Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “cDNA sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,477 records · Page 82Linked to original sources
PubMed1998

Cloning and sequencing of cDNA encoding the LS-12 antifreeze protein in the longhorn sculpin, Myoxocephalus octodecimspinosis.

cDNA coding for an antifreeze protein (LS-12) in the longhorn sculpin, Myoxocephalus octodecimspinosis, was prepared from liver mRNA using reverse transcriptase-polymerase chain reaction coupled with 3' and 5' RACE procedures. This cDNA contains 609 base pairs, including a 384-bp open reading frame which codes for a 128-residue LS-12 precursor protein. The predicted amino acid sequence of the mature LS-12 corresponds exactly to the amino acid sequence obtained from Edman degradation [G. Deng, D.W. Andrews, R.A. Laursen, FEBS Lett., 402, 1997, pp. 17-20]. The 20 residues preceding mature LS-12 are predicted to be a signal sequence, which is presumably cleaved off before the mature, 108-residue protein is secreted into the circulatory system. This is the first report of a cDNA sequence from M. octodecimspinosis.

Amino Acid Sequence↗
PubMed1990

Amino acid sequence of silkworm (Bombyx mori) hemolymph antitrypsin deduced from its cDNA nucleotide sequence: confirmation of its homology with serpins.

A cDNA clone of silkworm (Bombyx mori) larval hemolymph antitrypsin (sw-AT) has been isolated from a fat body cDNA library. The cDNA has an open reading frame which codes a 392-amino acid residue polypeptide comprising a 16-residue signal peptide and a 376-residue mature sw-AT of Mr 41,805. The reactive site of sw-AT for inhibition of bovine trypsin [Sasaki, T. et al. (1987) J. Biochem. 102, 433-441] was identified as Lys343-Val344. Alignment of the sw-AT amino acid sequence with those of 11 members of the serpin superfamily of proteins clearly confirmed the homology of sw-AT with serpins. The amino acid sequence of sw-AT is 56% identical with that of the proteinase inhibitor from a lepidopteron, Manduca sexta [Kanost, M.R. et al. (1989) J. Biol. Chem. 264, 965-972], but the sequence around the reactive site shows no homology and the inhibitory specificity for proteinases is very different.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of a novel class I cytokine receptor.

The human gp130 cDNA sequence was used as a query to search an expressed sequence tag database (dbEST) to identify cDNA sequences with similarity to the cytokine class I receptor family. A novel class I cytokine receptor was identified in a human infant brain cDNA library and was named WSX-1. Full-length cDNA sequences for human and murine WSX-1 were isolated and characterized. The WSX-1 cDNA encodes a 636 amino acid transmembrane protein with an extracellular domain of 482 amino acids and a cytoplasmic domain of 96 amino acids. The structure of the WSX-1 protein most closely resembles that of gp130. Northern blot analysis indicates high levels of expression in thymus, spleen, lymph node, and peripheral blood leukocytes, suggesting a role for WSX-1 in modulation of the immune response.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing of a human pancreatic tumor mucin cDNA.

A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation.

Amino Acid Sequence↗
PubMedDate not supplied

Mouse Clara cell 10-kDa (CC10) protein: cDNA nucleotide sequence and molecular basis for the variation in progesterone binding of CC10 from different species.

A protein similar to the rat Clara cell 10-kDa protein (CC10) was isolated from mouse lung homogenate by conventional chromatography. cDNA for the mouse CC10 protein was identified in the mouse lung cDNA library by using radiolabeled rat CC10 cDNA as the probe. The isolated cDNA was sequenced and the deduced primary amino acid sequence was compared to the known sequences of rabbit and hare uteroglobins and human and rat CC10 proteins. The cDNA sequence was confirmed by N-terminal amino acid sequencing of the purified protein. The purified mouse CC10 was tested for its ability to bind progesterone, and the binding was found to be 27% lower than rat CC10 and 48% lower than rabbit uteroglobin. The relative binding of mouse, rat, and human CC10 may reflect subtle structural perturbations. The only notable difference between mouse and rat CC10 is in the beta bend between helices 1 and 2, at residue 16. This difference also exists between rat and human CC10. The mouse CC10 sequence compares favorably with human CC10, which does not bind progesterone; however, the mouse CC10 does not contain M60, which has been proposed to block the binding of progesterone with human CC10. The wide variation in progesterone binding among this family of proteins casts doubt on the importance of such binding as a physiologic function.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of the cDNA coding sequence of IL-2 receptor-gamma (gammac) from human and murine forebrain: expression in the hippocampus in situ and by brain cells in vitro.

IL-2 has been implicated in various neurobiological processes of the mammalian CNS. To understand how IL-2 acts in the brain, our lab has sought to determine the molecular pharmacological characteristics of brain IL-2 receptors (IL-2R). The lymphocyte IL-2Rgamma, an essential subunit for IL-2 signaling, is also a common subunit (gammac) for multiple immune cytokine receptors (e.g., IL-4R, IL-7R, IL-9R, IL-15R). Having previously cloned the alpha and beta subunits of the IL-2R heterotrimer complex from normal murine forebrain, we examined the hypothesis that the brain IL-2Rgamma is derived from the same or a closely related gene coding sequence as that expressed by lymphocytes. In this study, we cloned and sequenced the full-length IL-2Rgamma coding region from saline-perfused mouse forebrain and from a human hippocampal library. The cDNA sequences of IL-2Rgamma from human and murine brain were 100% homologous to their lymphocyte sequences. Northern blot analysis showed that the mRNA transcripts in murine brain were the expected size, but the predominant transcript expressed in the brain was different than in the spleen. Compared to the spleen, very low levels of IL-2Rgamma were expressed in the forebrain. In the murine hippocampus, a region where a number of neurobiological actions of IL-2 have been reported, IL-2Rgamma mRNA was detected over the dentate gyrus and CA1-CA4 by in situ hybridization histochemistry. IL-2Rgamma was found to be constitutively expressed by murine HN33.dw hippocampal neuronal cells, murine NB41A3 neuroblastoma cells, astrocyte-enriched mixed glial cell cultures, and in SCID mouse forebrain. The human cortical neuronal cell lines, HCN-1A and HCN-2, did not express the IL-2Rgamma gene. These data suggest the possibility that, in addition to being essential in IL-2 signaling in brain, IL-2Rgamma could be a common subunit (gammac) for multiple cytokine receptors which may be operative in the mammalian CNS.

Animals↗
PubMedDate not supplied

Ovine glucose transporter-1 and -3: cDNA partial sequences and developmental gene expression in the placenta.

Transplacental glucose transfer utilizes specific glucose transporter (GLUT) proteins. cDNAs encoding ovine placental GLUT1 and GLUT3 genes were isolated, cloned and sequenced and then used to investigate their developmental regulation in ovine placenta. A cDNA of approximately 2.2 kb was isolated from a Clontech lambda gt10 ovine adult liver cDNA library using a 436-bp rat GLUT1 cDNA probe. Sequence data obtained from this clone (1600 bp) demonstrated 97 per cent homology to nucleotides 477-2079 of bovine GLUT1. The deduced amino acid sequence of the ovine cDNA presented 99 per cent identity to amino acid 103-493 of bovine GLUT1, and 97-98 per cent identity to corresponding regions in human and rat GLUT1 deduced amino acid sequences. Reverse transcription-PCR (RT-PCR) was used to isolate an ovine cDNA fragment from placental total RNA. Forward and reverse primers (16 mer) were designed to amplify a predicted 483-bp fragment between the second transmembrane-spanning domain (M2) and intracellular loop of GLUT3. The deduced 161 amino acid sequence of the ovine cDNA demonstrated 84 and 88 per cent identity with murine and human GLUT3. These cDNAs were used to investigate the ontogeny of placental oGLUT1 and oGLUT3 gene expression by Northern analysis. Total RNA was extracted from ovine placenta at gestational days 45, 60, 90, 120 and 138 (n=6 per time point). Ovine GLUT1 gene expression increased significantly from days 45 to 60 (P<0.05), peaked at around day 120 of gestation, then decreased to about two-thirds of maximal levels by day 138 of gestation (term 147 +/- 2). Ovine GLUT3 gene expression increased throughout gestation with significant increases from days 45 to 60, 60 to 120 and 120 to 138 (P<0.05). This study confirms the presence of both GLUT1 and GLUT3 genes in the ovine placenta and demonstrates ontogenic regulation of gene expression. The difference in temporal gene expression between oGLUT1 and oGLUT3 suggests distinct roles for each transporter during development. The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL Data Bank under accession numbers U89029 (oGLUT1) and U89030 (oGLUT3).

Amino Acid Sequence↗
PubMed1985

Cloning and sequencing of cDNA encoding the complete mouse liver alcohol dehydrogenase.

The main ethanol-active alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) in mouse liver (ADH-AA) is similar in catalytic and molecular properties to horse liver ADH-EE and to the human class I ADHs. We have isolated cDNA clones encoding the entire mouse liver enzyme plus flanking regions. A mixture of 16 different oligonucleotides, each 14 bases long, was used to screen a liver cDNA library made from a DBA/2J mouse. A strongly hybridizing clone was found and identified as an ADH-encoding cDNA by partial DNA sequencing. This clone was used as a probe to identify others. Two overlapping cDNA clones together contained the entire protein-encoding region plus 100 nucleotides of the 5' noncoding region and 133 nucleotides of the 3' noncoding region culminating in a short poly(dA) tail. The amino acid sequence of the mouse liver enzyme deduced from this cDNA closely resembles that of horse liver ADH-E: 316 of 374 residues are identical, and 29 of the differences are conservative substitutions. The 5' region of this cDNA is interesting: the AUG that initiates the ADH polypeptide is preceded by an AUG that would encode the first amino acid of a tripeptide. Presumably termination of this tripeptide is followed by reinitiation at the AUG immediately preceding the sequence of the mature ADH polypeptide.

Alcohol Dehydrogenase↗
PubMed2002

Single-strand conformation polymorphism of sequence-tagged site markers based on partial sequences of cDNA clones in Cryptomeria japonica.

Sixty-seven sequence-tagged site (STS) markers were identified from partial sequences of cDNA clones obtained from the inner bark of Cryptomeria japonica. Polymorphisms of the STSs were investigated for the parental clones of a mapping population, Haara and Kumotooshi, using both single-strand conformation polymorphism (SSCP) and sequencing analysis. Twenty-two STSs showed nucleotide differences between Haara and Kumotooshi, of which 19 STS differences were detectable under the electrophoresis conditions we used here. We also analyzed SSC-polymorphism in 10 additional clones derived from various Japanese regions to evaluate the usefulness of the STSs developed here among other populations of C. japonica. Twenty-five, about 40%, of the STSs showed polymorphism under selected electrophoresis conditions. The genotype segregation for 19 STSs was investigated among the Haara x Kumotooshi F(1) population, and these STS markers were mapped on a linkage map. SSCP analysis of STSs was efficient in terms of cost and time, and it allows detection of a sufficiently high proportion of polymorphisms to provide a convenient means for mapping of expressed sequences on a linkage map and for studying various aspects of population genetics.

Chromosome Mapping↗
PubMed1995

Diversity of T cell receptor delta-chain cDNA in the thymus of a one-month-old pig.

Pig TCR delta-chain cDNA sequences were obtained from the anchored PCR products of the reverse-transcribed RNA from the thymus of a 1-mo-old germfree pig. Among 43 sequences analyzed, 4 different J delta sequences were found, and in the cDNA sequences of rearranged V-D-J junctional regions, three putative D delta sequences were assigned. Also, the V delta region sequences were grouped into five families. One family, V delta 1, consisted of a large number of members and the other families consisted of limited members as determined by cDNA sequences as well as genomic Southern blots. Using PCR, V delta 1 gene segments were also obtained from liver genomic DNA of the same individual pig. Thirty-one distinct sequences were found for the V delta 1 family including the sequences obtained from both the thymic RNA and the PCR product of liver DNA. All the V delta 1 genes were found to be associated with almost identical leader gene sequences. The amino acid sequences of the complimentarity-determining region 3 coded by the V-D-J joints gave long and variable lengths with a high content of tryptophan and charged amino acids. The length of the complimentarity-determining region 3 in the porcine TCR delta-chain supports the idea that TCR delta-chain is designed to see Ags directly similar to the Ig heavy chain. Data presented from this study shows that the porcine TCR delta-chain has extensive V region diversity as well as junctional diversity. The combination of these two creates a level of diversity that is the highest of any lymphocyte Ag receptor studied so far.

Amino Acid Sequence↗
PubMed2002

Porcine gene discovery by normalized cDNA-library sequencing and EST cluster assembly.

Genetic and environmental factors affect the efficiency of pork production by influencing gene expression during porcine reproduction, tissue development, and growth. The identification and functional analysis of gene products important to these processes would be greatly enhanced by the development of a database of expressed porcine gene sequence. Two normalized porcine cDNA libraries (MARC 1PIG and MARC 2PIG), derived respectively from embryonic and reproductive tissues, were constructed, sequenced, and analyzed. A total of 66,245 clones from these two libraries were 5?-end sequenced and deposited in GenBank. Cluster analysis revealed that within-library redundancy is low, and comparison of all porcine ESTs with the human database suggests that the sequences from these two libraries represent portions of a significant number of independent pig genes. A Porcine Gene Index (PGI), comprising 15,616 tentative consensus sequences and 31,466 singletons, includes all sequences in public repositories and has been developed to facilitate further comparative map development and characterization of porcine genes (http://www.tigr.org/tdb/ssgi/). The clones and sequences from these libraries provide a catalog of expressed porcine genes and a resource for development of high-density hybridization arrays for transcriptional profiling of porcine tissues. In addition, comparison of porcine ESTs with sequences from other species serves as a valuable resource for comparative map development. Both arrayed cDNA libraries are available for unrestricted public use.

Animals↗
PubMed1986

Rainbow trout growth hormone: molecular cloning of cDNA and expression in Escherichia coli.

We have isolated several recombinant clones carrying the complementary DNA (cDNA) sequence of the rainbow trout (rt) Salmo gairdneri growth hormone (GH) mRNA by immunoblot screening using an antiserum to chum salmon (Oncorhyncus keta) GH. The nucleotide sequence of one of the rtGH cDNA clones (pAF51) was determined. The rt cDNA sequence in pAF51 encodes a hybrid polypeptide of 199 amino acid residues containing 9 amino acid residues of the bacterial beta-galactosidase, one residue from the codon at the junction of the beta-galactosidase gene, and the rtGH cDNA sequence, an additional residue from the presumptive signal peptide of the pre-rtGH and the entire sequence of the mature rtGH (188 amino acid residues). Pairwise matrix comparisons of the hydropathy profiles of bovine, human, rat, and rainbow trout GH polypeptides indicate that regions of similarity exist between the rtGH and mammalian GH. In particular, there are two major regions of similarity found near the amino-terminal region and at the carboxy-terminal region. These regions correspond to hydrophilic domains of the GH molecules. The possible significance of these domains is discussed.

Amino Acid Sequence↗
PubMed1983

Expression of Plasmodium falciparum blood-stage antigens in Escherichia coli: detection with antibodies from immune humans.

Many proteins produced by blood stages of the malaria parasite Plasmodium falciparum are natural immunogens in man. As an approach to determining which of these are relevant to protective immunity we have constructed an expression library of P. falciparum cDNA sequences, cloned in Escherichia coli. The cDNA sequences were inserted into the beta-galactosidase gene of an ampicillin-resistant derivative of the temperature-sensitive lysogenic bacteriophage lambda gt11. About 5% of the resulting clones expressed P. falciparum sequences as polypeptides fused to beta-galactosidase. We have identified many clones that express P. falciparum antigens by immunological screening in situ with antibodies from immune human sera that inhibit P. falciparum growth in vitro. The antigen-positive clones contain P. falciparum cDNA sequences, as determined by hybridization. Some express polypeptides that are larger than beta-galactosidase and react both with antibodies to beta-galactosidase and with antibodies from humans immune to P. falciparum. The cloned P. falciparum antigens should facilitate new approaches to the identification of potential vaccine molecules.

Animals↗
PubMed1990

Random silent mutagenesis in the initial triplets of the coding region: a technique for adapting human glutathione reductase-encoding cDNA to expression in Escherichia coli.

The introduction of random silent mutations into the 5'-coding region of a human cDNA as the basis for successful expression in Escherichia coli is demonstrated in four steps. (1) Plasmid pUB200 containing the pRpL promoters of phage lambda was found not to serve as an expression vector for a unchanged human glutathione reductase (hGR)-encoding cDNA. (2) When this cDNA was expressed in a two-cistron context using high-copy-number plasmids, recombinant protein was detected in low yield (0.03% of the total cell protein). (3) Silent mutations were introduced into the triplets coding for the N-terminal amino acids. When screening E. coli colonies transformed with expression plasmids containing cDNA mutants, we identified adapted clones that produced hGR in up to 70-fold higher yield than the clone containing the unchanged cDNA. Sequence analyses of adapted cDNA species revealed lower G + C contents in the modified regions, suggesting altered mRNA structures. (4) When the adapted cDNA sequences were recloned in the vector which had failed to express unchanged hGR cDNA in step 1, synthesis of recombinant protein was as high as in step 3. This means that the yield of expression for adapted cDNA was at least 1000-fold higher than for unchanged cDNA. In conclusion, random silent mutations introduced into the translation initiation region of cDNA might be a useful technique for designing sequence features which favour gene expression.

Amino Acid Sequence↗
PubMed1991

The nucleotide sequence of rabbit liver transferrin cDNA.

The cDNA sequence of rabbit liver transferrin has been determined. The largest cDNA was 2279 base pairs (bp) in size and encoded 694 amino acids consisting of a putative 19 amino acid signal peptide and 675 amino acids of plasma transferrin. The deduced amino acid sequence of rabbit liver transferrin shares 78.5% identity with human liver transferrin and 69.1% and 44.8% identity with porcine and Xenopus transferrins, respectively. At the amino acid level, vertebrate transferrins share 26.4% identity and 56.5% similarity. The most conserved regions correspond to the iron ligands and the anion binding region. Optimal alignment of transferrin sequences required the insertion of a number of gaps in the region corresponding to the N-lobe. In addition, the N-lobes of transferrins share less amino acid sequence similarity than the C-lobes.

Amino Acid Sequence↗
PubMed1992

Genes encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase in Phaseolus vulgaris L.: nucleotide sequence of cDNA clones and initial studies of expression.

The small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in the French bean Phaseolus vulgaris L. is encoded by a small gene family consisting of a minimum of three members. Three small subunit genes (rbcS genes) represented in a light-grown primary leaf cDNA library were characterised by sequencing two cDNAs which were full-length and one which was deficient in part of the sequence encoding the transit peptide. The cDNA clones are identical in their coding sequences, for both the transit peptide and the mature polypeptide, but divergent in their untranslated sequences. The derived amino acid sequence is very similar to that reported for other species, although the first amino acid of the mature polypeptide is isoleucine, which differs from the methionine found in all other higher plant rbcS genes. Surprisingly, one of the cDNA clones contains two introns, which are at positions conserved in rbcS genes from other species. It is concluded that this cDNA resulted from the cloning of an unprocessed transcript. Alternative polyadenylation sites are found for two of the genes. Expression of the rbcS genes in the primary leaves is stimulated by light, although transcripts can readily be detected in dark-grown leaves. Expression is also organ-specific, as in other species. The frequency of cDNA clones in the library indicates that the different genes show quantitative differences in expression and S1 nuclease analysis suggests that individual rbcS genes are photoregulated.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A.

Here we describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector lambda gt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH2-terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. We used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. We established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes.

Amino Acid Sequence↗
PubMed2001

Functional identification of sterol-4alpha-methyl oxidase cDNAs from Arabidopsis thaliana by complementation of a yeast erg25 mutant lacking sterol-4alpha-methyl oxidation.

Specific primers derived from both genomic sequence data and EST cDNA sequences were used to polymerase chain reaction amplify two full-length cDNA sequences (AtSMO1 and AtSMO2), 801 and 783 bp, respectively, from an Arabidopsis thaliana cDNA library. The predicted proteins show 32 and 29% identity to the ERG25 gene from Saccharomyces cerevisiae which encodes the sterol-4alpha-methyl oxidase (SMO), a membrane-bound non-heme di-iron oxygenase involved in lipid metabolism. Heterologous expression of AtSMO1 and AtSMO2 in a yeast erg25 ergosterol auxotroph, lacking SMO activity, restored growth and endogenous ergosterol synthesis. These results represent the first functional identification of SMO genes from plants.

Arabidopsis↗