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 775 records · Page 43Linked to original sources
PubMed1993

Purification and cDNA sequence of an inducible nitric oxide synthase from a human tumor cell line.

A combination of cytokines induced the expression of nitric oxide synthase (NOS) in a human colorectal adenocarcinoma cell line, DLD-1. We have purified the enzyme and examined some of its biochemical properties. An antiserum to an inducible NOS from murine macrophages cross-reacted with the DLD-1 NOS. The purified human and murine enzymes displayed a similar lack of dependence on exogenous calcium and calmodulin for activity, which contrasts with the requirement for calcium and calmodulin of purified brain and endothelial isoforms of NOS. We have also isolated a cDNA for a cytokine-induced NOS from DLD-1 cells. Sequence analysis of this cDNA and NOS cDNAs from human liver, smooth muscle, and macrophages suggests that, at the genetic level, there is a single isoform of human-inducible NOS.

Adenocarcinoma↗
PubMed1994

Alcohol dehydrogenase of class IV (sigma sigma-ADH) from human stomach. cDNA sequence and structure/function relationships.

Human stomach mucosa contains a characteristic alcohol dehydrogenase (ADH) enzyme, sigma sigma-ADH. Its cDNA has been cloned from a human stomach library and sequenced. The deduced amino acid sequence shows 59-70% identities with the other human ADH classes, demonstrating that the stomach enzyme represents a distinct structure, constituting class IV, coded by a separate gene, ADH7. The amino acid identity with the rat stomach class IV ADH is 88%, which is intermediate between constant and variable dehydrogenases. This value reflects higher conservation than for the classical liver enzymes of class I, compatible with a separate functional significance of the class IV enzyme. Its enzymic features can be correlated with its structural characteristics. The residues lining the substrate-binding cleft are bulky and hydrophobic, similar to those of the class I enzyme; this explains the similar specificity of both classes, compatible with the origin of class IV from class I. Position 47 has Arg, in contrast to Gly in the rat class IV enzyme, but this Arg is still associated with an extremely high activity (kcat = 1510 min-1) and weak coenzyme binding (KiaNAD+ = 1.6 mM). Thus, the strong interaction with coenzyme imposed by Arg47 in class I is probably compensated for in class IV by changes that may negatively affect coenzyme binding: Glu230, His271, Asn260, Asn261, Asn363. The still higher activity and weaker coenzyme binding of rat class IV (kcat = 2600 min-1, KiaNAD = 4 mM) can be correlated to the exchanges to Gly47, Gln230 and Tyr363. An important change at position 294, with Val in human and Ala in rat class IV, is probably responsible for the dramatic difference in Km values for ethanol between human (37 mM) and rat (2.4 M) class IV enzymes.

Alcohol Dehydrogenase↗
PubMed1988

Human hepatic lipase. Cloned cDNA sequence, restriction fragment length polymorphisms, chromosomal localization, and evolutionary relationships with lipoprotein lipase and pancreatic lipase.

Human hepatic lipase is an important enzyme in high density lipoprotein (HDL) metabolism, being implicated in the conversion of HDL2 to HDL3. Three human hepatic lipase cDNA clones were identified in two lambda gt11 libraries from human liver. The cDNA-derived amino acid sequence predicts a protein of 476 amino acid residues, preceded by a 23-residue signal peptide. Four potential N-glycosylation sites are identified, two of which are conserved in rat hepatic lipase. On alignment with human, mouse, and bovine lipoprotein lipase, the same two sites were also conserved in lipoprotein lipase in all three species. Stringent conservation of the cysteine residues was also evident. Comparative analysis of amino acid sequences shows that hepatic lipase evolves at a rapid rate, 2.07 x 10(-9) substitutions/site/year, about four times that in lipoprotein lipase and half that in pancreatic lipase. Further, hepatic lipase and pancreatic lipase appear to be evolutionarily closer to each other than either of them is to lipoprotein lipase. Southern blot analysis revealed high frequency restriction fragment length polymorphisms of the hepatic lipase gene for the enzymes HindIII and MspI. these polymorphisms will be useful for haplotype and linkage analysis of the hepatic lipase gene. Using cloned human hepatic lipase cDNA as a hybridization probe, we performed Southern blot analysis of a panel of 13 human-rodent somatic cell hybrids. Concordance analysis of the various hybrid clones indicates that the hepatic lipase gene is located on the long arm of human chromosome 15. Analysis of hybrids containing different translocations of chromosome 15 localized the gene to the region 15q15----q22.

Amino Acids↗
PubMed1993

SKALP/elafin: an elastase inhibitor from cultured human keratinocytes. Purification, cDNA sequence, and evidence for transglutaminase cross-linking.

SKALP/Elafin is a proteinase inhibitor found in psoriatic epidermis as a short polypeptide of 6 kDa. Here we present evidence that this protein is synthesized as a larger precursor molecule with distinct biological features. Purification and NH2-terminal sequencing of SKALP/elafin from cultured human keratinocytes and the cloning of its cDNA revealed the existence of a mature protein, which upon cleavage of a hydrophobic signal sequence of 22 amino acids has a calculated molecular mass of 9.9 kDa (95 amino acids). In addition to the known proteinase inhibitor domain, the mature protein contains a domain with 4 repeats which are homologous to putative transglutaminase substrate motifs. We were able to demonstrate on Western blots that immunoreactive SKALP is present in high molecular weight proteins extracted from psoriatic skin. This suggests that SKALP is covalently attached to epidermal proteins. In addition it was found that both the complete SKALP molecule and a synthetic peptide of the NH2-terminal portion of SKALP could be used as a transglutaminase substrate. We therefore speculate that SKALP/elafin, secreted by epidermal keratinocytes in inflamed skin, exists both as a free 6-kDa form and as an immobilized 9.9-kDa form covalently attached to the cornified envelopes by transglutaminase cross-linking.

Amino Acid Sequence↗
PubMed1993

cDNA sequence and localization of polymorphic human cytosolic phosphoenolpyruvate carboxykinase gene (PCK1) to chromosome 20, band q13.31: PCK1 is not tightly linked to maturity-onset diabetes of the young.

Complementary DNA clones encoding human cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) (PEPCK)] were isolated from a human kidney cDNA library. The nucleotide sequence of the 2.7 kb insert of one of these clones indicates that human PEPCK is a protein of 622 amino acids whose sequence shows 90% identity with that of the cognate rat enzyme. The human PEPCK gene (PCK1) was isolated by hybridization using a fragment of the hPEPCK cDNA as a probe. PCK1 was mapped to human chromosome 20 using DNA from a panel of reduced human-hamster somatic cell hybrids. This assignment was confirmed using fluorescence in situ chromosomal hybridization which localized PCK1 to chromosome 20, band q13.31. A simple tandem repeat DNA polymorphism in the 3'-untranslated region of the mRNA was characterized and used to localize PCK1 relative to the gene responsible for a form of non-insulin-dependent (Type 2) diabetes mellitus called maturity-onset diabetes of the young (MODY). Linkage studies showed that PCK1 is not tightly linked to MODY in one large pedigree and exclude this diabetes candidate gene as the cause of MODY in this family.

Alleles↗
PubMed1997

Isolation of a rat histidase cDNA sequence and expression in Escherichia coli--evidence of extrahepatic/epidermal distribution.

Histidase (histidine ammonia-lyase) is a cytosolic enzyme responsible for catalyzing the non-oxidative deamination of histidine to urocanic acid. Full-length cDNAs encoding rat histidase have been isolated from a lambdaZAP liver cDNA library using a partial cDNA fragment obtained by PCR. Whereas the initial description of the rat histidase 3' untranslated sequence contained a rare polyadenylation signal sequence, the data presented encompass a more distant 28-bp region, possessing a nucleotide stretch (AATATAAA), identical to that in the mouse histidase cDNA. Dideoxynucleotide chain-termination sequencing of two clones obtained by in vivo excision yielded an additional 376 bp and 105 bp of 5' and 3' untranslated sequences, respectively. A selected rat histidase cDNA clone was introduced into the pET-16b prokaryotic vector and expressed in BL21(DE3)pLysS Escherichia coli. After purification by nickel-chelation chromatography, recombinant histidine-tagged protein was employed to raise anti-(rat histidase) immunoglobulin in a Japanese white rabbit. The polyclonal rabbit antibody recognized and formed immune complexes with rat and recombinant human histidase proteins. Immunoblots of crude rat organ extracts detected a spectrum of histidase expression extending beyond that observed in liver and skin. Among other histidase-positive cells were those of the renal cortex tubular epithelium, fundic mucosal glands of stomach, gastric intramuscular (Auerbach's) plexus, and adrenal cortex. Immunohistochemical studies of histidase in rat liver produced discrete staining of hepatocytes in association with portal triads (Rappaport zone I). Furthermore, in contrast with previous reports of activity confined to epidermal stratum corneum, our findings demonstrate immunoreactive protein within and limited to the adjacent stratum granulosum.

Amino Acid Sequence↗
PubMedDate not supplied

cDNA sequence and organization of the immunoglobulin light chain gene of the duck, Anas platyrhynchos.

A cDNA was cloned which encoded an immunoglobulin (Ig) light (L) chain of the White Pekin duck. The organization of the variable (V) and constant (C) domains was analyzed by genomic Southern blotting. The duck L chain gene has a similar chromosomal organization to that of the chicken, with a single lambda-like C region and multiple VL, hybridizing elements. The amino acid sequence of the VL region of the White Pekin duck L chain showed 88% identity with the Muscovy duck and 87% identity with the chicken, the JL region showed 92% identity with these species, and the CL region showed 88% identity with Muscovy duck and 66% with chicken. The constraints imposed by the gene-conversion mechanism of generating antibody diversity might account for the similarities of the avian V region sequences.

Amino Acid Sequence↗
PubMed1992

cDNA sequence of the pregnancy-specific beta 1-glycoprotein-11s (PSG-11s).

Four cDNA clones representing the human pregnancy-specific beta 1-glycoprotein-11 (PSG-11) gene have been characterised. All encoded a splice variant of the PSG-11 gene designated PSG-11s, which can encode a secreted protein of 426 amino acids, containing six potential N-linked glycosylation sites, with a domain structure L-N-AI-AII-BII-C. Minor differences between the four clones sequenced included a restriction site polymorphic for ApaI that may differentiate between alleles of the PSG-11 gene.

Amino Acid Sequence↗
PubMed1994

cDNA sequence and tissue-specific expression of an anionic flax peroxidase.

A flax (Linum usitatissimum L.) lambda gt10 cDNA library was screened with a probe coding for the amino terminus of a flax peroxidase. The probe was generated by PCR amplification of the library with one of the lambda gt10 sequencing primers and a mixed oligonucleotide derived from a well-conserved amino acid region (distal heme ligand) found in all plant peroxidases. A positive clone (FLXPER2) was isolated and characterized. The cDNA contains 1153 nucleotides, excluding the poly(A) tail, and encodes a mature protein of 297 amino acids with a molecular mass of 31.9 kDa. The mature protein's amino acid sequence contains three highly conserved regions, two of which contain histidine ligands for the heme group. The deduced amino acid sequence has nine cysteine residues. Eight are identically located to those of horseradish C peroxidase, which participate in four disulfide bridges; these cysteines are highly conserved in all plant peroxidases. One potential N-glycosylation site (Asn-X-Thr/Ser) is present. The predicted pI value of 4.5 identifies FLXPER2 as an anionic peroxidase. Northern blot analysis shows that its mRNA expression is unique to stem tissue. Amino acid sequence comparisons show high similarity between FLXPER2 and peanut, rice, and tobacco peroxidases.

Amino Acid Sequence↗
PubMed1991

Evidence for different human tracheobronchial mucin peptides deduced from nucleotide cDNA sequences.

Highly glycosylated regions or glycopeptides were obtained by proteolysis of human tracheobronchial mucins. They were chemically deglycosylated and the resulting products were used to raise a rabbit antiserum. This antiserum specifically recognized the superanuclear region of respiratory and colonic goblet cells as areas around and below the nucleus of mucin-secreting cells in tracheobronchial mucous glands. A lambda gt11 cDNA library constructed from human tracheobronchial mucosa was screened with this antiserum. Ten positive clones were obtained from screening half of the library (about 10(6) recombinants). The antibodies were purified by absorption to each positive clone; some purified antibodies were specific for goblet cells and others recognized both goblet and mucous cells, indicating that there is differential cellular expression of mucin peptides. The total or partial amino acid sequences deduced from these cDNA clones could be classified into three groups. The first group contained repetitive sequences of eight amino acid residues, almost perfectly identical, and in different arrangements. The second type exhibited homology at their amino and carboxy-terminal ends. The last group had no distinctive feature except for a high content of hydroxy amino acids typical of mucins. Five different clones could correspond to the carboxy-terminal end of tracheobronchial apomucins. These results indicate that human tracheobronchial apomucins consist of a family of different proteins.

Amino Acid Sequence↗
PubMed1988

The cDNA sequence coding for prepro-PGS (prepro-magainins) and aspects of the processing of this prepro-polypeptide.

Amphibian skin is well known as a source of peptides homologous to bioactive peptides found in mammalian gut and brain. A systematic investigation of the skin secretions from Xenopus laevis revealed several peptides not derivable from known precursors. The sequence elucidation, utilizing fast atom bombardment/mass spectrometry, of two peptides, PGS and PGS Gly-10;Lys-22, is reported. These have been independently characterized and named magainins and found to have antimicrobial activity. A mixed sequence oligonucleotide probe complementary to the mRNA sequence coding for PGS was synthesized and used to screen a Xenopus skin cDNA library. A full length cDNA species encoding prepro-PGS was isolated and characterized, and its sequence is reported. The deduced precursor sequence was found to contain one copy of PGS Gly-10;Lys-22 and five copies of PGS. The proteolytic processing of this prepro-polypeptide is discussed.

Amino Acid Sequence↗
PubMed1992

Human inter-alpha-trypsin inhibitor: full-length cDNA sequence of the heavy chain H1.

Inter-alpha-trypsin inhibitor (ITI), called inter-alpha-inhibitor, is a 220 kDa serine proteinase inhibitor found in human serum. It is composed of at least three distinct polypeptide chains. These chains, named H1, H2 and L, are an independently synthesized and proteolytically processed precursor protein. Only the complete structure of H2 and L has been established so far. We used a PCR-based cloning approach and a cDNA screening library to isolate the full-length cDNA H1. The amino acid sequences of the two heavy chains deduced from the cDNA are highly similar (40% identity). Nevertheless, the structure of the signal peptide and propeptide in the N-terminal region is different in these two chains. A complex posttranslational cleavage at both ends of H1 and H2 may be proposed prior to assembly of the ITI chains.

Alpha-Globulins↗
PubMed1994

The c-erbA beta thyroid hormone receptor. Expression and cDNA sequence analysis of the hormone-binding domain in human cancer cell lines.

The human c-erbA beta protooncogene encodes a thyroid hormone receptor (comprising a hormone-binding domain and a DNA-binding domain) which modulates expression of specific genes, such as cell differentiation genes. Using the reverse transcription and polymerase chain reaction (RT-PCR) assay, significant expression of the c-erbA beta gene was detected in the SiHa, CaSki, HeLa cervical carcinoma; Hep3B, PLC/PRF/5, Mahlavu hepatocellular carcinoma; HT-1080 fibrosarcoma cell lines; as well as in normal MRC-5 embryo lung and FS-4 foreskin fibroblast cell lines. However, the Molt-4 leukaemia and Raji Burkitt's lymphoma cell lines exhibited very low levels of c-erbA beta expression. Single-strand conformation polymorphism analysis and direct sequencing of PCR products of the c-erbA beta hormone-binding domain cDNAs of these cell lines revealed identical sequences, but differed from the published human placental c-erbA beta sequence by five single base disparities. Sequencing of an aberrant fragment fortuitously amplified from the HT-1080 cDNA library demonstrated concordance with the cDNA of pregnancy-specific glycoprotein 4, which is related to the tumour marker, carcinoembryonic antigen.

Base Sequence↗
PubMed2001

Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses.

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.

Alkaline Phosphatase↗
PubMed1989

Expression, cloning and cDNA sequence of a fibroblast serum-regulated gene encoding a putative actin-associated protein (p27).

A cDNA clone for a basic putative actin microfilament-associated protein, p27, highly induced in serum-stimulated NIH 3T3 cells, has been isolated by polyclonal antibodies and sequenced. p27 mRNA is a 1.2-kb molecule which is very low in resting NIH 3T3 cells but can be induced at least 100 times after 8 h of fetal calf serum stimulation. In contrast to other inducible mRNAs, p27 mRNA is stable, and its levels can be superinduced by cycloheximide mainly by prolonging transcription. The lack of expression of this messenger in mouse tissues, as well as in all cell lines so far tested, suggests that p27 may be an fibroblast-specific protein. One major open reading frame found in p27 cDNA codes for a 201 amino acid polypeptide not related to any previously described actin-binding protein. Interestingly, it shows alternative hydrophilic and hydrophobic domains of amino acids symmetrically arranged from the middle of the protein. The coordinate induction of p27 and actin mRNAs suggest that p27 may be involved in the cytoskeletal rearrangements induced early in cell growth and proliferation.

Amino Acid Sequence↗
PubMed1991

Demonstration of apolipoprotein CII in guinea pigs. Functional characteristics, cDNA sequence, and tissue expression.

In contrast to plasma from other mammals, guinea pig plasma does not stimulate the activity of lipoprotein lipases in vitro. This had led previously to the conclusion that guinea pigs lack an analogue to apolipoprotein CII (apoCII). By adsorption of lipid-binding proteins to lipid droplets, thereby separating them from other plasma components, we could demonstrate apoCII-like activity in guinea pig plasma. On electrophoresis, the CII-like activity co-migrated with one isoform of guinea pig apolipoprotein CIII, identified by amino-terminal amino acid sequence determination (40 residues). By isoelectric focusing in a narrow pH gradient, the activating protein was separated sufficiently from the dominating apoCIII isoform to allow sequence determination of 8 residues from the amino terminus. Six of these were identical to corresponding residues in apoCII from dog and monkey. With the aid of a human apoCII cDNA probe we identified one cross-hybridizing mRNA species (approximately 600 nucleotides) on Northern blots of guinea pig liver. Three positive clones were isolated from a guinea pig liver cDNA library using the same cDNA probe. The nucleotide sequence showed extensive similarities to the previously known human, monkey, and canine sequences, but the signal peptide was 3 amino acid residues longer in the guinea pig protein, and there was a deletion of 4 residues in the putative lipid binding domain. Northern blot analyses indicated that guinea pig apoCII is mainly expressed in the liver with little or no contribution from the intestine.

Adsorption↗
PubMed1999

RasGRP, a Ras activator: mouse and human cDNA sequences and chromosomal positions.

We recently reported the molecular cloning of a novel transforming rat brain cDNA, rbc7, that encodes a Ras activator (Ebinu et al. Science 280, p. 1082, 1998). We proposed that this cDNA is a 5' and 3' truncated version of a larger normal transcript that encodes a predicted 90-kDa protein which we called RasGRP (Ras guanyl nucleotide releasing protein). We have now studied the structure of the mouse and human sequences and confirmed our conclusions about the nature of the 5' truncation. The human gene has been localized to 15q15 by an in situ hybridization technique, while the mouse gene has been positioned on Chr 2 near thrombospondin by linkage analysis. The relatedness of RasGRP to another human sequence and a hypothetical nematode protein are also discussed.

Amino Acid Sequence↗