUMO-1 over development stages in L. Vammamei is distinguished by a low level in nauplius stage and relatively high level in postlarva stage with continuous expression until juvenile stage. The LvSUMO-1 protein and its conjugated proteins are detected in both cytoplasm and nucleus in several tissues. Interestingly, LvSUMO-1 mRNA levels are high in abdominal muscle during the premolt stage, wherein it has significant activities of protein degradation, suggesting its possible role in the regulation of shrimp muscle protein degradation.

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  5. Conversion of cDNA differential display results (DDRT-PCR into quantitative transcription profiles

    Directory of Open Access Journals (Sweden)

    Koopmann Birger

    2005-04-01

    Full Text Available Abstract Background Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far. Results We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii intensity of bands is determined by densitometry, (iii densitometric values are normalized, and (iv intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans. Conclusion We developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in reseach on organisms for which microarray analysis is not available or economical.

  6. 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.

  7. 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.

  8. Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

    Science.gov (United States)

    Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

    2016-08-02

    Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

  9. [Rapid construction of full-length MnSOD cDNA of chickens by one-step 3'RACE].

    Science.gov (United States)

    Bu, You-Quan; Luo, Xu-Gang; Liu, Bin; Li, Su-Fen

    2004-07-01

    RACE (rapid amplification of cDNA ends) is a popular technique to rapidly obtain the full-length cDNA. After obtaining the 3' cDNA and 5' cDNA fragments with a overlapped region by 3' RACE and 5' RACE, the full-length cDNA could be generated by end-to-end PCR or subcloning. In this study, 3' RACE combined with touch-down PCR was successfully used for the rapid construction of full-length MnSOD cDNA of chickens. Compared with the conventional end-to-end PCR or subcloning, this method, called one-step 3' RACE, is fast, economical and highly specific. It especially fits the rapid construction of full-length cDNA by RACE method.

  10. 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

  11. Cloning and screening of cDNA of Psilgramma menephorn allergen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fragments less than 400 bp were removed by using GHROMA SPIN-400 column. The remaining fragments longer than 400 bp were ligated with Uni-ZAP XR vector. The recombinants were packaged in vitro and a small portion of the packaged phage was used to infect E.coli XL1-Blue MRF′ for titration. The recombinants were examined by color selection. The size of cDNA inserts and the diversity of library were analyzed by PCR. The library was screened using SPT positive sera from patients with Psilgramma menephorn allergy repeatedly. Results The cDNA expression library consisting of a 5×105 recombinant bacteriophages was constructed with the recombinant ratio of 67%. The average length of recombinant exogenous inserts was about 1.49 kb. Five positive cDNA clones were obtained. Conclusion The constructed cDNA expression library shows appropriate contents and size of cDNA fragments and the related genes of Psilgramma menephorn major allergens were harbored successfully, which lays the foundation for the positive clone identification and further analysis.

  12. The effect of column purification on cDNA indirect labelling for microarrays

    Directory of Open Access Journals (Sweden)

    Kiss John Z

    2007-06-01

    Full Text Available Abstract Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive

  13. 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.

  14. 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.

  15. 人促红细胞生成素受体胞外区基因的克隆 及其在大肠肝菌中的表达%Cloning cDNA of Extracellular Domain of Human Erythropoietin Receptor and its Expression in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    张映辉; 卢一凡; 刘一平; 邓继先

    2000-01-01

    Human erythropoietin receptor (hEPOR) plays an important role in regulating the red blood cell production by promoting the proliferation and differentiation of RBC from erythroid precursors, hEpoR is a transmembrane protein, and its extrocellular domain (sEpoR) is of great importance in Epo signal transduction pathway. We cloned the gene of sEpoR by RT-PCR from the total RNA of human fetal liver and expressed it in E. coli after insertion of the gene in the expression vector pBV220. The cloned gene was confirmed by sequencing analysis and gene product was confirmed by both Western blot and its first 11 amino acid residues sequence of the N-terminal. In vitro bioassay showed that the purified gene product can repress the growth of TF-cells in the presence of Epo.%以人胎肝为材料,通过RT-PCR的方法扩增出人促红细胞生成素受体(hEpoR)的胞外区基因。将获得的成熟受体胞外区基因起始密码子改构后克隆到原核表达载体pBV220中,进行原核温控诱导表达。表达产物经蛋白N端测序及Western-blot实验证实表达产物是hEpoR胞外区蛋白。利用上罐发酵培养获得的包涵体蛋白经复性纯化后,体外生物学活性检测表明表达产物可特异地抑制TF-1细胞在Epo刺激下的生长,证实了复性表达产物具有人促红细胞生成素受体胞外区结合Epo的生物活性。

  16. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.

    Science.gov (United States)

    Yang, G P; Ross, D T; Kuang, W W; Brown, P O; Weigel, R J

    1999-03-15

    Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

  17. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

    Science.gov (United States)

    Honda, A; Sugimoto, Y; Namba, T; Watabe, A; Irie, A; Negishi, M; Narumiya, S; Ichikawa, A

    1993-04-15

    A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

  18. 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.

  19. Study on Wusan Granule Anti-tumor Related Target Gene Screened by Cdna Microarray

    Institute of Scientific and Technical Information of China (English)

    YOU Zi-li; SHI Jin-ping; CHEN Hai-hong

    2006-01-01

    To screen Wusan Granule anti-tumor related target gene using cDNA microarray technique, both mRNA from Lewis lung carcinoma tissues treated by Wusan Granule and untreated control are reversibly transcribed to prepare cDNA probes which are labeled by Cy5 and Cy3. Then, the probes are hybridized to the mice cDNA microarray type MGEC-20S. After hybridization, the cDNA microarray is scanned by ScanArray 3 000 scanner and the data is analyzed by ImaGene 3 software to screen the differentially expressed genes. There are 45 differentially expressed genes including 18 known genes and 27 unknown genes between the two groups, and among them, 20 elevated genes and 25 reduced genes are identified. Additionally, the genes related to invasion and metastasis of malignant carcinomas are down-regulated and the genes related to apoptosis are up-regulated. The cDNA microarray technique is a high-throughput approach to screen the Wusan Granule anti-tumor related target genes, which allow us to explore the molecular biological mechanism on a genomic scale.

  20. Gene expression profile of esophageal cancer in North East India by cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    Indranil Chattopadhyay; Sujala Kapur; Joydeep Purkayastha; Rupkumar Phukan; Amal Kataki; Jagadish Mahanta; Sunita Saxena

    2007-01-01

    AIM: To identify alterations in genes and molecular functional pathways in esophageal cancer in a high incidence region of India where there is a widespread use of tobacco and betel quid with fermented areca nuts.METHODS: Total RNA was isolated from tumor and matched normal tissue of 16 patients with esophageal squamous cell carcinoma. Pooled tumor tissue RNA was labeled with Cy3-dUTP and pooled normal tissue RNA was labeled with Cy5-dUTP by direct labeling method.The labeled probes were hybridized with human 10K cDNA chip and expression profiles were analyzed by Genespring GX V 7.3 (Silicon Genetics).RESULTS: Nine hundred twenty three genes were differentially expressed. Of these, 611 genes were upregulated and 312 genes were downregulated. Using stringent criteria (P ≤ 0.05 and ≥ 1.5 fold change),127 differentially expressed genes (87 upregulated and 40 downregulated) were identified in tumor tissue. On the basis of Gene Ontology, four different molecular functional pathways (MAPK pathway,G-protein coupled receptor family, ion transport activity,and serine or threonine kinase activity) were most significantly upregulated and six different molecular functional pathways (structural constituent of ribosome,endopeptidase inhibitor activity, structural constituent of cytoskeleton, antioxidant activity, acyl group transferase activity, eukaryotic translation elongation factor activity)were most significantly downregulated.CONCLUSION: Several genes that showed alterations in our study have also been reported from a high incidence area of esophageal cancer in China. This indicates that molecular profiles of esophageal cancer in these two different geographic locations are highly consistent.

  1. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    Directory of Open Access Journals (Sweden)

    CLAUDIA TEREZIA SOCOL

    2013-12-01

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

  2. 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.

  3. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  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. Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

    Directory of Open Access Journals (Sweden)

    Woodruff Teresa K

    2004-10-01

    Full Text Available Abstract Background Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA. Methods The subunit cDNAs were cloned by a combination of reverse transcriptase polymerase chain reaction (RT-PCR and 5' rapid amplification of cDNA ends (RACE RT-PCR from adult rhesus monkey testis RNA. Results Both the inhibin alpha and betaB subunit nucleotide and predicted protein sequences are highly conserved with other mammalian species, particularly with humans. During the course of these investigations, a novel inhibin alpha mRNA isoform was also identified. This form, referred to as rhesus monkey inhibin alpha-variant 2, appears to derive from both alternative transcription initiation as well as alternative splicing. rmInhibin alpha-variant 2 is comprised of a novel 5' exon (exon 0, which is spliced in-frame with exon 2 of the conventional inhibin alpha isoforms (variant 1. Exon 1 is skipped in its entirety such that the pro-alpha and part of the alpha N regions are not included in the predicted protein. rmInhibin alpha -variant 2 is of relatively low abundance and its biological function has not yet been ascertained. Conclusion The data show that the predicted inhibin B protein is very similar between monkeys and humans. Therefore, studies in monkeys using recombinant human inhibins are likely to reflect actions of the homologous ligands. In addition, we have observed the first inhibin alpha subunit mRNA variant. It is possible that variants will be observed in other species as well and this may lead to novel insights into inhibin action.

  7. 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...

  8. Identification of a Herbicide Safener AD-67 Inducible cDNA in Rice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A herbicide safener AD-67 inducible cDNA was identified in an indica rice variety 9311 by mRNA differential display. The transcript was increased 6 h after sprayed with the safener solution, and 4 days later, the expression still could be detected. The fragment was recycled from the poly-gel and sequenced, and homologous analysis revealed the cDNA was 100% identical to some ESTs and cDNAs in rice database, and the amino acid sequence was 60-84% homologous to those of the Yippee genes in several eukaryotes. The fragment was extended to the whole long cDNA, and thus a primer pair was designed. RT-PCR analysis for the designed primer supported the induction result.

  9. Design and Screening of M13 Phage Display cDNA Libraries

    Directory of Open Access Journals (Sweden)

    Yuliya Georgieva

    2011-02-01

    Full Text Available The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS will be presented.

  10. Isolation and Characterization of Phytoene Desaturase cDNA from Stigma of Crocus sativus

    Institute of Scientific and Technical Information of China (English)

    Bai Jie(白洁); Xu Ying; Tang Lin; Zeng Yu; Feng Yun; Wang Shenghua; Chen Fang

    2004-01-01

    Phytoene desaturase (PDS) has recently been identified as an important enzyme in carotenoid biosynthesis pathway. A cDNA clone encoding phytoene desaturase gene is isolated from stigma of saffron (Crocus sativus L.) using RT-PCR technique. Sequence analysis shows 83% similarity to Narcissus pseudonarcissus, 79% to Zea mays, 78% to Arabidopsis thaliana, 77% to Lycopersicon esculentum. A new full-length cDNA is obtained by 5'-RACE and 3' -RACE techniques. The cDNA is 2149bp long with an open reading frame of 1697bp, which encodes a polypeptide of 565 amino acids. Southern analysis shows that the PDS gene is a single copy in saffron. Northern blot analysis shows higher expression level of PDS gene in stigma and anther than in leaves and stem.

  11. cDNA macroarray for analysis of gene expression profiles in prostate cancer

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Early diagnosis and timely treatment are important for improving therapeutic efficiency of prostate cancer. DNA array is a new bio-technology for disease diagnosis. This study was conducted to diagnose prostate cancer with cDNA macroarray and analysis gene expression profiles of some selective genes in prostate cancer.Methods Total RNA was isolated from patients with prostate cancer and from normal people, and poly(A) RNA was further purified. Then it was analyzed for differentially expressed genes in prostate cancer and normal prostate by cDNA macroarray system.Results There were different expressions in the nine prostate-associated specific genes in prostate cancer as compared with normal prostate, in which, 7 were significantly upregulated and 2 were down-regulated.Conclusion As a diagnostic approach at molecular level, the cDNA macroarray is an effectively diagnostic method for prostate cancer.

  12. LD-RTPCR:\tA NEW METHOD FOR LABELLING TRACE cDNA MICROARRAY PROBE

    Institute of Scientific and Technical Information of China (English)

    范保星; 孙敬芬; 梁好; 王升启; 周平坤; 吴德昌

    2002-01-01

    Objective: To explore the usefulness of long distance reverse transcript combining linear amplification (LD-RTPCR) in labeling slight trace probe used for cDNA microarray. Methods: Total RNA from BEP2D cells was extracted and labeled by two different methods, LD-RTPCR with Cy3-dCTP as fluorescent dye and traditionally used RNA reverse transcript (RT) with Cy5-dCTP as fluorescent dye. Then, the probes labeled by two methods were mixed equally and hybridized with the cDNA microarray. Results: Scan and analysis of the microarray showed that the two methods labeled probes had consistent results. Conclusion: LD-RTPCR was proved useful for labeling cDNA microarray probe, especially for limited RNA material.

  13. First characterization of infectious cDNA clones of Olive mild mosaic virus

    Directory of Open Access Journals (Sweden)

    Joana M.S. CARDOSO

    2012-09-01

    Full Text Available Full-length cDNA clones of an Olive mild mosaic virus (OMMV isolate were constructed in order to find infectious cDNA clones. The sequencing of three individual full-length clones revealed some differences between them. In vitro transcription of these clones was performed and the effect of spontaneous mutations in the biological behaviour of the in vitro transcripts was evaluated by symptomatology, RNA accumulation and virus replication in inoculated plants. In vitro synthesized RNA from one of these clones was found to mimic the wild-type OMMV, making it useful in future studies on protein structure and function by site directed mutagenesis of individual genes. This is the first report on constructing full-length cDNA clones of OMMV from which infectious RNAs can be transcribed in vitro.

  14. Profiling gene expression patterns of nasopharyngeal carcinoma and normal nasopharynx tissues with cDNA microarray

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    5 μg of total RNAs from normal nasopharynx and nasopharyngeal carcinoma tissue have been labeled with α-32P-dCTP during reverse transcription. The synthesized cDNA probes have been hybridized to high-density cDNA microarray containing 5184 genes or expression sequence tags (ESTs). Then image analysis software has been applied to comparing their expression profiles. Results show that 187 ESTs were of density value above 200 in nasopharyngeal carcinoma tissue while there were 307 such ESTs in normal nasopharynx tissue; 38 ESTs were strongly expressed in nasopharynx, but weakly expressed in nasopharyngeal carcinoma; 48 ESTs were strongly expressed in nasopharyngeal carcinoma, but weakly expressed in normal nasopharynx. These results suggest that there may exist some new differentially expressed genes involved in nasopharyngeal carcinoma development. Furthermore, the results strongly indicate that high-density cDNA microarray is a powerful and efficient tool for large-scale screening differentially expressed genes.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Establishment of Self-incompatibility Gene cDNA Microarray to Identify S-genotypes of Pyrus pyrifolia

    Directory of Open Access Journals (Sweden)

    Jiang Nan

    2015-11-01

    Full Text Available Based on the cDNA sequences from hyper variable (HV regions of identified 52 S-alleles in Oriental pear cultivars, S-RNase cDNA probes were designed, and a cDNA microarray for S-RNase detections was established. Each microarray contained 240 sites from 55 cDNA probes, including all specific cDNA sequences from the HV regions of the S-alleles. Using the cDNA of pistils of tested pear cultivars as template and Cy3 fluorescently labeling primers by PCR amplification, microarray hybridization detected the S-genotype of each pear cultivar. The genotypes inferred from the cDNA microarray hybridization signals of pear cultivars such as ‘Lijiang Huangsuanli’, ‘Xiuyu’, ‘Midu Yuli’, ‘Baimianli’, and ‘Deshengxiang’ were similar to the known genotypes of all tested cultivars. The S-RNase cDNA microarrays and the oligonucleotide gene chips were then used to conduct parallel testing of 24 P. pyrifolia cultivars with unknown S-genotypes. In conclusion, the construction of cDNA microarrays has further improved the pear S-RNase detection platform.

  17. Analysis of Metastatic-Related Gene Expression in Gastric Cancer by Low-Density cDNA Microarrays

    Institute of Scientific and Technical Information of China (English)

    Baojun Huang; Huimian Xu; Yujie Zhao; Zhenning Wang; Shaocheng Wang

    2006-01-01

    OBJECTIVE To screen metastatic-related genes in human gastric cancer by a low-density cDNA microarray technique.METHODS A total of 18 paired gastric cancer and adjacent normal mucosa were examined by a low-density cDNA microarray containing 23genes. RT-PCR was used for further verification.RESULTS The mRNA expression of MMP-7, heparanase, S100A4,hTERT, hRad17 in gastric cancers was higher than that in coupled normal mucosa (P =0.002, 0.00011, 0.000072, 0.002, 0.00016 respectively),whereas nm23H1, and CDH1 were lower (P=0.003, 0.012 respectively).The concordance was verified further by RT-PCR with a correlation coefficient of 0.774. In gastric primary lesions the mRNA expression of MMP-7, heparanase and S100A4 was higher in the serosa involved compared to non-involved (P=0.003, 0.009, 0.012 respectively), whereas nm23H1,CDH1, KAI1 were lower (P=0.001, 0.001, 0.006 respectively). With respect to the area of serosa involvement, MMP-7 and heparanase expressions were higher in an area of more than 20 cm2 compared to an area of less than 20 cm2 (P=0.001, 0.02 respectively), whereas nm23H1,CDH1 and KAI1 were lower (P=0.030, 0.041, 0.031 respectively). MMP-7and hTERT expressions were higher in the heavier lymph node metastatic cases (no less than 7) than in the lighter lymph node metastatic cases(no more than 6, P=0.001, 0.005 respectively).CONCLUSION Expression of MMP-7, S100A4, heparanase, hTERT,KAI1, CDH1 and nm23H1 correlated closely with invasion and metastasis in gastric carcinomas. The low-density cDNA microarrays can be used to examine the expression of many genes simultaneously, parallely and quickly.

  18. Cloning and screening of cDNA of Psilgramma menephorn allergen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To construct a cDNA expression library of Psilgramma menephorn to screen its major allergen so as to provide the basis for producing recombinant allergen vaccine of Psilgramma menephorn. Methods Total RNA was extracted from the whole body of Psilgramma menephorn with Trizol and mRNA was purified with Oligo (dT) Spin-Column. And dscDNA was synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoRⅠ adapters. Then the cDNAs were digested by XhoⅠ, and the fra...

  19. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end.......PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end....

  20. 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...

  1. Index of /data/medaka-full-length-cdna-db/20110331 [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Index of /data/medaka-full-length-cdna-db/20110331 Name Last modified Size Descript...ion Parent Directory - README.html 06-Sep-2012 16:47 13K medaka_full_length_c..> 26-Aug-2011 15:54 99M medaka_full_leng...th_c..> 26-Aug-2011 15:52 814K medaka_full_length_c..> 26-Aug-2011 15:52 8.7M Index of /data/medaka-full-length-cdna-db/20110331 ...

  2. Characterization of cDNA from the miracidial antigen family of Schistosoma japonicum (Chinese strain)

    Institute of Scientific and Technical Information of China (English)

    余传信; 平山謙二; 朱荫昌; 菊池三惠子; 殷旭仁

    2003-01-01

    Objective To identify the egg antigens related to the formation of hepatic granulomas and fibrosis of Schistosomiasis japonica.Methods The egg cDNA library of Schistosoma japonicum (S.japonicum) was constructed and screened by immunological methods with the pooled sera of advanced schistosomiasis patients. The inserted foreign DNA fragments of positive clones were sequenced. The sequence data were analyzed using Wdnasis 2.5 and compared with Genebank data using blast software. Conclusion The cDNA sequence of the miracidial antigen of S.japonicum (Chinese strain) was obtained for the first time.

  3. 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

  4. An annotated cDNA library of juvenile Euprymna scolopes with and without colonization by the symbiont Vibrio fischeri

    Directory of Open Access Journals (Sweden)

    Tong Deyan

    2006-06-01

    Full Text Available Abstract Background Biologists are becoming increasingly aware that the interaction of animals, including humans, with their coevolved bacterial partners is essential for health. This growing awareness has been a driving force for the development of models for the study of beneficial animal-bacterial interactions. In the squid-vibrio model, symbiotic Vibrio fischeri induce dramatic developmental changes in the light organ of host Euprymna scolopes over the first hours to days of their partnership. We report here the creation of a juvenile light-organ specific EST database. Results We generated eleven cDNA libraries from the light organ of E. scolopes at developmentally significant time points with and without colonization by V. fischeri. Single pass 3' sequencing efforts generated 42,564 expressed sequence tags (ESTs of which 35,421 passed our quality criteria and were then clustered via the UIcluster program into 13,962 nonredundant sequences. The cDNA clones representing these nonredundant sequences were sequenced from the 5' end of the vector and 58% of these resulting sequences overlapped significantly with the associated 3' sequence to generate 8,067 contigs with an average sequence length of 1,065 bp. All sequences were annotated with BLASTX (E-value Conclusion Both the number of ESTs generated from each library and GO categorizations are reflective of the activity state of the light organ during these early stages of symbiosis. Future analyses of the sequences identified in these libraries promise to provide valuable information not only about pathways involved in colonization and early development of the squid light organ, but also about pathways conserved in response to bacterial colonization across the animal kingdom.

  5. Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides

    Energy Technology Data Exchange (ETDEWEB)

    Robakis, N.K.; Ramakrishna, N.; Wolfe, G.; Wisniewski, H.M.

    1987-06-01

    Deposits of amyloid fibers are found in large numbers in the walls of blood vessels and in neuritic plaques in the brains of patients with Alzheimer disease and adults with Down syndrome. The authors used the amino acid sequence of the amyloid peptide to synthesize oligonucleotide probes specific for the gene encoding this peptide. When a human brain cDNA library was screened with this probe, a clone was found with a 1.7-kilobase insert that contains a long open reading frame coding for 412 amino acid residues including the 28 amino acids of the amyloid peptide. RNA gel blots revealed that a 3.3-kilobase mRNA species was present in the brains of individuals with Alzheimer disease, with Down syndrome, or with not apparent neurological disorders. Southern blots showed that homologous genes are present in the genomic DNA of humans, rabbits, sheep, hamsters, and mice, suggesting that this gene has been conserved through mammalian evolution. Localization of the corresponding genomic sequences on human chromosome 21 suggest a genetic relationship between Alzheimer disease and Down syndrome, and it may explain the early appearance of large numbers of neuritic plaques in adult Down syndrome patients.

  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. A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase II.

    Science.gov (United States)

    Finkelstein, A; Kostrub, C F; Li, J; Chavez, D P; Wang, B Q; Fang, S M; Greenblatt, J; Burton, Z F

    1992-01-30

    RAP30/74 (also known as TFIIF, beta gamma and FC is one of several general factors required for initiation by RNA polymerase II. The small RAP30 subunit of RAP30/74 binds directly to polymerase and appears structurally and functionally homologous to bacterial sigma factors in their RNA polymerase-binding region. RAP30/74 or recombinant RAP30 suppresses nonspecific binding of RNA polymerase II to DNA and is required for RNA polymerase II to assemble stably into a preinitiation complex containing promoter DNA and the general factors TFIID, TFIIA and TFIIB; both RAP30 and RAP74 are physical components of the preinitiation complex. A complementary DNA encoding human RAP30 has been isolated, and here we report the isolation of a cDNA encoding human RAP74. RAP30 and RAP74 produced in Escherichia coli can be used in place of natural human RAP30/74 to direct accurate transcription initiation by RNA polymerase II in vitro.

  8. 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.

  9. 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.

  10. Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

    NARCIS (Netherlands)

    Platteel, Anouk C M; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M

    2016-01-01

    Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA va

  11. 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...

  12. 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 ...

  13. cDNA - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available English ]; } else { document.getElementById(lang).innerHTML= '[ Japanese | English ]'; } } window.onload = ...ies Species name Chr. No. Chromosome No. Strand Strand Gene name Gene name UniGene ID UniGene ID cDNA UniGen

  14. Observation of intermittency in gene expression on cDNA microarrays

    CERN Document Server

    Peterson, L E

    2002-01-01

    We used scaled factorial moments to search for intermittency in the log expression ratios (LERs) for thousands of genes spotted on cDNA microarrays (gene chips). Results indicate varying levels of intermittency in gene expression. The observation of intermittency in the data analyzed provides a complimentary handle on moderately expressed genes, generally not tackled by conventional techniques.

  15. CLONING AND CHARACTERIZATION OF CDNA ENCODING GIARDIA LAMBLIA d-GIARDIN

    Science.gov (United States)

    A cDNA coding for d-giardin was cloned from Giardia lamblia trophozoites in order to localize the protein and study its function in mediating surface attachment. Recombinant d-giardin antigen was produced in Escherichia coli as a poly-histidine fusion protein and was purified by affinity chromatogr...

  16. Isolation of carrot Argonaute1 from subtractive somatic embryogenesis cDNA library.

    Science.gov (United States)

    Takahata, Kiminori

    2008-03-01

    Carrot Argonaute1 (C-Ago1) was isolated from a subtractive cDNA library to obtain somatic embryogenesis related genes. C-Ago1 has three conserved domains, which are found in all other Argonautes. C-Ago1 has specific expression during somatic embryogenesis, which indicates that microRNA gene expression controlling system is required for somatic embryogenesis.

  17. 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.

  18. Construction of full-length cDNA library of white flower Salvia ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    May 18, 2009 ... base for further study on the structure and function of these cDNAs. Key words: white flower ..... precipitated by ethylene glycol monobutyl ether is high purity. The A260/A230 ... gives high feasibility for cloning full length cDNA.

  19. Transcription analysis of apple fruit development using cDNA microarrays

    NARCIS (Netherlands)

    Soglio, V.; Costa, F.; Molthoff, J.W.; Weemen-Hendriks, M.; Schouten, H.J.; Gianfranceschi, L.

    2009-01-01

    The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and

  20. Isolation and cDNA cloning of somatolactin in rabbitfish (Siganus guttatus).

    Science.gov (United States)

    Ayson, F G; de Jesus, E G; Amemiya, Y; Moriyama, S; Hirano, T; Kawauchi, H

    1999-08-01

    We report the isolation and cDNA cloning of somatolactin (SL) from rabbitfish, Siganus guttatus. Rabbitfish SL was isolated from an alkaline extract of the pituitary glands by gel filtration chromatography on Sephadex G-100 and reversed-phase high-performance liquid chromatography. SL was monitored by immunoblotting with flounder SL antiserum. The preparation (yield: 0.86 mg/g wet tissues) contained two immunoreactive bands of 24 and 28 kDa on SDS-PAGE. Overlapping partial cDNA clones corresponding to teleost SLs were amplified by PCR from single-strand cDNA from pituitary glands. Excluding the poly(A) tail, rabbitfish SL cDNA is 1605 bp long. It contains a 693-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a mature protein of 207 aa. Rabbitfish SL has two possible N-glycosylation sites at positions 11 and 121 and seven half Cys residues. The deduced amino acid sequence shows over 80% identity with those of advanced teleosts like sea bream, red drum, and flounder, 76% with the salmonids, 57% with the eel, and 46% with the goldfish SL.

  1. Data of evolutionary structure change: 1CDNA-1CLMA [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1CDNA-1CLMA 1CDN 1CLM A A KSPEELKGIFEKYAAKE--GDPNQLSKEELKLLLQTEFP...--- LTE-EQIAEFKEAFALFDKDGDGTITTKELGTVMRSL-----GQNPTEAELQDMINEVDADGNGTIDFPEFLSLMARKMKEQDSEEELIEA...line> 1CLM A 1CLMA 1CLM A 1CLMA...fEVID> 2 1CLM A 1CLMA

  2. 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.

  3. 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 ...

  4. FIELD VALIDATION OF A SHEEPSHEAD MINNOW ESTROGEN-RESPONSIVE CDNA MACROARRAY

    Science.gov (United States)

    Hemmer, Michael J., Iris Knoebl, Becky L. Hemmer, Patrick Larkin, Peggy S. Harris and Nancy D. Denslow. In press. Field Validation of a Sheepshead Minnow Estrogen-Responsive cDNA Macroarray (Abstract). To be presented at the SETAC Fourth World Congress, 14-18 November 2004, Portl...

  5. Consistent errors in first strand cDNA due to random hexamer mispriming

    NARCIS (Netherlands)

    Van Gurp, T.P.; McIntyre, L.M.; Verhoeven, K.J.F.

    2013-01-01

    Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as

  6. Characterization of mouse Clpp protease cDNA, gene, and protein

    DEFF Research Database (Denmark)

    Andresen, B S; Corydon, T J; Wilsbech, M

    2000-01-01

    characterized the mouse Clpp cDNA sequence, the organization of the mouse gene, the chromosomal localization, and the tissue-specific expression pattern. Moreover. the cellular localization and processing of mouse Clpp was studied by overexpression in transfected eukaryotic cells. Our results indicate...

  7. EXPRESSION PROFILING OF ESTROGENIC COMPOUNDS USING A SHEEPSHEAD MINNOW CDNA MACROARRAY

    Science.gov (United States)

    Larkin, Patrick, Leroy C. Folmar, Michael J. Hemmer, Arianna J. Poston and Nancy D. Denslow. 2003. Expression Profiling of Estrogenic Compounds Using a Sheepshead Minnow cDNA Macroarray. Environ. Health Perspect. 111(6):839-846. (ERL,GB 1171). A variety of anthropogenic c...

  8. Construction of full-length cDNA library of white flower Salvia ...

    African Journals Online (AJOL)

    African Journal of Biotechnology. Journal Home · ABOUT ... Double strand cDNA was cloned into pDNR-LIB vector. The number of clones, ... After BLAST analysis of some cDNAs, their possible functions were predicted. It is found that most of ...

  9. 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...

  10. Characterization of the soybean early nodulin cDNA clone GmENOD55.

    NARCIS (Netherlands)

    Blank, de C.; Mylona, P.; Katinakis, P.; Bisseling, T.; Franssen, H.; Yang, W.C.

    1993-01-01

    Two cDNA clones of the soybean early nodulin GmENOD55 were characterized. These clones may represent two members of the soybean early nodulin gene family GmENOD55. GmENOD55 has an N-terminal signal peptide and it contains an internal domain consisting of proline and serine residues. Analyses of

  11. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end....

  12. 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.

  13. KLONING cDNA HORMON PERTUMBUHAN DARI IKAN GURAME (Osphronemus gouramy

    Directory of Open Access Journals (Sweden)

    Estu Nugroho

    2016-11-01

    Full Text Available Penelitian mengenai kloning cDNA pengkode hormon pertumbuhan ikan gurame telah dilakukan. Tujuan dari penelitian ini adalah untuk memperoleh sekuens DNA komplemen hormon pertumbuhan sebagai langkah awal dalam rangka pengembangan teknologi rekayasa genetik ikan gurame. Empat buah kelenjar hifopisa ikan gurame digunakan sebagai bahan bakunya dan dilakukan proses ekstraksi RNA total dari kelenjar hipofisa, dilanjutkan dengan sintesis cDNA, amplifikasi PCR, purifikasi fragmen DNA dari gel, ligasi produk PCR dengan vektor kloning, transformasi dan inkubasi bakteri, seleksi koloni bakteri putih, isolasi plasmid, dan sekuensing. Hasil sekuensing menunjukkan bahwa panjang produk amplifikasi PCR adalah 843 bp yang menyandikan 204 asam amino residu dan mengandung sekuens-sekuens yang konserf untuk gen hormon pertumbuhan (GH. Analisis homologi menunjukkan kesamaan sekuens hasil isolasi antara 52,4%--97,6% dengan gen GH ikan lainnya, dengan persentase homologi tertinggi adalah dengan ikan sepat. Dengan demikian dapat disimpulkan bahwa sekuens hasil isolasi merupakan sekuens gen GH. Dari hasil analisis sekuens terlihat bahwa gen GH ikan gurame secara evolusi adalah konserf. Research on cDNA cloning encoded the gouramy growth hormone was conducted. The aim of the research was to get complementary DNA, cDNA, sequences of growth hormone as an initial step to develop genetic engineering of gouramy fish. Four pituitary glands of the gouramy were taken and then processed with total RNA extraction, and continued with cDNA synthesis, PCR amplification, DNA fragment purification from the gel, PCR product legation with cloning vector, transformation and incubation of bacteria, white colony bacteria selection, plasmid isolation and sequencing analysis. Sequencing result showed that the amplified PCR product length had 834 bp, encoding 204 amino acid residue and contained conserve sequence for GH (growth hormone gen. Homolog analysis showed sequence similarity of

  14. Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA

    Institute of Scientific and Technical Information of China (English)

    李惠芳; 杨永红; 石永进; 王逸群; 朱平

    2004-01-01

    Background Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to erdiacate metastatic malignant cells. So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells. Methods CIK cells were obtained from peripheral blood and induced by IFN-γ, anti-CD3 monoclonal antibody, IL-2 and IL-1. CIK cells were transfected with plasmid PHaMDR containing human mdr1 cDNA by electroporation. RT-PCR was used to detect mdr1 mRNA in transfected CIK cells. P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry. Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method.Results mdr1 mRNA was detected in transfected CIK cells. P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21%-37% of the total CIK cells after transfection. The IC50 to doxorubicin increased to 22.3-45.8 times, and that to colchicines to 6.7-11.35 times, as compared to those of untransfected CIK cells. However, the cytotoxic activity to MCF7 cell line remained unaltered.Conclusions CIK cells were successfully transfected with mdr1 cDNA by using electroporation. The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells.

  15. Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

    Directory of Open Access Journals (Sweden)

    MacFarlane Amanda J

    2009-07-01

    Full Text Available Abstract Background Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D. Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened. Results Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies. Conclusion Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health.

  16. Analysis of beta-carotene hydroxylase gene cDNA isolated from the American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library.

    Science.gov (United States)

    Bhore, Subhash J; Kassim, Amelia; Loh, Chye Ying; Shah, Farida H

    2010-09-20

    It is well known that the nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is of important to identify the genetic features for its superior value. This could be achieved through the genome sequencing of the oil-palm. However, the genome sequence is not available in the public domain due to commercial secrecy. Hence, we constructed a cDNA library and generated expressed sequence tags (3,205) from the mesocarp tissue of the American oil-palm. We continued to annotate each of these cDNAs after submitting to GenBank/DDBJ/EMBL. A rough analysis turned our attention to the beta-carotene hydroxylase (Chyb) enzyme encoding cDNA. Then, we completed the full sequencing of cDNA clone for its both strands using M13 forward and reverse primers. The full nucleotide and protein sequence was further analyzed and annotated using various Bioinformatics tools. The analysis results showed the presence of fatty acid hydroxylase superfamily domain in the protein sequence. The multiple sequence alignment of selected Chyb amino acid sequences from other plant species and algal members with E. oleifera Chyb using ClustalW and its phylogenetic analysis suggest that Chyb from monocotyledonous plant species, Lilium hubrid, Crocus sativus and Zea mays are the most evolutionary related with E. oleifera Chyb. This study reports the annotation of E. oleifera Chyb. ESTs - expressed sequence tags, EoChyb - Elaeis oleifera beta-carotene hydroxylase, MC - main cluster.

  17. Construction and Screening of an Expression cDNA Library from the Triactinomyxon Spores of Myxobolus cerebralis, the causative agent of Salmonid Whirling Diseases

    OpenAIRE

    Soliman, Hatem Mohamed Touhan

    2005-01-01

    The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract...

  18. Molecular cloning and characterization of a novel human kinase gene, PDIK1L

    Indian Academy of Sciences (India)

    Lingchen Guo; Chaoneng Ji; Shaohua Gu; Kang Ying; Haipeng Cheng; Xiaoghua Ni; Jianping Liu; Yi Xie; Yumin Mao

    2003-04-01

    We isolated a 4301-bp cDNA from a human foetal brain cDNA library by high-throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the putative kinase may interact with PDZ and LIM domain proteins. Therefore the protein and its cDNA were named ‘PDLIM1 interacting kinase 1 like’ (PDIK1L; nomenclature approved by the HUGO Gene Nomenclature Committee). Ensembl Genome Browser located PDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be localized to the nucleus.

  19. The murine decorin. Complete cDNA cloning, genomic organization, chromosomal assignment, and expression during organogenesis and tissue differentiation.

    Science.gov (United States)

    Scholzen, T; Solursh, M; Suzuki, S; Reiter, R; Morgan, J L; Buchberg, A M; Siracusa, L D; Iozzo, R V

    1994-11-11

    Decorin, a proteoglycan known to interact with collagen and growth factors, may play key roles during ontogenesis, tissue remodeling, and cancer. We have deciphered the complete protein sequence of the murine decorin by cDNA cloning, elucidated its gene structure and chromosomal location, and investigated its expression in the developing embryo. The decorin protein and the gene were highly conserved vis à vis the human counterpart; however, the murine gene lacked a leader exon, exon Ib, which was found only in the human. Using interspecific backcrossing, we assigned the gene to chromosome 10 just proximally to the Steel gene locus. In situ hybridization studies of developing mouse embryos showed a distinct pattern of expression with a progressive increase of decorin mRNA during ontogenesis. At early stages (day 11 postconception), decorin was detectable only in the floor plate region. Subsequently (days 13-16 postconception), decorin expression was especially prominent in the meninges and mesothelial linings of pericardium, pleura, and coelomic cavity, as well as in the dermis and subepithelial layers of the intestine and urinary bladder. In contrast, the major parenchymal organs were only weakly positive for decorin mRNA. These findings suggest that decorin may play a role in epithelial/mesenchymal interactions during organ development and shaping.

  20. Splicing variants of SERPINA1 gene in ovine milk: characterization of cDNA and identification of polymorphisms.

    Science.gov (United States)

    Marchitelli, Cinzia; Crisà, Alessandra; Mostarda, Elisa; Napolitano, Francesco; Moioli, Bianca

    2013-01-01

    The serine protease inhibitor, clade A, member 1 (SERPINA1) is the gene for a protein called alpha-1-antitrypsin (AAT), which is a member of the serine protease inhibitor (serpin) superfamily of proteins. By conformational change, serpins control several chemical reactions inhibiting the activity of proteases. AAT is the most abundant endogenous serpin in blood circulation and it is present in relatively high concentration in human milk as well as in bovine and porcine colostrum. Here we report for the first time the molecular characterization and sequence variability of the ovine SERPINA1 cDNA and gene. cDNAs from mammary gland and from milk were PCR amplified, and three different transcripts (1437, 1166 and 521bp) of the SERPINA1 gene were identified. We amplified and sequenced different regions of the gene (5' UTR, from exon 2 to exon 5 and 3' UTR), and we found that the exon-intron structure of the gene is similar to that of human and bovine. We detected a total of 97 SNPs in cDNAs and gene sequences from 10 sheep of three different breeds. In adult sheep tissues a SERPINA1 gene expression analysis indicated a differential expression of the three different transcripts. The finding reported in this paper will aid further studies on possible involvement of the SERPINA1 gene in different physiological states and its possible association with production traits.

  1. Splicing variants of SERPINA1 gene in ovine milk: characterization of cDNA and identification of polymorphisms.

    Directory of Open Access Journals (Sweden)

    Cinzia Marchitelli

    Full Text Available The serine protease inhibitor, clade A, member 1 (SERPINA1 is the gene for a protein called alpha-1-antitrypsin (AAT, which is a member of the serine protease inhibitor (serpin superfamily of proteins. By conformational change, serpins control several chemical reactions inhibiting the activity of proteases. AAT is the most abundant endogenous serpin in blood circulation and it is present in relatively high concentration in human milk as well as in bovine and porcine colostrum. Here we report for the first time the molecular characterization and sequence variability of the ovine SERPINA1 cDNA and gene. cDNAs from mammary gland and from milk were PCR amplified, and three different transcripts (1437, 1166 and 521bp of the SERPINA1 gene were identified. We amplified and sequenced different regions of the gene (5' UTR, from exon 2 to exon 5 and 3' UTR, and we found that the exon-intron structure of the gene is similar to that of human and bovine. We detected a total of 97 SNPs in cDNAs and gene sequences from 10 sheep of three different breeds. In adult sheep tissues a SERPINA1 gene expression analysis indicated a differential expression of the three different transcripts. The finding reported in this paper will aid further studies on possible involvement of the SERPINA1 gene in different physiological states and its possible association with production traits.

  2. Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies.

    Science.gov (United States)

    Lewis, A P; Parry, N; Peakman, T C; Crowe, J S

    1992-07-01

    Human monoclonal antibody production has been hampered for many years by the instability of cell lines and low levels of expression of the antibodies. We describe here the rescue of human immunoglobulin genes utilizing micro-mRNA preparation from a small number of human hybridoma cells and conventional cDNA cloning. This allows cloning and immediate high-level expression from full-length human heavy and light chain cDNA molecules and provides a mechanism to rescue whole human monoclonal antibodies of proven efficacy.

  3. 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.

  4. [Construction of subtractive cDNA libraries of the sporogony stage of Eimeria tenella by suppression subtractive hybridization].

    Science.gov (United States)

    Han, Hong-Yu; Lin, Jiao-Jiao; Zhao, Qi-Ping; Dong, Hui; Jiang, Lian-Lian; Wang, Xin; Han, Jing-Fang; Huang, Bing

    2007-11-01

    In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

  5. 3' rapid amplification of cDNA ends (RACE) walking for rapid structural analysis of large transcripts.

    Science.gov (United States)

    Ozawa, Tatsuhiko; Kondo, Masato; Isobe, Masaharu

    2004-01-01

    The 3' rapid amplification of cDNA ends (3' RACE) is widely used to isolate the cDNA of unknown 3' flanking sequences. However, the conventional 3' RACE often fails to amplify cDNA from a large transcript if there is a long distance between the 5' gene-specific primer and poly(A) stretch, since the conventional 3' RACE utilizes 3' oligo-dT-containing primer complementary to the poly(A) tail of mRNA at the first strand cDNA synthesis. To overcome this problem, we have developed an improved 3' RACE method suitable for the isolation of cDNA derived from very large transcripts. By using the oligonucleotide-containing random 9mer together with the GC-rich sequence for the suppression PCR technology at the first strand of cDNA synthesis, we have been able to amplify the cDNA from a very large transcript, such as the microtubule-actin crosslinking factor 1 (MACF1) gene, which codes a transcript of 20 kb in size. When there is no splicing variant, our highly specific amplification allows us to perform the direct sequencing of 3' RACE products without requiring cloning in bacterial hosts. Thus, this stepwise 3' RACE walking will help rapid characterization of the 3' structure of a gene, even when it encodes a very large transcript.

  6. RT-Bst: an integrated approach for reverse transcription and enrichment of cDNA from viral RNA.

    Science.gov (United States)

    Kabir, M S; Clements, M O; Kimmitt, P T

    2015-01-01

    The synthesis of cDNA from RNA is challenging due to the inefficiency of reverse transcription (RT). In order to address this, an RT-Bst method was developed for sequential RT of RNA and Bst DNA polymerase amplification for enrichment of cDNA in a single-tube reaction. Using genomic RNA from bacteriophage MS2, the yield of cDNA produced by RT alone and RT-Bst were compared by analysis of polymerase chain reaction (PCR)-amplified products. A superior performance was observed when amplifying MS2 cDNA with random primers following RT-Bst compared to RT alone, indicating greater quantities of cDNA were present after RT-Bst. RT-Bst was also compared with RT alone for their relative ability to produce sufficient cDNA to amplify eight target regions spanning the respiratory syncytial virus (RSV) genome. Six out of eight targets were amplified consistently by PCR subsequent to RT-Bst amplification, whereas only three out of eight targets could be amplified after RT alone. The RSV sequences were selectively amplified using RSV-specific primers from a mixed template containing an excess of MS2 RNA without amplifying MS2 sequences. This suggests that RT-Bst can be used to amplify RNA sequences non-specifically using random primers and specifically using sequence-specific primers, and enhances the yield of cDNA when compared to RT alone.

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

    Directory of Open Access Journals (Sweden)

    Erma Sulistyaningsih

    2015-10-01

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

  8. Molecular cloning of GA-suppressed G2 pea genes by cDNA RDA

    Institute of Scientific and Technical Information of China (English)

    朱玉贤; 张翼凤; 李慧英

    1997-01-01

    GA-treated and non-treated G2 pea cDNAs were compared using a newly developed method called cDNA representational difference analysis (cDNA-RDA), and several GA-suppressed mRNAs were found. After cloning of the larger fragments PGAS1-3 ( pea GA-suppressed cDNA 1-3), they were demonstrated to be expressed only in pea tissue not treated with GA3 through Northern analysis. Compared with subtractive hybridization and differ-ential display techniques, this method not only can be easily manipulated but also has a relatively low rate of false posi-tive and is highly repetitive. It is the major progress in molecular cloning techniques.

  9. Identification of brassinosteroid responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    胡玉欣; 汪政科; 王永红; 包方; 李凝; 彭镇华; 李家洋

    2001-01-01

    We have systematically monitored brassinosteroid (BR) responsive genes in a BR-deficient mutant det2 suspension culture of Arabidopsis by using a cDNA array approach. Among 13000 cDNA clones arrayed on filters, 53 BR responsive clones were identified and designated BRR1-BRR53. Sequence analysis of 43 clones showed that 19 clones are novel genes, 3 clones are genes involved in the control of cell division, 4 clones are genes related to plant stress responses, 4 clones are transcriptional factor or signal transduction component genes, and 3 clones are genes involved in RNA splicing or structure forming. In addition, we also found that BR regulated the transcription of genes related to many physiological processes, such as photoreaction, ion transportation and some metabolic processes. These findings present molecular evidence that BR plays an essential role in plant growth and development.

  10. Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Soumen Naskar

    2012-01-01

    Full Text Available In the present study, water buffalo MHC (Bubu-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

  11. Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    徐永忠; 谢建平; 李瑶; 乐军; 陈建平; 淳于利娟; 王洪海

    2003-01-01

    Objective To examine the global effects of Mycobacterium tuberculosis (M.tuberculosis) infection on macrophages. Methods The gene expression profiling of macrophage U937, in response to infection with M.tuberculosis H37Ra, was monitored using a high-density cDNA microarray. Results M.tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M.tuberculosis infection and intracellular survival. Conclusions M.tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M.tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

  12. CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C......-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed...

  13. Molecular cloning of a cDNA related to vernalization(verc203) in winter wheat

    Institute of Scientific and Technical Information of China (English)

    种康; 谭克辉; 黄华梁; 梁厚果

    1995-01-01

    A cDNA clone related to the vernalization in winter wheat(verc203)was harvested from the en-riched cold-induced cDNA library of 10~4 pfu with differential screening.The insert of verc203 in λ gt10 vector wassubcloned into the sites between BamH Ⅰ and Hind Ⅲ in pUC19 plasmid after being amplified with PCR.the analysis of the Northern blotting with a probe of verc203 indicated that the verc203 has a negative signalfor the control and the devernalized mRNA and a positive signal for the vernalized winter wheat and non-vernalized spring wheat at about 2.6 kb.

  14. Construction of cDNA Library of Pyrocystis lunula(Pyrophyta)

    Institute of Scientific and Technical Information of China (English)

    SUI Zhenghong; Klaus V.Kowallik

    2004-01-01

    Complementary DNA library of a dinoflagellate Pyrocystis lunula was constructed for the purpose of expression sequence tags analysis. The RNA isolated from this alga was about 20 μg g-1 net cells, and the band intensity ratio of 28 S/18 S in electrophoresis pattern was nearly 1 to 1. Different cDNA/vector molar ratios were exploited in the ligating reaction to be optimized. The clones produced by cDNA/vector molar ratio of 3.75 to 1 were desirable, most of whose inserts were longer than 300 bp. The recombinants insert length of the unfractionation cDNA library was largely shorter than 500 bp. However, in the fractionation library made from high molecule weight cDNA parts, over seventy percent of the recombinants contained inserts longer than 1 kb, some of which were even longer than 3 kb. Operating concerns were discussed at the end.

  15. Identification of auxin responsive genes in Arabidopsis by cDNA array

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The plant hormone auxin influences a variety of developmental and physiological processes. But the mechanism of its action is quite unclear. In order to identify and analyze the expression of auxin responsive genes, a cDNA array approach was used to screen for genes with altered expression from Arabidopsis suspension culture after IAA treatment and was identified 50 differentially expressed genes from 13824 cDNA clones. These genes were related to signal transduction, stress responses, senescence, photosynthesis, protein biosynthesis and transportation. The results provide the molecular evidence that auxin influences a variety of physiological processes and pave a way for further investigation of the mechanism of auxin action. Furthermore,we found that the expression of a ClpC (regulation subunit of Clp protease) was repressed by exogenous auxin, but increased in dark-induced senescing leaves. This suggests that ClpC may be a senescence-associated gene and can be regulated by auxin.

  16. Consistent errors in first strand cDNA due to random hexamer mispriming.

    Directory of Open Access Journals (Sweden)

    Thomas P van Gurp

    Full Text Available Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.

  17. PolyA PCR amplification of cDNA from RNA extracted from formalin-fixed paraffin-embedded tissue.

    Science.gov (United States)

    Byers, Richard; Roebuck, Jamie; Sakhinia, Ebrahim; Hoyland, Judith

    2004-09-01

    RNA extraction still relies almost exclusively on the use of fresh or frozen tissue, limiting the number of samples that can be analyzed, and there is a growing need for means of global mRNA analysis of archived formalin-fixed paraffin-embedded tissue (FFPET). Previous reports of RNA extraction and amplification from FFPET are limited and do not enable global cDNA amplification. This study used polyA PCR to generate globally amplified cDNA from RNA extracted from formalin-fixed paraffin-embedded samples. RNA was extracted from nine routinely processed archival FFPET samples (lymph node, nasopharynx, prostate, lung and bone marrow) using an Ambion Paraffin Block RNA Isolation Kit. Global cDNA was generated by polyA RT-PCR and used in GAPDH specific PCR and PCR for CD33, c-myb, and SNF2. PolyA cDNA was reamplified by polyA PCR and the reamplified cDNA also used in GAPDH PCR. RNA was extracted from all nine samples, but was degraded. PolyA RT-PCR generated cDNA from all samples and was positive for GAPDH PCR in seven. PCR for CD33, c-myb, and SNF2 was positive in all samples tested. Following reamplification, the polyA cDNA remained positive for GAPDH by PCR. The results demonstrate the feasibility of globally amplifying RNA isolated from archival FFPET samples using polyA RT-PCR, which generates a renewable cDNA pool that can be probed for any cDNA species and reamplified as necessary.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Induction of mosquitocidal activity in mice immunized with Anopheles gambiae midgut cDNA.

    Science.gov (United States)

    Foy, B D; Magalhaes, T; Injera, W E; Sutherland, I; Devenport, M; Thanawastien, A; Ripley, D; Cárdenas-Freytag, L; Beier, J C

    2003-04-01

    Vaccines that induce mosquito-killing (mosquitocidal) activity could substantially reduce the transmission of certain mosquito-borne diseases, especially vaccines against African malaria vectors, such as the mosquito Anopheles gambiae. To generate and characterize antimosquito immunity we immunized groups of mice with two individual A. gambiae midgut cDNAs, Ag-Aper1 (a secreted peritrophic matrix protein) and AgMuc1 (a midgut-bound mucin), and an A. gambiae midgut cDNA library from blood-fed mosquitoes. We observed significantly increased mortality among mosquitoes that fed on either the AgMuc1- or the cDNA library-immunized mice compared to that of controls, but no differences were observed among those fed on Ag-Aper1-immunized mice. Analysis of the humoral and cellular immune responses from mice showed that the induced mosquitocidal effect was associated with immune profiles characterized by elevated tumor necrosis factor alpha and gamma interferon cytokine levels and very low antibody titers. Furthermore, an additional immunization of cDNA library-immunized mice with midgut protein shifted immunity toward a Th2-type immune response, characterized by elevated antibody titers and high interleukin-5 and interleukin-10 cytokine levels; importantly, mosquitoes feeding on these mice exhibited no undue mortality. Finally, when immune sera was ingested by mosquitoes through a membrane feeder, no effect on mosquito mortality was observed, indicating that serum factors alone were not responsible for the mosquitocidal effect. Our results demonstrate that mosquitocidal immunity in mice can be consistently generated by midgut cDNA immunization and suggest this cDNA-induced mosquitocidal immunity is cell mediated.

  3. RAPID SCREENING OF AN ARRAYED cDNA LIBRARY BY IMPROVED PCR-BASED METHOD

    Institute of Scientific and Technical Information of China (English)

    杜光伟; 潘美辉; 袁建刚; 周彦; 强伯勤; 梁植权

    1998-01-01

    Tbe present study reports an improved PCR-based technique that allows quick and effecfive screening of eDNA libraries. First, the eDNA library was arrayed as follows: about 3×106 cDNA clones were multiplied as individual plsques on solid medium in 24-well culture dishes at 1 200 plaque forming units per well.The phage suspension of each well was transferred to an individual micrccentrifuge tube in 72-tube box. Then,box pool, row pools and column pools were set up that respectively represent a 72-tube box,rows and columns within the box. To screen a specific target cDNA,primers specific for novel ESTs obtained in our laboratory were eznployed to conduct PCR in a fiierarchy mode. PCR began with the box pools, resulting in the identification of some positive box pools. Then PCR went down to the row and column pools of the positive box. Tbe intersection of the positive row(s) and column(s) revealed the candidate positive tubes. The specificity of PCR products were meanwhile checked hy restriction enzyme digestion. Finally, hybridization was carried out to get single specific eDNA clones-from the positive tlabes. This PCR-hased technique features high specificity, high efficiency and Les-cost in large-scale cDNA library screening. Our initial implementation of the technique resulted in the isolation of three longer different cDNA clones from a hnman fetal brain eDNA library. Thus this improved technique can serve as an alternative to the time-consuming and laborious conventional hybridization-hased metfiod for screening cDNA library.

  4. Single primer amplification (SPA) of cDNA for microarray expression analysis

    OpenAIRE

    2003-01-01

    The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplific...

  5. Obtaining reliable information from minute amounts of RNA using cDNA microarrays

    OpenAIRE

    2002-01-01

    Abstract Background High density cDNA microarray technology provides a powerful tool to survey the activity of thousands of genes in normal and diseased cells, which helps us both to understand the molecular basis of the disease and to identify potential targets for therapeutic intervention. The promise of this technology has been hampered by the large amount of biological material required for the experiments (more than 50 μg of total RNA per array). We have modified an amplification procedu...

  6. Isolation of cDNA Fragment of Gene Encoding for Actin from Melastoma malabthricum.

    Directory of Open Access Journals (Sweden)

    Suharsono

    2010-11-01

    Full Text Available Isolation of cDNA Fragment of Gene Encoding for Actin from Melastoma malabthricum. M. malabathricumgrows well in acidic soil with high Al solubility, thereby it can be used as a model plant for tolerance to aluminum andacid stresses. Actin is housekeeping gene used as an internal control for gene expression analysis. The objective of thisresearch was to isolate and clone the cDNA fragments of MmACT encoding for actin of M. malabathricum. Total RNAwas isolated and used as the template for cDNA synthesis by reverse transcription. Four cDNA fragments of MmACT,called MmACT1, MmACT2, MmACT3, and MmACT4, had been isolated and inserted into pGEM-T Easy plasmid.Nucleotide sequence analysis showed that the size of MmACT1 and MmACT2 is 617 bp, whereas MmACT3 andMmACT4 is 735 bp. The similarity among these four MmACT is about 78%-99% based on nucleotide sequence andabout 98%-100% based on amino acid sequence. Phylogenetic analysis based on amino acid sequence showed that at1% dissimilarity, the MmACT1, MmACT2, MmACT3 and the ACT5 Populus trichocarpha are clustered in one group,while the MmACT4 is grouped with ACT9 P. trichocarpa and ACT1 Gossypium hirsutum, and these two groups areseparated from actin group of monocotyledonous plants. The sequence of MmACT fragments were registered inGenBank/EMBL/DDBJ database with accession numbers AB500686, AB500687, AB500688, and AB500689.

  7. Cloning and roles of goldfish maternal factor β-Catenin cDNA in embryonic development

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jingpu; WANG Weixian; ZHU Shaoxia

    2004-01-01

    Interaction between nucleus and cytoplasm has been focused in the field of animal embryonic development, in which study of maternal factors is required positively. Β-Catenin, an important maternal factor in early embryogenesis, has been analyzed in its expression pattern and functions in this paper. We have cloned goldfish β-Catenin cDNA gene and compared it with zebrafish β-Catenin cDNA. High homology was found in cDNA and in amino acid sequences between them, 93% (2227/2384 bp) and 98.5% (768/780 aa) respectively. The expression pattern of β-Catenin by in situ hybridization and the roles of β-Catenin on embryonic development by co-injection of anti-sense RNA and reporter gene, EGFP have been investigated in the whole process of goldfish embryonic development. The results suggest that β-Catenin presents dynamic distribution, mainly locates at body axis, dorsal tissues, head and tail structures after being fertilized. The loss of β-Catenin activity would cause serious destruction of embryo in dorsal tissues and in anteroposterior axes, and leads embryos to die before larva get hatched.

  8. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    Directory of Open Access Journals (Sweden)

    Zhongwei Wu

    2010-01-01

    Full Text Available In this paper we describe an isothermal rolling-circle amplification (RCA protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP.The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  9. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu.

    Science.gov (United States)

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group.

  10. cDNA cloning and expression of a collectin from red-spotted grouper (Epinephelus akaara)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhiwen; DING Shaoxiong; WANG Ying; MAO Yong; SU Yongquan; WANG Jun

    2009-01-01

    Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin (EALec1) from Epinephelus akaara using RACE. The complete EALec1 cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly(A) tail. The EALec1 cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain (CRD), indicating that EALec1 is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALec1 in 12 different tissues using RT-PCR. EALec1 was expressed in all tissues, though at different levels. In addition, we inserted EALec1 into an expression vector (pET-28a) for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALec1 recombinant expression vector.

  11. cDNA cloning and expression of a collectin from red-spotted grouper ( Epinephelus akaara)

    Science.gov (United States)

    Zhang, Zhiwen; Ding, Shaoxiong; Wang, Ying; Mao, Yong; Su, Yongquan; Wang, Jun

    2009-09-01

    Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin (EALec1) from Epinephelus akaara using RACE. The complete EALec1 cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly(A) tail. The EALec1 cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain (CRD), indicating that EALec1 is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALec1 in 12 different tissues using RT-PCR. EALec1 was expressed in all tissues, though at different levels. In addition, we inserted EALec1 into an expression vector (pET-28a) for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALec1 recombinant expression vector.

  12. Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Frisch, S.M.; Clark, E.J.; Werb, Z.

    1987-05-01

    Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. The authors studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggest coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-12-31

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

  14. [Construction and analysis of subtractive cDNA library of Phellodendron amurense under drought stress].

    Science.gov (United States)

    Wang, Huimei; Wang, Yanbing; Zu, Yuangang; Sun, Lianhui

    2008-02-01

    With cDNA from Phellodendron amurense seedlings treated with drought stress as tester and cDNA from this plant in normal growth as driver, we construct cDNA subtracted library using suppression subtractive hybridization (SSH). In the library, the rate of recombination was 95%, the size of inserts was 300-800 bp. Two hundred and sixty-five new genes were obtained by DNA sequencing 816 positive clones picked randomly, and partitioned to 16 classes after nucleotide Blast and BlastX homological analysis against NT, NR, SWISSPROT, KEGG database. Forty-four drought stress associated genes, such as heat shock protein cognate 70, dehydration responsive protein 22, universal stress protein, metallothionein II, late embryogenesis abundant protein, were obtained, which made 16.6% of the overall genes. These genes included osmotic regulator, signal component regulatory protein and antioxidant enzyme. The research had established a basis for cloning stress resistance genes and further studying genes expression in P. amurense seedlings under drought stress.

  15. Isolation of a cDNA Encoding a Protease from Perinereis aibuhitensis Grube

    Institute of Scientific and Technical Information of China (English)

    Rong-Gui LI; Dong-Meng QIAN; Dao-Sen GUO; Gui-Cai DU; Zhi-Yong YAN; Bin WANG

    2006-01-01

    The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMALPPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-β-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.

  16. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  17. Detection of HIV cDNA point mutations with rolling-circle amplification arrays.

    Science.gov (United States)

    Wu, Lingwei; Liu, Quanjun; Wu, Zhongwei; Lu, Zuhong

    2010-01-27

    In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP).The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  18. CTL responses to Leishmania mexicana gp63-cDNA vaccine in a murine model.

    Science.gov (United States)

    Ali, S A; Rezvan, H; McArdle, S E; Khodadadi, A; Asteal, F A; Rees, R C

    2009-07-01

    Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term (51)Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.

  19. Isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus) growth hormone.

    Science.gov (United States)

    Ayson, F G; de Jesus, E G; Amemiya, Y; Moriyama, S; Hirano, T; Kawauchi, H

    2000-02-01

    We report the isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus; Teleostei; Perciformes; Siganidae) growth hormone (GH). Rabbitfish GH was extracted from pituitary glands under alkaline conditions, fractionated by gel filtration chromatography on Sephadex G-100, and purified by high-performance liquid chromatography. The fractions containing GH were identified by immunoblotting with bonito GH antiserum. Under nonreducing conditions, the molecular weight of rabbitfish GH is about 19 kDa as estimated by SDS-PAGE. The purified hormone was potent in promoting growth in rabbitfish fry. Weekly intraperitoneal injections of the hormone significantly accelerated growth. This was evident 3 weeks after the start of the treatment, and its effect was still significant 2 weeks after the treatment was terminated. Rabbitfish GH cDNA was cloned to determine its nucleotide sequence. Excluding the poly (A) tail, rabbitfish GH cDNA is 860 base pairs (bp) long. It contained untranslated regions of 94 and 175 bp in the 5' and 3' ends, respectively. It has an open reading frame of 588 bp coding for a signal peptide of 18 amino acids and a mature protein of 178 amino acid residues. Rabbitfish GH has 4 cysteine residues. On the amino acid level, rabbitfish GH shows high identity (71-74%) with GHs of other perciforms, such as tuna, sea bass, yellow tail, bonito, and tilapia, and less (47-49%) identity with salmonid and carp GHs.

  20. 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.

  1. 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

  2. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA.

    Science.gov (United States)

    Michael, A J; Furze, J M; Rhodes, M J; Burtin, D

    1996-02-15

    A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present.

  3. Development and validation of a bovine macrophage specific cDNA microarray

    Directory of Open Access Journals (Sweden)

    Waddington David

    2006-09-01

    Full Text Available Abstract Background The response of macrophages to danger signals is an important early stage in the immune response. Our understanding of this complex event has been furthered by microarray analysis, which allows the simultaneous investigation of the expression of large numbers of genes. However, the microarray resources available to study these events in livestock animals are limited. Results Here we report the development of a bovine macrophage specific (BoMP cDNA microarray. The BoMP microarray contains 5026 sequence elements (printed in duplicate and numerous controls. The majority of the clones incorporated on the microarray were derived from the BoMP cDNA library generated from bovine myeloid cells subjected to various stimuli, including over 900 sequences unique to the library. Additional clones representing immunologically important genes have been included on the BoMP microarray. The microarray was validated by investigating the response of bovine monocytes to stimulation with interferon-γ and lipopolysaccharide using amplified RNA. At 2 and 16 hours post stimulation 695 genes exhibited statistically significant differential expression, including; 26 sequences unique to the BoMP library, interleukin 6, prion protein and toll-like receptor 4. Conclusion A 5 K cDNA microarray has been successfully developed to investigate gene expression in bovine myeloid cells. The BoMP microarray is available from the ARK-Genomics Centre for Functional Genomics in Farm Animals, UK.

  4. Chitinase cDNA cloning and mRNA induction by fungal elicitor, wounding, and infection.

    Science.gov (United States)

    Hedrick, S A; Bell, J N; Boller, T; Lamb, C J

    1988-01-01

    Chitinase, which catalyzes the hydrolysis of beta-1,4 N-acetylglucosamine linkages of the fungal cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgaris L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colletotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a lambdagt11 cDNA library containing sequences complementary to poly A(+) RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, transient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase of rapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome.

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

    Science.gov (United States)

    Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

    2011-01-01

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

  6. Cloning and expression of a cDNA encoding ribosomal protein S4 from Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A cDNA clone, pS4, has been isolated from a cDNA library prepared from rice anthers of about 1.0 mm in length. DNA sequence analysis and database search show that the cDNA encodes a protein which is highly homologous to eukaryotic 80S ribosomal protein subunit 4 (S4). Northern hybridization indicates that this gene expresses in all tissues analyzed although the expression level varies and it cannot be induced by mechanical wounding in leaves. Southern blot analysis demonstrates that this rice S4 gene is from a multigene family.

  7. 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

  8. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis.

    Science.gov (United States)

    Rho, Jaerang; Altmann, Curtis R; Socci, Nicholas D; Merkov, Lubomir; Kim, Nacksung; So, Hongseob; Lee, Okbok; Takami, Masamichi; Brivanlou, Ali H; Choi, Yongwon

    2002-08-01

    Bone homeostasis is maintained by the balanced action of bone-forming osteoblasts and bone-resorbing osteoclasts. Multinucleated, mature osteoclasts develop from hematopoietic stem cells via the monocyte-macrophage lineage, which also give rise to macrophages and dendritic cells. Despite their distinct physiologic roles in bone and the immune system, these cell types share many molecular and biochemical features. To provide insights into how osteoclasts differentiate and function to control bone metabolism, we employed a systematic approach to profile patterns of osteoclast-specific gene expression by combining suppression subtractive hybridization (SSH) and cDNA microarray analysis. Here we examined how gene expression profiles of mature osteoclast differ from macrophage or dendritic cells, how gene expression profiles change during osteoclast differentiation, and how Mitf, a transcription factor critical for osteoclast maturation, affects the gene expression profile. This approach revealed a set of genes coordinately regulated for osteoclast function, some of which have previously been implicated in several bone diseases in humans.

  9. cDNA Microarrays Detect Activation of a Myogenic Transcription Program by the PAX3-FKHR Fusion Oncogene

    National Research Council Canada - National Science Library

    Javed Khan; Michael L. Bittner; Lao H. Saal; Ulrike Teichmann; David O. Azorsa; Gerald C. Gooden; William J. Pavan; Jeffrey M. Trent; Paul S. Meltzer

    1999-01-01

    .... To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA micro...

  10. Difference in gene expression of macrophage between normal spleen and portal hypertensive spleen idendified by cDNA microarray

    OpenAIRE

    2007-01-01

    AIM: To identify the difference in gene expression of microphage (Mφ) between normal spleen and portal hypertensive spleen using cDNA microarrays and find new gene functions associated with hypersplenism in portal hypertension.

  11. Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast

    National Research Council Canada - National Science Library

    Riesmeier, J.W; Willmitzer, L; Frommer, W.B

    1992-01-01

    ...‐symport, but so far no sucrose carrier gene has been identified. Using an engineered Saccharomyces cerevisiae strain, a cDNA from spinach encoding a sucrose carrier was identified by functional expression...

  12. 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.

  13. Molecular cloning of the feline thymus and activation-regulated chemokine cDNA and its expression in lesional skin of cats with eosinophilic plaque.

    Science.gov (United States)

    Maeda, Sadatoshi; Okayama, Taro; Ohmori, Keitaro; Masuda, Kenichi; Ohno, Koichi; Tsujimoto, Hajime

    2003-02-01

    Thymus and activation-regulated chemokine (TARC) is a member of CC chemokine and plays an essential role in recruitment of CC chemokine receptor 4 positive Th2 cells to allergic lesion. To investigate the association of TARC in allergic inflammation of cats, a TARC cDNA was cloned from feline thymus by RT-PCR with 3' rapid amplification of cDNA ends (RACE) method. The feline TARC clone contained a full length open reading frame encoding 99 amino acids which shared 80.8%, 72.5%, 65.6% and 67.8% homology with dog, human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lung, lymph node, kidney, small intestine, colon and skin of the normal cat tissues examined. Furthermore, it was found that TARC mRNA was strongly expressed in lesional skin of cats with eosinophilic plaque. The present results demonstrated that TARC might be involved in the pathogenesis of eosinophilic plaque in cats.

  14. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.

    OpenAIRE

    1988-01-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex...

  15. cDNA cloning and life-cycle stage-specific expression of coronin from Physarum polycephalum.

    Science.gov (United States)

    Minami, Yoshiko; Ishihara, Masaaki; Hayase, Masato; Sakaguchi, Tomohisa; Yubisui, Toshitsugu

    2009-03-23

    Coronin cDNA was cloned from the plasmodia of Physarum polycephalum. The amino acid sequence deduced from the cDNA was comprised of 449 residues and showed 60% identity to that of Dictyostelium discoideum coronin. Southern blot analysis suggested that the coronin gene present in the P. polycephalum genome might be a single copy. Coronin was expressed in diploid plasmodia, while it was not detected in haploid amoebae or spores.

  16. Rapid Amplification of 5′ cDNA End of S. Liaotungensis Choline Monooxygenase Using Inverse PCR RACE

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Based on part of a known cDNA sequence of Suaeda Liaotungensis choline monooxygenase, the authors successfully cloned the 5′ cDNA end of Suaeda Lianotungensis choline monooxygenase using Inverse PCR RACE with a specially designed 5′-phosphated RT primer and two pairs of specific inverse PCR primers. Compared with the anchored PCR RACE, inverse PCR RACE has better specificity and higher amplification.

  17. Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies

    DEFF Research Database (Denmark)

    Gottwein, Judith; Scheel, Troels; Callendret, Benoit

    2010-01-01

    Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic...... analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees...

  18. ISOLATION AND CLONING OF cDNA OF GENE ENCODING FOR METALLOTHIONEIN TYPE 2 FROM MELASTOMA AFFINE

    Directory of Open Access Journals (Sweden)

    UTUT WIDYASTUT

    2009-01-01

    Full Text Available Metallothionein is an important protein for detoxifying heavy metal ions. h is research was conducted to isolate and clone cDNA of gene encoding for metallothionein type 2 from Melastoma affi ne . Total RNA was isolated from young leaves. Total cDNA was synthesized from the total RNA by reverse transcription. h e MaMt2 cDNA was successfully isolated by PCR technique. h e MaMt2 cDNA was inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into Escherichia coli DH5 α . DNA sequencing analysis showed that this cDNA is full length consisting of 246 pb encoding 81 amino acid residues. h is cDNA is identical to mRNA of AtMt2 from Arabidopsis thaliana. It does not contain any restriction sites found in the cloning sites of pGEM-T Easy. h e deduced protein of MaMT2 contains 14 cysteine residues distributed in the Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys motifs

  19. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Marcelo Bento (New York, NY); Bonaldo, Maria de Fatima (New York, NY)

    1998-01-01

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

  20. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Soares, M.B.; Fatima Bonaldo, M. de

    1998-12-08

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods. 25 figs.

  1. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Science.gov (United States)

    Soares, Marcelo Bento; Bonaldo, Maria de Fatima

    1998-01-01

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

  2. RAI,one candidate gene associated with differentiation of human lung adenocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    王雪皎; 张睿; 刘芝华; 王秀琴; 丁芳; 郭明洲; 吴旻

    2000-01-01

    From all-trans retinoic acid (ATRA)-treated human lung adenocarcinoma GLC-82 cells and control, subtractive cDNA library has been constructed using subtractive hybridization technique in our laboratory. The screening of the cDNA subtractive library resulted in identification of a clone containing cDNA fragment of one ATRA-induced gene (RAI) in GLC-82 cells. The positive clone with full-length cDNA of RAI was identified by screening fetal brain cDNA library using colony hybridization technique, and then sequenced. RT-PCR results showed that RAI was expressed in many different human fetal tissues. These results suggest that RAI may be involved in cell differentiation and play an important role in vital activities of cells.

  3. RAI, one candidate gene associated with differentiation of human lung adenocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    From all-trans retinoic acid (ATRA)-treated human lung adenocarcinoma GLC-82 cells and control, subtractive cDNA library has been constructed using subtractive hybridization technique in our laboratory. The screening of the cDNA subtractive library resulted in identification of a clone containing cDNA fragment of one ATRA-induced gene (RAI) in GLC-82 cells. The positive clone with full-length cDNA of RAI was identified by screening fetal brain cDNA library using colony hybridization technique, and then sequenced. RT-PCR results showed that RAI was expressed in many different human fetal tissues. These results suggest that RAI may be involved in cell differentiation and play an important role in vital activities of cells.

  4. Cancer immunotherapy using novel tumor-associated antigenic peptides identified by genome-wide cDNA microarray analyses.

    Science.gov (United States)

    Nishimura, Yasuharu; Tomita, Yusuke; Yuno, Akira; Yoshitake, Yoshihiro; Shinohara, Masanori

    2015-05-01

    Recent genome-wide cDNA microarray analysis of gene expression profiles in comprehensive tumor types coupled with isolation of cancer tissues by laser-microbeam microdissection have revealed ideal tumor-associated antigens (TAAs) that are frequently overexpressed in various cancers including head and neck squamous cell cancer (HNSCC) and lung cancer, but not in most normal tissues except for testis, placenta, and fetal organs. Preclinical studies using HLA-transgenic mice and human T cells in vitro showed that TAA-derived CTL-epitope short peptides (SPs) are highly immunogenic and induce HLA-A2 or -A24-restricted CTLs. Based on the accumulated evidence, we carried out a phase II clinical trial of the TAA-SP vaccine in advanced 37 HNSCC patients. This study showed a significant induction of TAA-specific CTLs in the majority of patients without serious adverse effects. Importantly, clinical responses including a complete response were observed in this study. Another phase II clinical trial of therapeutic TAA-SP vaccine, designed to evaluate the ability of prevention of recurrence, is ongoing in HNSCC patients who have received curative operations. Further studies in human preclinical studies and in vivo studies using HLA class I transgenic mice showed TAA-derived long peptides (TAA-LPs) have the capacity to induce not only promiscuous HLA class II-restricted CD4(+) T helper type 1 cells but also tumor-specific CTLs through a cross-presentation mechanism. Moreover, we observed an augmentation of TAA-LP-specific T helper type 1 cell responses and tumor antigen-spreading in HNSCC patients vaccinated with TAA-SPs. This accumulated evidence suggests that therapeutic TAA-SPs and LPs vaccines may provide a promising cancer immunotherapy.

  5. Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2

    Directory of Open Access Journals (Sweden)

    Murphy Brian R

    2004-10-01

    Full Text Available Abstract Background A dengue virus type 2 (DEN-2 Tonga/74 isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Δ30 in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. Methods A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2 and rDEN2Δ30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice, in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. Results The rDEN2Δ30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Δ30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Δ30, designated rDEN2Δ30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Δ30 in SCID-HuH-7 mice. Conclusions The rDEN2Δ30 and rDEN2Δ30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.

  6. Full-length cDNA cloning and structural characterization of preproinsulin in Alligator sinensis.

    Science.gov (United States)

    Zhang, R; Zhang, S Z; Li, E; Wang, C; Wang, C L; Wu, X B

    2014-10-27

    Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure were analyzed. The full-length of preproinsulin cDNA sequence consists of 528 base pairs (bp), comprising a 34-bp 5'-untranslated region, a 170-bp 3'-untranslated region and an open reading frame that is 324 bp in length. The open reading frame encodes a 107-amino acid preproinsulin with a molecular weight of approximately 12,153.8 Da, theoretical isoelectric point of 5.68, aliphatic index of 92.06, and grand average of hydropathicity of -0.157, from which a signal peptide, a B-chain, a C-peptide, and an A-chain are derived. Online analysis suggested that the deduced preproinsulin amino acid sequence contains a transmembrane region, and that it has a signal peptide whose cleavage site occurs between alanine 24 and alanine 25. Comparative analysis of preproinsulin amino acid sequences indicated that the A-chain and B-chain sequences of preproinsulins are highly conserved between reptiles and birds, and that the preproinsulin amino acid sequence of Alligator sinensis shares 89% similarity to that of Chelonia mydas, but low similarity of 48-63% to those of mammals and fishes. The phylogenetic tree constructed using the neighbor-joining method revealed that preproinsulin of Alligator sinensis had high homology with reptiles and birds, such as Chelonia mydas, Gallus gallus, and Columba livia.

  7. Construction of the subtractive cDNA library of injured adult and fetal rabbit skins

    Institute of Scientific and Technical Information of China (English)

    张波; 刘大维; 王正国; 朱佩芳; 周继红; 蒋建新

    2004-01-01

    Objective: Early gestational mammalian fetuses possess the amazing ability to heal cutaneous wounds in a scarless fashion. Over the past years, scientists have been working to decipher the mechanisms underlying this regenerative repair. The remarkable phenotypic differences between fetal and adult healings behoves us to learn their characteristics in genetics, which represents potentially important mechanisms involved in wound repair observed in fetal versus adult tissues. In this sense, it is reasonable to construct subtractive cDNA library for future research.Methods: Middle laparotomy and hysterotomy were performed on pregnant rabbits at 20-day gestation to expose the fetal back, and a longitudinal incision through the skin was made on the back of the fetus. The traumatized fetal skin was harvested 12 hours post-operation, the fetus control and traumatized adult skin specimens were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. Taking one of the three samples as Tester respectively and the other two as Drivers, we obtained 1 forward and 2 reverse hybridization products. After being amplified with selective polymerase chain reaction, the products were inserted into a vector, and then transferred into E.coli HB101. The colonies were screened afterwards. Results: The wounded fetuses were alive for a long time even after birth. Every determinant step, such as RNA isolation, cDNA synthesis, Rsa I digestion, adaptor ligation and hybridization, was well-operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization (SSH) was successful. Insertion into vector and transferring to E.coli were satisfactory. Conclusions: Instead of classic SSH, an improved SSH with 2 Drivers was applied for the experiment. Results confirmed that the improved program was reasonable and correct in both theory and practice. The subtractive cDNA library we have obtained is going to be used for

  8. Screening for candidate genes related to breast cancer with cDNA microarray analysis

    Institute of Scientific and Technical Information of China (English)

    Yu-Juan Xiang; Zhi-Gang Yu; Ming-Ming Guo; Qin-Ye Fu; Zhong-Bing Ma; De-Zong Gao; Qiang Zhang; Yu-Yang Li; Liang Li; Lu Liu; Chun-Miao Ye

    2015-01-01

    Objective: The aim of this study was to reveal the exact changes during the occurrence of breast cancer to explore significant new and promising genes or factors related to this disease. Methods: We compared the gene expression profiles of breast cancer tissues with its uninvolved normal breast tissues as controls using the cDNA microarray analysis in seven breast cancer patients. Further, one representative gene, named IFI30, was quanti-tatively analyzed by real-time PCR to confirm the result of the cDNA microarray analysis. Results: A total of 427 genes were identified with significantly differential expression, 221 genes were up-regulated and 206 genes were down-regulated. And the result of cDNA microarray analysis was validated by detection of IFI30 mRNA level changes by real-time PCR. Genes for cell proliferation, cell cycle, cell division, mitosis, apoptosis, and immune response were enriched in the up-regulated genes, while genes for cell adhesion, proteolysis, and transport were significantly enriched in the down-regulated genes in breast cancer tissues compared with normal breast tissues by a gene ontology analysis. Conclusion: Our present study revealed a range of differentially expressed genes between breast cancer tissues and normal breast tissues, and provide candidate genes for further study focusing on the pathogenesis and new biomarkers for breast cancer. Copyright © 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  9. A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

    Directory of Open Access Journals (Sweden)

    I. da Silva

    2002-08-01

    Full Text Available In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8 showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries. Plants submitted to stress (wounding, virus infection and ethylene treatment presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

  10. Application of restriction display PCR technique in the preparation of cDNA microarray probes

    Institute of Scientific and Technical Information of China (English)

    Zhao-Hui Sun; Wen-Li Ma; Bao Zhang; Yi-Fei Peng; Wen-Ling Zheng

    2005-01-01

    AIM: To develop a simplified and efficient method for the preparation of hepatitis C virus (HCV) cDNA microarray probes.METHODS: With the technique of restriction display PCR (RD-PCR), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and re-amplified by RD-PCR. We separated the differential genes by polyacrylamide gel electrophoresis and silver staining. Single bands cut out from the polyacrylamide gel were isolated. The third-round PCR was performed using the single bands as PCR template.The RD-PCR fragments were purified and cloned into the pMD18-T vector. The recombinant plasmids were extracted from positive clones, and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting RD-PCR products to the surface of amino-modified glass slides using a robot. We validated the detection of microarray by hybridization and sequence analysis.RESULTS: A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced,which were the specific gene fragments of HCV. These fragments could be further used as probes in microarray preparation. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The results of hybridization and sequence analysis showed that the specificity, sensitivity, accuracy, reproducibility,and linearity in detecting HCV RNA were satisfactory.CONCLUSION: The RD-PCR technique is of great value in obtaining a large number of size-comparable gene probes, which provides a speedy protocol in generating probes for the preparation of microarrays. Microarray prepared as such could be further optimized and applied in the clinical diagnosis of HCV.

  11. Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays.

    Directory of Open Access Journals (Sweden)

    Christine M Costello

    2005-08-01

    Full Text Available BACKGROUND: The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD, Crohn disease (CD, and ulcerative colitis (UC are only partially understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression. METHODS AND FINDINGS: High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11, CD patients (n = 10 and UC patients (n = 10. 33P-radiolabeled cDNA from purified poly(A+ RNA extracted from biopsies (unpooled was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome and CDH11 (cadherin 11, type 2. By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology. CONCLUSION: A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches.

  12. cDNA cloning, prokaryotic expression and purification of rat α-synuclein

    Institute of Scientific and Technical Information of China (English)

    Xin LI; Yao-Hua LI; Jun-Yan HAN; Shun YU; Biao CHEN

    2006-01-01

    Objective To clone the cDNA of rat α-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat α-Syn protein. Methods Rat α-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotie expressing vector. The recombinant plasmid containing rat α-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat α-Syn protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat α-Syn protein. The recombinant rat α-Syn protein was further purified using Superdex S200 gel filtration.Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat α-Syn. After transformation, the recombinant plasmid pGEX-raSyn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat α-Syn.Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against α-Syn. Conclusion The rat α-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat α-Syn recombinant protein was produced.

  13. 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.

  14. 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

  15. 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

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

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    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

  17. Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Rat calcineurin (CaN) A α isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemiareperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG-FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared.The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results veri fied that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A α (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

  18. 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.

  19. Annexin A11 (ANXA11) gene structure as the progenitor of paralogous annexins and source of orthologous cDNA isoforms.

    Science.gov (United States)

    Bances, P; Fernandez, M R; Rodriguez-Garcia, M I; Morgan, R O; Fernandez, M P

    2000-10-01

    The genomic organization of the annexin A11 gene was determined in mouse and human to assess its congruity with other family members and to examine the species variation in alternative splicing patterns. Mouse annexin A11 genomic clones were characterized by restriction analysis, Southern blotting, and DNA sequencing, and the homologous human gene (HGMW-approved gene symbol ANXA11) was deciphered from high-throughput genomic sequence with coanalysis of expressed sequence tags. Exons 6-15 of the tetrad core repeat region differ from annexins A7 and A13 but are spliced identically to other phylogenetic descendents, making annexin A11 the putative primary progenitor of up to nine paralogous human annexins. The 5' regions consist of untranslated exon 1, followed by an extensive intron 1 comprising almost half the total gene length of >40 kb, and additional GC-rich exons 2-5 encoding the proline- and glycine-rich amino-terminus. Distinct cDNA isoforms in cow and human were determined to be unique to each species and hence of dubious general significance for this gene's function. Multiple transcription start sites were revealed by primer extension analysis of the mouse gene, and transfection constructs containing the prospective promoter generated transcriptional activity comparable to that of the SV40 promoter. Internal repetitive elements and vicinal gene markers were mapped for the complete human annexin A11 gene sequence to characterize the surrounding genomic environment. Copyright 2000 Academic Press.

  20. 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.