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At least 991 records · Page 55Linked to original sources
PubMed1991

Molecular cloning and nucleotide sequence of cDNA encoding the human liver S-adenosylmethionine synthetase.

cDNA clone for human liver S-adenosylmethionine synthetase (liver-specific isoenzyme) was isolated from a cDNA library of human liver poly(A)+ RNA. The cDNA sequence encoded a polypeptide consisting of 395 amino acid residues with a calculated molecular mass of 43675 Da. Alignment of the predicted amino acid sequence of this protein with that of rat liver S-adenosylmethionine synthetase showed a high degree of similarity. The coding region of the human liver S-adenosylmethionine synthetase cDNA sequence was 89% identical at the nucleotide level and 95% identical at the amino acid level to the sequence for rat liver S-adenosylmethionine synthetase.

Amino Acid Sequence↗
PubMed1999

Identification of betaIII- and betaIV-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): antibody mapping and cDNA sequencing.

Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptation we have further characterized cod tubulins using alpha- and beta-tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain beta-tubulin isotype has been sequenced. In mammals there are five beta-tubulins (betaI, betaII, betaIII, betaIVa and betaIVb) expressed in brain. A cod betaIII-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The betaIII-like tubulin accounted for more than 30% of total brain beta-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated beta(x), which was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was identified as a betaIV-tubulin, and a betaIV-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubulin. Based on the estimated amounts of betaIII- and betaIV-tubulins in cod brain, our results indicate that cod has at least one additional beta-tubulin isotype and that beta-tubulin diversity evolved early during fish evolution. The sequenced cod betaIV-tubulin had four unique amino acid substitutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.

Adaptation, Physiological↗
PubMed1994

Characterization of one novel venom protease with beta-fibrinogenase activity from the Taiwan habu (Trimeresurus mucrosquamatus): purification and cDNA sequence analysis.

Several fibrinogenolytic proteases were isolated from the venom of Taiwan habu, Trimeresurus mucrosquamatus, a snake species belonging to the Crotalidae family. One protease with strong fibrinogenolytic activity was further purified to homogeneity through multiple-step chromatographies including ion-exchange chromatography, gel permeation and reversed-phase HPLC. In vitro, the purified enzyme cleaved beta-chain of fibrinogen molecules efficiently and showed relatively lower activity on alpha-chain, with almost no activity on gamma-chain even after a long period of incubation. Further characterization indicated that it is a single-chain polypeptide with molecular weight of about 28,000. Its stability at high temperatures (> 90 degrees C) distinguished it from the previously reported venom fibrinogenases. N-Terminal sequence analysis revealed that it is similar to batroxobin and ancrod, which were shown to possess either fibrinogen-clotting or antithrombotic effect. Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of fresh venom glands of the same snake species to facilitate the cloning and sequencing of this important fibrinogenase. Sequencing several positive clones corresponding to the coding sequence of the enzyme revealed the existence of a family of novel thrombin-like fibrinogenases in the Taiwan habu, which are heat-stable and may be useful as strong antithrombotic agents.

Amino Acid Sequence↗
PubMed1996

cDNA sequence analysis and expression of cardiotoxins from Taiwan Cobra.

The cDNAs encoding cardiotoxins I, III and N were constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription-polymerase chain reaction. A high degree of sequence homology was observed with the three cardiotoxins. The cardiotoxins were subcloned in the expression vector pET 20b(+) and transformed in BL 21(DE3) E. coli strain. The expressed protein was isolated from the inclusion bodies of E. coli and purified by reversed phase high performance liquid chromatography. The purified recombinant cardiotoxin III showed an immunoreactivity with anti-cardiotoxin III antibodies as revealed by immunoblot analysis.

Amino Acid Sequence↗
PubMed1999

Porcine guanylin and uroguanylin: cDNA sequences, deduced amino acid sequences, and biological activity of the chemically synthesized peptides.

Guanylin and uroguanylin are structurally related intestinal peptide hormones which were purified from a limited number of mammals and are capable of activating the particulate guanylate cyclase-C. Although the biological functions of guanylin and uroguanylin are not yet clarified in detail, they are involved in the regulation of the intestinal water and electrolyte balance. In order to verify the general importance of this hormone system in mammals, we cloned the corresponding cDNAs from pig. Here, we present the nucleotide sequences and the deduced amino acid sequences representing porcine guanylin and uroguanylin. The expression patterns of the corresponding genes, as shown by Northern hybridization and RT-PCR analysis, resemble those of the human homologues. Further, we demonstrate the bioactivity of both porcine peptide hormones by inducing the intracellular cGMP production in human T84 cells and by ion transport experiments using porcine intestinal mucosa in the Ussing chamber.

Amino Acid Sequence↗
PubMed2000

Structural organization of the human glutathione reductase gene: determination of correct cDNA sequence and identification of a mitochondrial leader sequence.

The primary structure of human glutathione reductase gene (GSR) was determined by genomic cloning. The gene structure of human GSR spans 50 kb, consists of 13 exons, and was found to be highly similar to the mouse GSR gene. The coding sequence of human GSR resides on all 13 exons. An N-terminal arginine-rich mitochondrial leader sequence was present, with high homology to the murine leader sequence, between two in-frame start codons in the first exon. The 5' and 3' intron/exon splice junctions, with one exception, followed the general consensus sequences for intron spliced donor and acceptance sites.

Animals↗
PubMed2000

The complete cDNA sequences of IL-2, IL-4, IL-6 AND IL-10 from the European rabbit (Oryctolagus cuniculus).

The cDNAs for four rabbit cytokine genes [interleukin 2 (IL-2), IL-4, IL-6 and IL-10] have been cloned from primary lymphocytes by polymerase chain reaction (PCR) methods. IL-2 and IL-10 are both highly conserved between rabbit and other species. IL-4 and IL-6 are less strongly conserved, at both nucleotide and amino acid levels, and exhibit structural differences. An extension of the coding region of rabbit IL-6 relative to all other reported IL-6 genes results from a mutation in the usual stop codon which allows translation to continue for a further 27 amino acids. Analysis of IL-6 from four other lagomorph species suggests that this mutation is specific to the European rabbit. Sequence and structural differences of IL-4 and IL-6, while presumably not altering function, may render them highly species-specific. Several alternatively spliced variants of IL-2 and IL-4 are also reported.

Amino Acid Sequence↗
PubMed2000

A group of expressed cDNA sequences from the wheat fungal leaf blotch pathogen, Mycosphaerella graminicola (Septoria tritici).

A group of expressed sequence tags (ESTs) from the wheat fungal pathogen Mycosphaerella graminicola utilizing ammonium as a nitrogen source has been analyzed. Single pass sequences of complementary DNAs from 986 clones were determined. Contig analysis and sequence comparisons allowed 704 unique ESTs (unigenes) to be identified, of which 148 appeared as multiple copies. Searches of the nrdb95 protein database at EMBL using the BLAST2x algorithm revealed 407 (57.8%) sequences that generated high to moderate high scoring pairs with proteins of known and unknown function. The rest of the sequences (297) showed either weak or no similarities to database entries. Among the unigenes with assigned function, 26.7% were involved in primary metabolism and 17.9% were associated with protein and RNA metabolism. Fewer clones were ascribed roles in signal transduction (4.9%), transport and secretion (6.1%), cell structure (3.1%), and cell division (3.6%). Approximately 18.1% of the identities found were to hypothetical or unknown proteins mainly from the yeasts Saccharomyces cerevisiae and Schizosaccaromyces pombe. Comparison of the 297 sequences with no clear function to other fungal ESTs in the public domain revealed 12 sequences that had high to moderate similarity to Neurospora crassa, Emericella (Aspergillus) nidulans, or Magnaporthe grisea sequences.

Ascomycota↗
PubMed1994

The HNF-3 gene family of transcription factors in mice: gene structure, cDNA sequence, and mRNA distribution.

The rat HNF-3 (hepatocyte nuclear factor 3) gene family encodes three transcription factors known to be important in the regulation of gene expression in liver and lung. We have cloned and characterized the mouse genes and cDNAs for HNF-3 alpha, beta, and gamma and analyzed their expression patterns in various adult tissues and mouse embryonic stages. The HNF-3 proteins are highly conserved between mouse and rat, with the exception of the amino terminus of HNF-3 gamma, which in mouse is more similar to those of HNF-3 alpha and beta than to the amino termini of the rat HNF-3 gamma protein. The mouse HNF-3 genes are small and contain only two or three (HNF-3 beta) exons with conserved intron-exon boundaries. The proximal promoter of the mouse HNF-3 beta gene is remarkably similar to that of the previously cloned rat HNF-3 beta gene, but is different from the promoters of the HNF-3 alpha and gamma genes. The mRNA distribution of the mouse HNF-3 genes was analyzed by quantitative RNase protection with gene-specific probes. While HNF-3 alpha and beta are restricted mainly to endoderm-derived tissues (lung, liver, stomach, and small intestine), HNF-3 gamma is more extensively expressed, being present additionally in ovary, testis, heart, and adipose tissue, but missing from lung. Transcripts for HNF-3 beta and alpha are detected most abundantly in midgestation embryos (Day 9.5), while HNF-3 gamma expression peaks around Day 15.5 of gestation.

Amino Acid Sequence↗
PubMed1996

cDNA sequence, map, and expression of the murine homolog of GTBP, a DNA mismatch repair gene.

DNA mismatch repair genes (MMR) have been actively studied in humans for their role in cancer susceptibility. Recently, GTBP, a new member of the MMR gene family, has been cloned and found to encode a 160-kDa protein that forms a heterodimer with hMSH2. Our group has isolated and characterized the mouse homolog of GTBP. The transcript, termed Gtmbp (G/T mismatch-binding protein), encodes a protein highly similar to its human counterpart. The gene is markedly conserved across species and maps to the distal portion of mouse chromosome 17, in a region homologous to human 2p. One Gtmbp-related sequence maps to proximal mouse chromosome 4. Studies of the expression of Gtmbp suggest that its regulated transcription is tightly linked to the level of DNA replication, consistent with the protein's DNA proofreading function.

Animals↗
PubMed1997

Cloning and mapping of human Rab7 and Rab9 cDNA sequences and identification of a Rab9 pseudogene.

Rab GTPases reside in specific intracellular compartments and are key regulators of vesicular transport. To facilitate studies of the mechanism of lysosomal integral membrane protein (LAMP-1) transport, cDNAs for human Rab7 and Rab9 were isolated, and their nucleotide sequences were determined. During isolation and characterization of these cDNAs a Rab9 pseudogene was identified. The sequences are highly homologous to other mammalian Rab proteins and also share homology with proteins of the Rab GTPase family. Rab7 and the Rab9 pseudogene were mapped to chromosomes 3 and 5, respectively, by amplification of their sequences from human monochromosomal somatic cell hybrids. In addition, preliminary studies using antisense expression indicate that down-regulation of either Rab7 or Rab9 proteins induces severe cell vacuolation that resembles the phenotype seen in fibroblasts from patients with Chediak-Higashi syndrome.

Animals↗
PubMed1999

Human peroxisome proliferator activated receptor gamma coactivator 1 (PPARGC1) gene: cDNA sequence, genomic organization, chromosomal localization, and tissue expression.

Brown adipose and muscle tissues can increase energy expenditure via adaptive thermogenesis, thereby protecting against obesity. Mouse peroxisome proliferator activated receptor gamma coactivator 1 (Pgc1) has been reported to enhance the expression of uncoupling protein-1, a key mediator of thermogenesis in brown adipose tissue (Puigserver et al., 1998, Cell 92, 829-839). We report here the characterization of the human PPARGC1 gene. PPARGC1 spans a genomic region of approximately 67 kb, is composed of 13 exons, and encodes a 91-kDa protein that exhibits 94% amino acid identity with the mouse ortholog. mRNA species, transcribed from the TATA-less promoter, are 6.4 and 5.3 kb in length due to utilization of two polyadenylation signals. Northern blotting revealed expression of both transcripts in heart, skeletal muscle, and kidney and to a lesser extent in liver, brain, and pancreas as well as in the perirenal adipose tissue of a pheochromocytoma patient. PPARGC1 was mapped to chromosome 4p15.1, a region that has been associated with basal insulin levels in Pima Indians. Hence, PPARGC1 expression might influence insulin sensitivity as well as energy expenditure, thereby contributing to the development and pathophysiology of human obesity.

Amino Acid Sequence↗
PubMed2000

Four genes encode acetylcholinesterases in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. cDNA sequences, genomic structures, mutations and in vivo expression.

We report the full coding sequences and the genomic organization of the four genes encoding acetylcholinesterase (AChE) in Caenorhabditis elegans and Caenorhabditis briggsae, in relation to the properties of the encoded enzymes. ace-1 and ace-2, located on chromosome X and I, respectively, encode two AChEs (ACE-1 and ACE-2) that present 35% identity. The C-terminal end of ACE-1 is homologous to the C terminus of T subunits of vertebrate AChEs. ACE-1 oligomerizes into amphiphilic tetramers. ACE-2 has a hydrophobic C terminus of H type. It associates into glycolipid-anchored dimers. In C. elegans and C. briggsae, ace-3 and ace-4 are organized in tandem on chromosome II, with only 356 nt and 369 nt, respectively, between the stop codon of ace-4 (upstream gene) and the ATG of ace-3. ace-3 produces only 5 % of the total AChE activity. It encodes an H subunit that associates into dimers of glycolipid-anchored catalytic subunits, which are highly resistant to the usual AChE inhibitors, and which hydrolyze butyrylthiocholine faster than acetylthiocholine. ACE-4 is closer to ACE-3 (54 % identity) than to ACE-1 or ACE-2. The usual sequence FGESAG surrounding the active serine residue in cholinesterases is changed to FGQSAG in ace-4. ACE-4 was not detected by our current biochemical methods, although the gene is transcribed in vivo. However the level of ace-4 mRNAs is far lower than those of ace-1, ace-2 and ace-3. The ace-2, ace-3 and ace-4 transcripts were found to be trans-spliced by both SL1 and SL2, although these genes are not included in typical operons. The molecular bases of null mutations g72 (ace-2), p1304 and dc2 (ace-3) have been identified.

Acetylcholinesterase↗
PubMed1999

cDNA sequence and characterization of the gene that encodes human myotrophin/V-1 protein, a mediator of cardiac hypertrophy.

The predominant response of the heart to sustained increased work load is development of ventricular hypertrophy, principally as a result of hypertrophy of cardiomyocytes. The molecular mechanisms and factors involved in cardiomyocyte hypertrophy are poorly understood. Myotrophin is a novel 12-kilodalton protein recently implicated as a factor associated with and able to induce cardiac hypertrophy. Cloning of rat myotrophin revealed that this protein is identical to the functionally undefined rat, murine and chicken V-1 proteins. Although human myotrophin has been purified to homogeneity, its gene has not been characterized. In this report we describe the cloning, expression, purification and characterization of the human homolog of myotrophin/V-1 protein. Sequence analysis indicators high homology (>90%) between all species at both the nucleotide and amino acid levels, and Southern blot analysis of genomic DNA from diverse species verifies that myotrophin/V-1 is a highly conserved gene. Northern analysis indicates wide-spread expression of a single human transcript, and examination of mRNA distribution in 50 human tissues by dot blot analysis indicates ubiquitous expression with relatively high expressioon in adult and fetal heart. We verify that recombinant human myotrophin produces cardiomyocyte hypertrophy, and we demonstrate for the first time that elevated levels of myotrophin/V-1 protein mRNA are expressed in human dilated cardiomyopathic hearts. We report the novel findings that myotrophin expression is elevated in ischemic hearts, and that myotrophin expression correlates positively with ventricular mass in a hypoxic rat model of induced right ventricular hypertrophy.

Amino Acid Sequence↗