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 829 records · Page 46Linked to original sources
PubMed1998

A female-specific cDNA sequence of Schistosoma mansoni encoding an amidase that is expressed in the gastrodermis.

A female-specifically expressed cDNA clone was obtained by screening of a subtractive cDNA library enriched for RNA from Schistosoma mansoni females. The deduced protein shows significant homology to a class of enzymes functioning as amidases. Northern blots revealed a transcript of 4.0 kb which is absent in larval stages, but is expressed in adult female worms. By in situ hybridization, the expression site of the gene was exclusively localized in the gastrodermis of female schistosomes. This is the first report of a female-specifically transcribed sequence of S. mansoni that is not expressed in the reproductive organs.

Animals↗
PubMed1989

cDNA sequence, predicted primary structure, and evolving amphiphilic helix of human aspartyl-tRNA synthetase.

Eight of the mammalian aminoacyl-tRNA synthetases associate as a multienzyme complex, whereas prokaryotic and low eukaryotic synthetases occur only as free soluble enzymes. Association of the synthetases may result in effective compartmentalization of synthetases and suggests the association of the entire protein biosynthetic machinery. To elucidate the structural elements and the nature of the molecular interactions involved in the association of the synthetases, we have cloned and sequenced the complementary DNA coding human aspartyl-tRNA synthetase. The full length cDNA encodes an open reading frame of 500 amino acids with 56% identity with yeast aspartyl-tRNA synthetase. The similarity with yeast aspartyl-tRNA synthetase is unevenly distributed with a high percent of identity at the C-terminus and relatively low identity at the N-terminus. The N-terminal sequence strongly prefers an alpha-helical secondary structure and shows amphiphilic characteristics. Further comparison with the yeast synthetases showed that the basic positively charged helixes in yeast synthetases are evolved to a neutral amphiphilic helix in this mammalian synthetase. The mammalian neutral amphiphilic helix is so far unique among all known sequences of bacterial, yeast, and mammalian synthetases and may account for the association of synthetases in the synthetase complex.

Amino Acid Sequence↗
PubMed1997

Variant cDNA sequences of human ATP:citrate lyase: cloning, expression, and purification from baculovirus-infected insect cells.

ATP:citrate lyase (ACL) is a major generator of cytosolic acetyl-coenzymeA, which is required for both fatty acid and cholesterol biosynthesis. The human ACL (hACL) cDNA was cloned by RT-PCR, and our results indicate the existence of previously unknown sequence variations in hACL. Expression of the hACL cDNA in Spodoptera frugiperda 9 insect cells resulted in the production of high levels of soluble, active enzyme. The recombinant protein (re-hACL) was purified to homogeneity from the soluble lysate of infected cells and was observed to exist as a tetramer by gel filtration chromatography. Kinetic analyses indicated that the re-hACL and rat ACL have very similar enzymological properties. The facile preparation of milligram quantities of purified, active re-hACL affords the opportunity to characterize the enzyme for structure-based design of hypolipidemic drugs, and to further examine the functional significance of the sequence variations.

ATP Citrate (pro-S)-Lyase↗
PubMed1986

Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins.

The complete nucleotide and primary structure (1276 amino acids) of a full length mdr cDNA capable of conferring a complete multidrug-resistant phenotype is presented. The deduced amino acid sequence suggests that mdr is a membrane glycoprotein which includes six pairs of transmembrane domains and a cluster of potentially N-linked glycosylation sites near the amino terminus. A striking feature of the protein is an internal duplication that includes approximately 500 amino acids. Each duplicated segment includes a consensus ATP-binding site. Amino acid homology is observed between the mdr gene and a series of bacterial transport genes. This strong homology suggests that a highly conserved functional unit involved in membrane transport is present in the mdr polypeptide. We propose that an energy-dependent transport mechanism is responsible for the multidrug-resistant phenotype.

Amino Acid Sequence↗
PubMed1992

Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme.

Two functional human dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) cDNAs were isolated from a cDNA expression library by genetic complementation in Escherichia coli. These cDNAs differed in size but exhibited a common overlapping DNA sequence. Contained within this sequence was a single long open reading frame sufficient to encode a polypeptide of 141 amino acids with a calculated molecular mass of 16.6 kDa. The amino acid sequence of this protein exhibits 35% identity with the E. coli dUTPase and 53% identity with the Saccharomyces cerevisiae enzyme. The human dUTPase was found to contain five characteristics amino acid sequence motifs that are common to the dUTPases of E. coli, yeast, and herpesviruses and to dUTPase-like sequences encoded by some retrovirus gag and pol genes. A high degree of amino acid sequence identity (greater than 60%) was also observed between the human dUTPase and the putative pseudoproteases of two poxviruses, indicating that these virus proteins are dUTPases. Northern hybridization analysis reveals that dUTPase is encoded by at least two species of poly(A)+ mRNA and possibly a third, smaller species. All of these mRNAs are present in a variety of human tissues but their relative levels vary between tissues. Southern analysis indicates that the dUTPase gene has been conserved to some extent throughout vertebrate evolution; however, the gene may be very large, or its organization somewhat complex in some systems. We suggest that dUTPase may generally perform an essential role in DNA replication and therefore could serve as a target enzyme for the development of chemotherapeutic compounds.

Amino Acid Sequence↗
PubMed1998

Characterization of the wound-induced metallocarboxypeptidase inhibitor from potato. cDNA sequence, induction of gene expression, subcellular immunolocalization and potential roles of the C-terminal propeptide.

A partial cDNA clone for the potato wound-inducible metallocarboxypeptidase inhibitor (PCI) was isolated from a cDNA library constructed from mRNA of abscisic acid (ABA)-treated potato leaves. The full 5' region of the cDNA was obtained through a RACE-PCR protocol. PCI mRNA encodes a precursor polypeptide which comprises a 29 residue N-terminal signal peptide, a 27 residue N-terminal pro-region, the 39 residue mature PCI protein, and a 7 residue C-terminal extension. Northern blot analysis demonstrates that the PCI gene is transcriptionally activated by wounding, and wound signaling can be induced by ABA and jasmonic acid. Subcellular localization of the protein was investigated by immunocytochemistry and electron microscopy, showing that PCI accumulates within the vacuole. A partial PCI precursor form, comprising the mature protein and the C-terminal extension, has been expressed in Escherichia coli and characterized. Its inability to inhibit carboxypeptidases, and stability to carboxypeptidase digestion, suggest that the C-terminal pro-domain may have, besides a probable vacuolar sorting function, a role in modulation of the inhibitory activity of PCI.

Abscisic Acid↗
PubMed1996

Partial cDNA sequencing of mouse dentine sialoprotein and detection of its specific expression by odontoblasts.

Dentine sialoprotein (DSP), a 53-kDa acidic glycoprotein, is expressed by odontoblasts and secreted into the dentine extracellular matrix. Although little is known about its biological function, it might play a part in dentinogenesis. Because DSP has only been shown to occur in rat dentine, it is important to demonstrate its existence in another species. Here, the presence of DSP gene in the mouse genome, and the cloning of a mouse DSP cDNA coding for about one-fifth of the molecule with a nucleotide sequence similar to that for rat cDNA, are reported. Using in-situ hybridization, DSP mRNA was uniquely detected in mouse odontoblasts.

Animals↗
PubMed2006

Complete cDNA sequences and phylogenetic analyses of the Th1 and Th2 cytokines of the bactrian camel (Camelus bactrianus).

The complementary DNAs of the Th1 (IL-2, IL-12p35, and IFN-gamma) and Th2 (IL-4, IL-10 and IL-13) cytokine genes of the bactrian camel (Camelus bactrianus) were cloned, sequenced, and analyzed. IL-2, IL-4, IL-10, IL-12p35, IL-13, and IFN-gamma were found to have 465, 402, 537, 669, 411, and 501 bp length open reading frames with 154, 133, 178, 222, 136, and 166 amino acid encodings, respectively. The homology ranged from 58.8% to 100% between the nucleotide sequences of the camel cytokine genes and the published sequences of other mammalian genes, including the llama, pig, cow, horse, human, and mouse. The cDNA had highest homology with orders Artiodactyla (pigs and cattle) and Perissodactyla (horses), especially to the recently cloned llama sequences.

Amino Acid Sequence↗
PubMed1998

Cloning, cDNA sequence analysis and patch clamp studies of a toxin from the venom of the armed spider (Phoneutria nigriventer).

The cDNAs (Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.

Amino Acid Sequence↗
PubMed1997

cDNA sequence of a translational elongation factor Ts homologue from Caenorhabditis elegans: mitochondrial factor-specific features found in the nematode homologue peptide.

The cDNA for a homologue of elongation factor Ts which probably functions in mitochondria has been sequenced from a nematode Caenorhabditis elegans. The deduced amino acid sequence (316 amino acids long) has a possible transit peptide sequence at the amino terminus and several common specific features for mammalian mitochondrial EF-Ts. The amino acid identities in the protein from C. elegans compared with those of bovine mitochondria and Escherichia coli are 29.5% and 24.0%, respectively. The C. elegans sequence was classified as a long EF-Ts (ca. 280 amino acids long) similar to peptides from mammalian mitochondria and eubacteria other than Thermus and cyanobacteria (except Spirulina platensis), rather than short EF-Ts (ca. 200 amino acids long) as those of Thermus, cyanobacteria and plastids.

Amino Acid Sequence↗
PubMed1998

cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide.

The spinach pullulanase gene was cloned and sequenced using peptide sequences of the purified enzyme as a starting point and employing PCR techniques and cDNA library screening. Its open reading frame codes for a protein of 964 amino acids which represents a precursor of the pullulanase. The N-terminal transit peptide consists of 65 amino acids, and the mature protein, comprising 899 amino acids, has a calculated molecular mass of 99kDa. Pullulanase is a member of the alpha-amylase family. In addition to a characteristic catalytic (beta/alpha)8-barrel domain, it contains a domain, F, that is specific for branching and debranching enzymes. Pullulanase cDNA was expressed in Escherichia coli, and the purified protein was compared with the enzyme from spinach leaves. Identity of the two proteins was confirmed in terms of catalytic properties, N-terminal amino acid sequences and molecular masses. The pullulanase produced by E. coli showed the same microheterogeneity as the spinach leaf enzyme: it could be resolved into two substrate-induced forms by electrophoresis in amylopectin-containing polyacrylamide gels, and, in the absence of substrate, into several free forms (charge isomers) by isoelectric focusing or chromatofocusing. Rechromatofocusing of single free forms resulted in the originally observed pattern of molecular forms. However, heterogeneity of the protein disappeared on isoelectric focusing under completely denaturing conditions when only one protein band was observed. Post-translational modifications such as glycosylation and phosphorylation could be excluded as potential explanations for the protein heterogeneity. Therefore the microheterogeneity of spinach leaf pullulanase results from neither genetic variation nor post-translational modifications, but is a property of the single unmodified gene product. The different interconvertible forms of the pullulanase represent protein populations of different tertiary structure of the same polypeptide.

Amino Acid Sequence↗
PubMed1997

Analysis of a sequenced cDNA library from multiple sclerosis lesions.

To identify genes that are expressed in MS pathogenesis, we have analyzed a normalized cDNA library made from mRNA obtained from CNS lesions of a patient with primary progressive MS. Complementary DNA clones obtained from this library were subjected to automated DNA sequencing to generate expressed sequence tags. Analysis of this MS cDNA library revealed the presence of 54 cDNAs that were associated with immune activation and indicated the presence of an ongoing inflammatory response with evidence of both cell-mediated and humoral immune responses. The surprising finding was that 16 of the cDNAs encoded autoantigens associated with seven other autoimmune disorders, while only three of these 16 autoantigen cDNAs were present in a similarly constructed adult brain library. Such aberrant autoantigen expression could provide a source of secondary autoimmune stimulation that could contribute to the ongoing inflammatory response in MS. In addition, two cDNAs were found that mapped to a known MS susceptibility locus (5p14-p12): one encoded an excitatory amino acid transporter and the other a human homologue of the Drosophila disabled gene. This approach to the molecular biology of MS pathogenesis may help to illuminate previously unappreciated aspects of this disease.

Cloning, Molecular↗
PubMed1999

Complete cDNA sequence and genomic organization of a human pancreas-specific gene homologous to Caenorhabditis elegans sel-1.

We have isolated the complete cDNA of a human SEL-1L gene, termed TSA305, that is abundantly expressed only in the pancreas. The cDNA contained an open reading frame of 2382 nucleotides, encoding a deduced protein of 794 amino acids whose predicted sequence showed 46% identity and 64% similarity with SEL-1 of Caenorhabditis elegans. SEL-1 is thought to be a negative regulator of the NOTCH, LIN-12, and GLP-1 receptors, which are required for differentiation and maturation of cells as well as cell-cell interactions during development in C. elegans. The degree of homology among these proteins suggests that the TSA305 gene product may be a member of the SEL-1 family and therefore involved in downregulation of mammalian Notch signaling. Direct sequencing revealed at least 20 coding exons in TSA305. We localized the gene to chromosome bands 14q24.3-q31 by radiation hybrid (RH) mapping and fluorescence in situ hybridization (FISH). The IDDM11 locus has been mapped in this region, and TSA305 may represent a candidate gene for predisposition in some families whose insulin-dependent diabetes is not linked to the HLA locus.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular and cell biology of porcine HSP47 during wound healing: complete cDNA sequence and regulation of gene expression.

Heat shock protein (HSP) 47 is a major stress-inducible protein that is localized to the endoplasmic reticulum of avian and mammalian cells and is thought to act as a molecular chaperone specific for the processing of procollagen. However, limited information is available regarding the regulation of HSP47 during wound healing. Using a polymerase chain reaction strategy, screening of a cDNA library, and RACE-polymerase chain reaction approaches, the sequence of a full-length porcine HSP47 cDNA has been identified. The cDNA contained 2096 bp that encodes for an 18 amino acid signal peptide and a mature protein coding region consisting of 401 amino acid residues. It also included 108 bp of the 5' noncoding region and a 731-bp 3' noncoding region. The deduced amino acid is 83% identical to chicken, 87% identical to mouse, 88% identical to rat, and 91% identical to human HSP47. It also shares between 26% and 30% identity with different members of the serine protease inhibitor superfamily. The protein contains a RDEL endoplasmic reticulum retention signal, and two potential glycosylation sites. All of these features are characteristic of HSP47 in higher vertebrates. Heat shock treatment of porcine fibroblasts led to up-regulation of HSP47 at both the transcriptional and translational levels. HSP47 protein levels were also up-regulated during skin wound healing in a pig model. Moreover, a higher molecular weight complex at approximately 140 Kda containing HSP47 was detected at the stage of healing that was coincident with the maximal transcriptional expression of HSP47 during wound healing in this animal model. Further investigation of how HSP47 is regulated during normal and abnormal skin wound healing may lead to new therapeutic approaches to improve the healing process.

Animals↗
PubMed1994

Pancreatic carboxylester lipase from Atlantic salmon (Salmo salar). cDNA sequence and computer-assisted modelling of tertiary structure.

We report the isolation and characterization of a 1795-bp cDNA fragment encoding Atlantic salmon pancreatic carboxylester lipase from salmon pancreas mRNA. The nearly full-length cDNA contained a 540-amino-acid open-reading frame, encompassing the mature protein (by similarity to mammalian carboxylester lipase enzymes). The salmon carboxylester lipase primary structure shared 58% identity with mammalian carboxylester lipases, lacking the proline-rich C-terminal repeats found in human and rat carboxylester lipases. Congruent with other esterase B type enzymes, the salmon carboxylester lipase contained a canonical serine-esterase catalytic triad motif consisting of serine, histidine and aspartic acid. Computer-assisted modelling of the tertiary structure for salmon carboxylester lipase was conducted using acetylcholine esterase (Torpedo californica) as a template structure. The model, in conjunction with sequence comparisons and available enzymological data, has been used to locate putative bile-salt-binding and lipid-binding sites. The carboxylester lipase enzymes contain a unique, highly conserved insert region that may be associated with bile-salt binding. In the model structure, this region is located close to the active site, and contains a tyrosine residue with an adjacent carboxylester-lipase-conserved arginine. These traits have previously been predicted for the non-specific (regarding bile-salt hydroxylation) bile-salt-binding site in carboxylester lipase enzymes. At this site, a dihydroxy or trihydroxy bile-salt molecule may bind the tyrosine via hydrophobic interactions, the anionic bile-salt head group may bind the arginine, while hydrogen bonding between the bile-salt 12 alpha hydroxy group and an adjacent aspargine residue is possible. The model does not contain an active site 'lid' structure as found in other lipases. The carboxylester lipase structural homolog to the 'flap' of the lipases from Geotrichum candidum and Candida rugosa contains a carboxylester-lipase-conserved deletion that renders this region unable to cover the active site. Instead, the shortening of this loop leads to solvent exposure of the carboxylester lipase insert region, an additional indication of the functional importance of this region.

Amino Acid Sequence↗
PubMed1993

cDNA sequence and expression of an intron-containing histone H2A gene from Norway spruce, Picea abies.

We have isolated a cDNA clone corresponding to a histone H2A gene from Norway spruce, Picea abies (L.) Karst. The clone was isolated on the basis of the preferential expression of the corresponding gene during germination. The identification of the clone was based on the high degree of nucleotide sequence identity (60-65%) to a range of eukaryotic histone H2A genes and the presence of a 9 amino acids long sequence identical to the conserved 'H2A box' in the deduced amino acid sequence. Like other plant histone genes, the spruce histone H2A gene encodes a polyadenylated transcript. Further, the spruce gene contains an intervening sequence of 891 bp in the coding region. The presence of introns is typical of a distinct class of replication-independent histone genes in other eukaryotes. However, the sequence of the spruce gene and its high expression in mitotically active tissues such as the apical meristem, strongly suggests that it belongs to the class of replication-dependent histone genes. This is the first documentation of an intervening sequence in this class of histone genes and the finding implies that introns were present in the ancestral histone H2A gene before the divergence of the two classes of histone genes.

Amino Acid Sequence↗
PubMed1996

cDNA sequence and expression of subunit E of the vacuolar H(+)-ATPase in the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum.

A cDNA coding for subunit E of the vacuolar H(+)-ATPase was cloned from Mesembryanthemum crystallinum, a plant which switches from C3-photosynthesis to Crassulacean acid metabolism under saline growth conditions. Sequence homology between the three subunit E-polypeptides of different higher plant species varied between 77.6 and 73.3%; peptide length was between 226 and 230 amino acid residues, 43 of which are invariant in all seven subunit E-polypeptides known so far from animals, fungi and plants. The deduced relative molecular mass of subunit E in Mesembryanthemum crystallinum is 26162 Da. Subunit E is present both in C3- and CAM-plants. mRNA levels increased severalfold in leaves of CAM-induced plants. This was accompanied by a less pronounced increase in subunit E protein. Obviously, expression is stimulated under conditions of increased requirement for tonoplast H(+)-pumping activity.

Amino Acid Sequence↗
PubMed1992

Primary Structure of Cytochrome b(5) from Cauliflower (Brassica oleracea L.) Deduced from Peptide and cDNA Sequences.

Cytochrome b(5) is a microsomal protein that functions as an intermediate electron donor in fatty acid desaturation and other oxidation/reduction reactions. cDNA clones were isolated from cauliflower (Brassica oleracea L.) by using oligonucleotides based on the partial amino acid sequence of the protein. The deduced amino acid sequence of the polypeptide exhibited approximately 30% sequence identity with the homologous protein from vertebrates.

Journal Article↗