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PubMed1994

Cloning and characterization of human phosphatase inhibitor-2 (IPP-2) sequences.

cDNA clones similar to rabbit muscle phosphatase inhibitor-2 (IPP-2) were isolated from human libraries. On Northern blots two transcripts of approximately 2kbp and approximately 4kbp were detected in all tissues tested. Analysis of cDNA sequences showed that the longer transcripts were similar to the shorter clones but contained extended 3' ends. The human nucleotide sequence was highly homologous (94% identity) to the rabbit IPP-2 sequence and encoded a peptide of 205 amino acids. IPP-2 sequences were highly conserved throughout vertebrates. Southern hybridization results were consistent with the existence of a family of related IPP-2 sequences in the human genome. Most of these are likely to be pseudogenes, since all of the cDNA clones examined could have originated from a single gene. By in situ hybridization IPP-2 sequences were mapped to several different human chromosomes. We sequenced one gene located in the major histocompatibility complex (MHC) on Chromosome (Chr) 6 that contained the entire coding region of IPP-2.

Amino Acid Sequence↗
PubMed1997

Immunoreactive growth hormone in human peripheral T lymphocytes: encoding sequence of cDNA identical to that of the pituitary human growth hormone.

OBJECTIVE: To detect the cDNA sequence of immunoreactive growth hormone (irGH) in human lymphocytes and to compare it with that of human growth hormone (hGH) in the pituitary. METHODS: The mRNA of irGH in human peripheral blood mononuclear cells (PBMC) was detected by Northern blot analysis. The cDNA of irGH was obtained by reverse-transcriptase and polymerase chain reaction amplification and obtained by screening from a cDNA library of human T lymphocytes. The sequence of irGH cDNA was determined and compared with that of the pituitary hGH. RESULTS: Very low level of irGH mRNA was detected in human PBMCs by Northern blot analysis. The cDNA sequence of the entire open reading frame of human irGH was 651 bp, which is identical to that of pituitary hGH. CONCLUSIONS: The same hGH molecule can be produced by human pituitary somatotrophic cells and human peripheral lymphocytes.

Amino Acid Sequence↗
PubMed1994

The delta-subunit of murine guanine nucleotide exchange factor eIF-2B. Characterization of cDNAs predicts isoforms differing at the amino-terminal end.

Protein synthesis in mammalian cells is regulated at the level of the guanine nucleotide exchange factor, eIF-2B, which catalyzes the exchange of eukaryotic initiation factor 2-bound GDP for GTP. We have isolated and sequenced cDNA clones encoding the delta-subunit of murine eIF-2B. The cDNA sequence encodes a polypeptide of 544 amino acids with molecular mass of 60 kDa. Antibodies against a synthetic polypeptide of 30 amino acids deduced from the cDNA sequence specifically react with the delta-subunit of mammalian eIF-2B. The cDNA-derived amino acid sequence shows significant homology with the yeast translational regulator Gcd2, supporting the hypothesis that Gcd2 may be the yeast homolog of the delta-subunit of mammalian eIF-2B. Primer extension studies and anchor polymerase chain reaction analysis were performed to determine the 5'-end of the transcript for the delta-subunit of eIF-2B. Results of these experiments demonstrate two different mRNAs for the delta-subunit of eIF-2B in murine cells. The isolation and characterization of two different full-length cDNAs also predicts the presence of two alternate forms of the delta-subunit of eIF-2B in murine cells. These differ at their amino-terminal end but have identical nucleotide sequences coding for amino acids 31-544.

Alternative Splicing↗
PubMed2001

Identification, cloning and characterisation of a novel copper-metallothionein in tetrahymena pigmentosa. Sequencing of cDNA and expression.

The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the induction of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducible MT-isoform isolated from Tetrahymena after exposure to a non-toxic dose of copper. The cDNA sequence was determined utilising the partial peptide sequence of purified protein. The Cu-MT cDNA encodes 96 amino acids containing 28 cysteine residues (29%) arranged in motifs characteristic of the metal-binding regions of vertebrate and invertebrate MTs. Both the amino acid and nucleotide sequences differ, not only from other animal MTs, but also from the previously characterised Tetrahymena Cd-MT. Both MTs contain the structural pattern GTXXXCKCXXCKC, which may be proposed as a conservative sequence of Tetrahymena MTs. Cu-dependent regulation of MT expression was also investigated by measuring MT-mRNA and MT levels. MT synthesis occurs very quickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological functions, are expressed according to the inducing metal, Cu or Cd.

Amino Acid Sequence↗
PubMed2005

Nucleotide sequence for cDNA of bovine mitochondrial ATP-dependent protease and determination of N-terminus of the mature enzyme from the adrenal cortex.

We have determined the cDNA sequence encoding bovine mitochondrial ATP-dependent Lon protease. Since the 5'-end region of the cDNA was highly GC-rich and thus could not be amplified by the 5'-RACE method, a genomic DNA fragment containing an in-frame ATG was isolated and sequenced. The translated amino acid sequence contained 961 amino acids with a calculated molecular weight 106,665. Sequence similarities of the bovine enzyme to human and E. coli orthologs were 92 and 27%, respectively. The N-terminal amino acid sequence seemed to be a mitochondrial targeting signal. To determine the cleavage site of the signal sequence we analyzed the mature enzyme purified from bovine adrenocortical mitochondria. Analysis of CNBr-digested peptides revealed that the N-terminus was heterogeneous. We suggest that nonspecific aminopeptidase might remove several amino acids from the N-terminus after mitochondrial processing peptidase has cleaved Gly(67)-Leu(68) or Leu(68)-Trp(69).

ATP-Dependent Proteases↗
PubMed1987

Molecular cloning and nucleotide sequence of cDNA for human liver arginase.

Arginase (EC 3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, we isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted Mr, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.

Amino Acid Sequence↗
PubMed1989

Human alpha-L-fucosidase: complete coding sequence from cDNA clones.

The human lysosomal storage disorder fucosidosis results from the deficiency of alpha-L-fucosidase, a lysosomal enzyme essential for the catabolism of oligosaccharides containing alpha-L-fucosides. cDNA clones coding for human alpha-L-fucosidase have been isolated from lambda gt10 and lambda gt11 cDNA libraries derived from human liver, placenta and colon. Compilation of cDNA sequences results in a nucleotide sequence of 2053 base pairs encoding alpha-L-fucosidase. The sequence contains an open reading frame of 461 amino acids beginning with the first in-frame methionine and includes 439 amino acids which comprise the mature protein in addition to a hydrophobic signal peptide sequence of 22 amino acids.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of cDNA coding for rat liver pI 6.1 esterase (ES-10), a carboxylesterase located in the lumen of the endoplasmic reticulum.

A commercial rat liver cDNA library in lambda gt11 was screened with a rabbit antiserum to native pI 6.1 esterase (ES-10). The inserts of the immunoreactive clones were short (0.9-1.1 kbp). One of these was used as a probe to rescreen the library, yielding 30 clones, two of which contained relatively long (approx. 1.9 kbp) and widely overlapping cDNA inserts. They did not contain the first two nucleotide residues of the initiator codon, nor the 5'-end untranslated portion of the mRNA. These were derived from a home-made rat liver cDNA library in lambda gt11, screened with an oligonucleotide corresponding to the 5'-end of the already known cDNA sequence. The nucleotide sequence consists of 48 bp of 5'-end non-coding region, 1695 bp of coding region and 212 bp of 3'-end non-coding region including a 20 bp poly(A) tail. The signal peptide and the mature protein subunit are 18 and 547 residues long respectively. Tyr is confirmed as N-terminal residue. The predicted amino acid sequence is highly similar to those of rabbit liver esterase forms 1 (77% identity) and 2 (56% identity), determined by protein sequencing [Korza & Ozols (1988) J. Biol. Chem. 263, 3486-3495; Ozols (1989) J. Biol. Chem. 264, 12533-12545]. The three enzymes share the Ser and His residues presumed to be part of the active site, four Cys residues and a high proportion of charged side chains at their C-terminus. The C-terminal tetrapeptides of the three esterases (-HVEL, -HIEL and -HTEL for pI 6.1 and forms 1 and 2 esterases respectively) are reminiscent of, but not identical with, the localization signal identified in other proteins of the endoplasmic-reticulum lumen (-KDEL in animal cells [Munro & Pelham (1987) Cell 48, 899-907]; -HDEL in yeast [Pelham, Hardwick & Lewis (1988) EMBO J. 7, 1757-1762]). We still lack direct evidence to decide whether or not these C-terminal tetrapeptides commit esterases to reside in the endoplasmic reticulum. In that case the antepenultimate residue (D, V, I or T) would be only weakly stringent, and some sequences primed by H instead of K would be recognized in animal as well as in yeast cells.

Amino Acid Sequence↗
PubMed1984

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor.

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.

Amino Acid Sequence↗
PubMed1986

[Nucleotide sequence of cDNA of glicinin A3 subunits: variability of different species detected by comparative analysis].

Several cDNA cloned which code for subfamily A3 subunits (A3B4 and A5A4B3) of soybean storage protein glycinin were analysed by means of restriction mapping and hybrid - selection. The comparison of A3B4 and A5A4B3 subunits cDNA sequences reveals the 90% homology. The nucleotide sequences obtained in the process of this work were compared with those reported about elsewhere, in order to study the interaspecific variability of homologous but not identical storage protein genes of subfamily A3 glycinin subunit. Nucleotide replacements were found to occur 6 times more frequently in A3B4 subunit gene, as compared to A5A4B3 subunit gene (48 replacements against 8).

Amino Acid Sequence↗
PubMed1992

Presence of three rice sucrose synthase genes as revealed by cloning and sequencing of cDNA.

By sequencing cDNA clones, we have concluded that three distinct sucrose genes are expressed in rice (Oryza sativa cv. Tainong 67). When the amino acid sequences deduced from these cDNAs as well as those of known sucrose synthase are compared, the highest divergence is found in the C-termini. The most suitable DNA sequences for use as specific for the mRNA derived from these genes have been suggested.

Amino Acid Sequence↗
PubMed1987

Prostatic spermine-binding protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and androgenic control of mRNA level.

cDNA for mRNA of an androgen-dependent spermine-binding protein (SBP) of rat ventral prostate was cloned by inserting cDNA into a dG-tailed expression vector, pUC8, and screening the expression library with anti-SBP antibodies. Hybrid-selected translation using plasmid DNA from positive clones yielded a 34-kDa protein which was immunoprecipitated by affinity-purified anti-SBP antibodies. SBP mRNA is about 1260 bases long as measured by Northern blot hybridization. An amino acid sequence deduced from the nucleotide sequence of the cDNA was identical to an amino acid sequence found in SBP. SBP is extremely rich in acidic residues. Aspartic and glutamic acids, which make up about 33% of the total sequence, comprise 89 of a stretch of 126 amino acids at the carboxyl-terminal end. By dot hybridization analysis, SBP mRNA was not detected in rat liver, kidney, brain, submaxillary gland, or uterus. The prostate levels of SBP mRNA were measured by mRNA translation and dot hybridization. SBP mRNA level decreased to less than 20% of normal 2 days after castration of rats, and this decrease was reversed by 5 alpha-dihydrotestosterone injection into castrated rats.

Amino Acid Sequence↗
PubMed1989

Sequencing of porcine growth hormone cDNA.

Sequences for two pGH cDNAs are reported in this paper. The cDNAs were obtained by reverse transcription from porcine pituitary gland mRNA, cloned, and screened. The results are discussed and the two cDNA sequencings are compared with one by Seeburg et al. and another by Vize et al.

Amino Acid Sequence↗
PubMed1989

Synthesis of an immunopathogenic fusion protein derived from a bovine interphotoreceptor retinoid-binding protein cDNA clone.

We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector. The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined. These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP. This cDNA sequence validates the intron: exon boundaries predicted from the gene. A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1. Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination. This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots. This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP. Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of cloned cDNA for human pancreatic kallikrein.

Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.

Amino Acid Sequence↗
PubMed1995

Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing.

We have isolated and sequenced cDNA clones that encode the core protein of PG-M-like proteoglycan produced by cultured mouse aortic endothelial cells (Morita, H., Takeuchi, T., Suzuki, S., Maeda, K., Yamada, K., Eguchi, G., and Kimata, K. (1990) Biochem. J. 265, 61-68). A homology search of the cDNA sequence has suggested that the core protein is a mouse equivalent of chick PG-M(V1), one of the alternatively spliced forms of the PG-M core protein, which may correspond to human versican. Northern blot analysis revealed three mRNA species of 10, 9, and 8 kilobases (kb) in size. The analysis of PG-M mRNA species in embryonic limb buds and adult brain revealed the presence of other mRNA species with different sizes; the one with the largest size (12 kb) was found in embryonic limb buds, and the ones with smaller sizes of 7.5 and 6.5 kb were in adult brain. Sequencing of cDNA clones for the smaller forms in the adult brain showed that they were different from PG-M(V1) in encoding the second chondroitin sulfate attachment domain (CS alpha) alone. Occurrence of the PCR products striding over the junction of the first and second chondroitin sulfate attachment domains suggested that a mRNA of 12 kb in size corresponded to a transcript without the alternative splicing (PG-M(V0)). It is likely, therefore, that multiforms of the PG-M core protein may be generated by alternative usage of either or both of the two different chondroitin sulfate attachment domains (alpha and beta) and that molecular forms of PG-M may vary from tissue to tissue by such an alternative splicing.

Alternative Splicing↗
PubMed1997

Molecular cloning and sequencing of the cDNA encoding the catalytic subunit of mouse glutamate-cysteine ligase.

Reverse transcription-polymerase chain reaction (RT-PCR) was used for the enzymatic synthesis of cDNA sequences encompassing the open reading frame for the catalytic subunit of mouse kidney glutamate-cysteine ligase (Glclc). Comparison of the mouse Glclc cDNA sequence and predicted protein sequence with that of rat Glclc and human GLCLC revealed between 94.8% and 88.4% cDNA homology and 98.4% to 95% amino acid identity, respectively.

Amino Acid Sequence↗
PubMed1995

Structure and organization of the human thrombospondin 3 gene (THBS3).

The promoter/5' flank sequence, cDNA sequence, and exon/intron structures of the human thrombospondin 3 (THBS3) gene have been determined. THBS3 cDNA clones were obtained by PCR amplification of human fetal lung cDNA using THBS3-specific primers. Analysis of cDNA and genomic sequences showed the THBS3 gene to be composed of 23 exons, 1 more than the number of exons in the previously characterized mouse TSP3 gene. The additional exon results from the division of mouse exon F into exons F1 and F2. The cDNA encodes a polypeptide of 956 amino acids that is highly acidic, with a clustering of acidic side chains in the third quarter of the polypeptide. This region corresponds to seven type III (Ca(2+)-binding) repeats, a feature shared with other thrombospondins. In addition to these type III repeats, four type II (EGF-like) repeats and NH2- and COOH-terminal domains are present in thrombospondin 3. The THBS3 and mouse TSP3 genes differ in intron sizes, but exon sequences and sizes and positions of insertion of introns are conserved to a high degree. The structural organization of the THBS3 gene is of interest because of its close proximity to that of metaxin, with which it shares a common promoter sequence, and to the gene encoding glucocerebrosidase, a deficiency in which causes Gaucher disease.

Amino Acid Sequence↗