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PubMed1991

The WDNM1 gene product is a novel member of the 'four-disulphide core' family of proteins.

It has been previously demonstrated that the WDNM1 gene is downregulated in metastatic rat mammary adenocarcinomas. Detailed analysis of the structure of WDNM1 mRNA reveals the presence of two transcripts generated by alternative splicing of a single exon. The unspliced version possesses an in-frame termination codon which encodes a predicted protein of 18 amino acids. The spliced version encodes a predicted protein of 60 amino acids which exhibits strong homology to a family of proteins possessing a conserved arrangement of cysteine residues. This family includes several proteinase inhibitors suggesting that WDNM1 could encode a product with proteinase inhibiting capacity. Possible modulation in the level of this protein product could be related to overall proteinase activity of specific tumour cells which, in turn, could influence the invasive and metastatic potential of such cell populations.

Adenocarcinoma↗

PubMed1991

Molecular cloning and sequencing of a cDNA clone encoding a new calcium binding protein, named calgizzarin, from rabbit lung.

We purified a new EF-hand type calcium binding protein from chicken gizzard smooth muscle, tentatively named calgizzarin (Todoroki, H., et al. J. Biol. Chem. (1991) in press. Based on the internal peptide sequence of calgizzarin, we isolated and sequenced a cDNA clone coding for calgizzarin from a rabbit lung cDNA library. This clone (pCALG) has 309 nucleotides of open reading frame including termination codon TGA, 621 nucleotides of the 5' leader and 186 nucleotides of the 3' noncoding region. The polypeptides deduced from the open reading frame were consisted of 102 amino acid residues with a molecular weight of 11,429. Computer aided homology analysis revealed that calgizzarin exhibits a 43.2% homology to S-100 alpha, 38.6% to S-100 beta and 40.0% to annexin II light chain, p10. By Northern blot analysis, with pCALG, a band of 1.1 kbp was detected in rabbit lung, suggesting pCALG contains nearly full length of mRNA.

Amino Acid Sequence↗

PubMed1992

Intermediate form of mucopolysaccharidosis type II (Hunter disease): a C1327 to T substitution in the iduronate sulfatase gene.

Hunter disease, an X-linked recessive lysosomal storage disorder, is caused by a deficiency in iduronate sulfatase activity. Sequence analysis of mRNA of fibroblasts of an intermediate phenotype patient showed a single C1327 to T nucleotide transition. This mutation resulted in a substitution of termination codon for normal arginine at position 443 of the peptide sequence. Expression studies with this abnormal cDNA in fibroblasts from the patient revealed a loss of enzymatic activity and instability of the mutated protein. We posturate that this mutation is probably the cause of the intermediate form of Hunter disease.

Base Sequence↗

PubMed1992

Vacuolar type H(+)-ATPase genes: presence of four genes including pseudogenes for the 16-kDa proteolipid subunit in the human genome.

Genes for the human vacuolar type H(+)-ATPase proteolipid (16-kDa) subunit were cloned and their nucleotide sequences were determined. Comparison of the deduced sequences indicated that at least four genes including pseudogenes are present in the human genome. One of them corresponded to that for the 16-kDa subunit expressed in HeLa cells. The coding sequence was separated by two introns. The second intron was located in the DNA segment giving a loop between the second and third transmembrane helices, supporting the idea that the 16-kDa subunit was evolved by gene duplication. The primary sequence determined from the second clone had a termination codon behind the third transmembrane helix. Possible translation products from the other two clones had no putative acidic residues essential for proton transport function of the 16-kDa subunit. Thus, it is interesting to know whether these genes are transcribed, since they may have unique cellular functions.

Base Sequence↗

PubMed1992

Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus.

We have isolated a cDNA clone encoding a novel calcium-binding protein of the recoverin family from rat brain cDNA library. This clone (PCB11) has 588 nucleotides in the open reading frame including the termination codon, 174 nucleotides of the 5' leader and 800 nucleotides of the 3' noncoding region. The complete amino acid sequence deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22,574 Daltons, and contains three putative calcium-binding domains of the EF-hand structure. The deduced amino acid sequence has a striking sequence homology to those of the retinal recoverin family (recoverin, visinin, P26, 23kD protein, S-modulin) and the brain-derived recoverin family (P23k, 21-kDa CaBP and neurocalcin). Northern blot, in situ hybridization, immunoblot and immunohistochemical analyses revealed that the protein is exclusively expressed in pyramidal layer of the hippocampus. The protein was therefore designated hippocalcin.

Amino Acid Sequence↗

PubMed1992

Molecular cloning of rabbit cytochrome b5 genes: evidence for the occurrence of two separate genes encoding the soluble and microsomal forms.

The rabbit genomic segments for the soluble cytochrome b5 (b5) and microsomal b5 were amplified and isolated, respectively, by means of the polymerase chain reaction using primers corresponding to various portions of the open reading frame of microsomal b5 cDNA. The DNA sequence analysis revealed that the soluble b5 gene has an extra 24 nucleotide long insert which encodes a C-terminal amino acid and a termination codon which are specific to the soluble b5. Except for the insert, the sequences of the soluble and microsomal b5 genes are identical with each other from the 5' end to the 3' end of the open reading frame of the microsomal b5 cDNA. Comparison of the genomic sequences with the cDNA sequences suggested that the soluble and microsomal genes are intronless within their open reading frames. These data indicate that rabbit soluble and microsomal b5 mRNAs are encoded by two highly conserved but separate genes.

Amino Acid Sequence↗

PubMed1986

Calmodulin gene expression during sea urchin development: persistence of a prevalent maternal protein.

Calmodulin gene expression during embryogenesis of the sea urchin Strongylocentrotus purpuratus was investigated. Several identical bacteriophages containing a cDNA insert encoding sea urchin calmodulin (CM-1) were identified by screening a lambda gt10 library of S. purpuratus gastrula-stage cDNAs with a chicken calmodulin cDNA sequence. A 1.2-kb cDNA fragment from CM-1 was subcloned into pUC-8 to give plasmid pCAL-8. pCAL-8 contains a single open reading frame encoding 79 amino acids, a termination codon, and 0.9 kb of 3'-untranslated message. This sea urchin amino acid sequence shows 95% homology to amino acid residues 69-148 of the predicted sequence of chicken calmodulin. Northern analysis showed that pCAL-8 hybridizes to a single size (3.2 kb) of mRNA in both embryonic and adult somatic tissues. Genome blots suggested that there is a single calmodulin gene in the S. purpuratus genome. We used pCAL-8 to study calmodulin mRNA accumulation in S. purpuratus embryos. Calmodulin mRNA is present in the unfertilized egg at the level of a typical rare-class mRNA (1000-2000 transcripts) and accumulates approximately 100-fold to levels representing about 1/10th of 1% of the total mRNA in pluteus-stage cells. Synthesis of calmodulin, identified by two-dimensional gel electrophoresis, shows a similar developmental pattern. However, in spite of the very active synthesis of calmodulin during embryogenesis, most of the calmodulin in the pluteus is apparently provided for by an enormous store of calmodulin in the egg, corresponding to about 2% of the mass of total protein.

Animals↗

PubMed1988

Molecular analysis of cDNA coding for ZP3, a sperm binding protein of the mouse zona pellucida.

At fertilization, mammalian sperm bind is a species-specific manner to the extracellular zona pellucida that surrounds ovulated eggs. ZP3, an 83,000-85,000 Da glycoprotein of the murine zona pellucida, has been shown to inhibit sperm binding via its O-linked oligosaccharide side chains. We have recently isolated cDNA clones coding for ZP3 and have demonstrated that ZP3 transcripts are accumulated in oocytes where their expression is developmentally regulated during oogenesis. We now report that ZP3 mRNA is 1317 nt long with an estimated poly(A) tail of 200-300 nt. The short 29-nt 5' untranslated region is followed by a single open reading frame coding for a polypeptide chain of 46,307 Da which includes six possible sites for N-linked oligosaccharides. The N-terminus of ZP3 contains a potential 22-amino acid signal peptide which upon cleavage would result in a secreted core protein of 43,943 Da. The termination codon is a part of the AATAAA polyadenylation signal and is contained in an unusually short 16-nt 3' untranslated region. Sequences homologous to ZP3 are conserved among mammals and are expressed in ovarian tissue as mature transcripts with indistinguishable molecular weights.

Amino Acid Sequence↗

PubMed1984

Nucleotide sequence of the lspA gene, the structural gene for lipoprotein signal peptidase of Escherichia coli.

The nucleotide sequence of the lspA gene coding for lipoprotein signal peptidase of Escherichia coli was determined and the amino acid sequence of the peptidase was deduced from it. The molecular mass and amino acid composition of the predicted lipoprotein signal peptidase were consistent with those of the signal peptidase purified from cells harboring the lspA gene-carrying plasmid. The peptidase most probably has no cleavable signal peptide. The lspA gene was preceded by the ileS gene coding for isoleucyl-tRNA synthetase and the tandem termination codons of the ileS gene overlapped with the initiation codon of the lspA gene.

Amino Acid Sequence↗

PubMed1986

Cloning and DNA sequence of the 5'-exonuclease gene of bacteriophage T5.

The nucleotide sequence of the BalI-PstI fragment of T5 DNA, 1347 bp in length, coding for 5'-exonuclease (D15 gene), has been determined. A coding region of the gene contains 873 bp and is preceded by a typical Shine-Dalgarno sequence. The D15 gene belongs to a cluster, consisting of at least 3 genes, in which a termination codon of a preceding gene overlaps an initiation codon of the following one. The sequence contains an open reading frame for 291 amino acid residues. The molecular mass of the 5'-exonuclease calculated from the predicted amino acid sequence is 33 400 Da.

Amino Acid Sequence↗

PubMed1986

Complete cDNA sequence for rabbit muscle glycogen phosphorylase.

The cDNA for the nearly full-length rabbit muscle glycogen phosphorylase mRNA has been isolated and sequenced. The cDNA is rich in G and C nucleotides. This feature is especially striking at the 3rd position of codons, where 86% of the 843 amino acid codons terminate with G or C. Methionine, presumably the initiation residue, is found at position-1, suggesting that the removal of only a single methionine residue precedes the amino-terminal acetylation at serine. Eight differences between the deduced amino acid sequence and the previously determined protein sequence are discussed.

Amino Acid Sequence↗

PubMed1988

The conversion of phosphoserine residues to selenocysteine residues on an opal suppressor tRNA and casein.

This study has been undertaken in order to elucidate the mechanisms of incorporation of Se into glutathione peroxidase (GSHPx), in which selenocysteine corresponds to the opal termination codon UGA on the mRNA. We studied the above mechanisms using an opal suppressor tRNA, prepared from bovine liver, and casein as a model protein for the GSHPx apo-enzyme which might contain phosphoserine. The results showed that opal suppressor tRNA did not accept selenocysteine (lower than 0.1 mmol/mol) under the standard conditions. A trace amount of phosphoseryl-tRNA was converted to selenocysteyl-tRNA by incubation with H2Se and some enzymes. Meanwhile, a number of phosphoserine residues in casein were converted to selenocysteine residues by incubation with H2Se and enzymes. These results suggest that opal suppressor tRNA plays a role in synthesizing GSHPx via co- and/or post-translational mechanisms.

Animals↗

PubMed1990

Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector.

To examine the validity of the strategy to express a foreign gene as a fusion protein with the coat protein (CP) of tobacco mosaic virus (TMV), we have constructed ENK RNA by using an in vitro transcription system of TMV RNA. ENK RNA differs from TMV RNA only in that ENK RNA carries an additional sequence coding for Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) (Enk) with a preceding in-frame methionine just before the termination codon of CP gene. In protoplasts inoculated with ENK RNA, CP + Enk fusion protein accumulated as the major protein.

Amino Acid Sequence↗

PubMed1991

Identification and characterization of a C-terminally extended form of recombinant murine IL-6.

Murine interleukin-6 (mIL-6) was expressed in Escherichia coli in the insoluble fraction of cell lysates. Approximately equal amounts of two polypeptide species, reactive with anti-IL-6 antibodies, were produced. The two forms of mIL-6 were isolated and found to have identical N-terminal sequences initiated by Met-Phe-Pro-Thr-Ser-Gln-. Peptide mapping after endoproteinase glu-C digestion led to isolation and characterization of the C-terminal peptides from each of the two forms and allowed the source of the heterogeneity to be identified as a C-terminal addition of three amino acids, Gln-Lys-Leu, to authentic mIL-6. Inspection of the nucleotide sequence of the plasmid containing the mIL-6 gene and expression of the plasmid in other strains suggested that the addition of three amino acids was caused by a readthrough of the termination codon arising from an unexpected suppressor mutation in the original host strain. Although the C-terminus of IL-6 is critical for the activity of this cytokine, the IL-6 variant with extended C-terminus was fully active in two separate bioassays. This suggests that the additional amino acids do not disrupt the structure or function of this important region of the molecule.

Amino Acid Sequence↗

PubMed1992

Molecular cloning and nucleotide sequence analysis of a cDNA encoding the main beta-neurotoxin from the venom of the South American scorpion Tityus serrulatus.

A cDNA encoding the main Tityus serrulatus beta-neurotoxin was isolated from a venom gland cDNA library by using an oligonucleotide probe. The amino acid sequence deduced from the cDNA nucleotide sequence indicated that the toxin is the processed product of a precursor containing: (i) a signal peptide of 20 residues; (ii) the amino acid sequence of the mature toxin; and (iii) an extra Gly-Lys-Lys tail at the C-terminal end before the termination codon. Thus, in addition to the removal of the signal peptide by a signal peptidase, the generation of the mature toxin requires both a post-translational cleavage by a carboxypeptidase specific for basic residues and the action of an alpha-amidating enzyme. These results also show that the biosynthetic pathway for beta-toxins of 'New World' scorpion venoms is similar to that already described for alpha-toxins of 'Old World' scorpion venoms.

Amino Acid Sequence↗

PubMed1996

Expression and pharmacological characterization of the human mu-opioid receptor in the methylotrophic yeast Pichia pastoris.

The human mu-opioid receptor cDNA from which the 32 amino-terminal codons were substituted by the Saccharomyces cerevisiae alpha-mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase-1 gene. Cell membranes exhibited specific and saturable binding of the opioid antagonist [3H]diprenorphine (Kd = 0.2 nM and Bmax = 400 fmol/mg protein or 800 sites/cell). Competition studies with non-selective, and mu-, delta- and kappa-selective opioid agonists and antagonists revealed a typical mu-opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.

Cell Membrane↗

PubMed1993

Cholesterol-lowering drug therapy in a patient with receptor-negative homozygous familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is caused by mutations in the gene for the low density lipoprotein (LDL) receptor. It is generally believed that homozygous FH patients do not respond well to lipid-lowering drug therapy with inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase because they cannot respond to an increased demand for hepatic cholesterol by up-regulation of LDL-receptor activity. In this paper we show that serum cholesterol in a homozygous FH patient with a receptor-negative LDL-receptor phenotype was reduced by 30% after treatment with simvastatin alone and by a further 11% with simvastatin in combination with probucol and nicotinic acid. The patient was a true homozygote, with two identical alleles of the LDL receptor gene in which a previously undescribed point mutation in exon 11 introduces a premature termination codon at residue 540 in the protein; the mutant protein is predicted to be truncated in the domain with homology to the epidermal growth factor precursor. Cultured cells from the patient were unable to bind, internalise or degrade LDL by the receptor pathway and there was no immunodetectable LDL receptor protein in the cells. Thus the lipid lowering effect of simvastatin in this individual must involve mechanisms other than stimulation of LDL receptors.

Adolescent↗

PubMed1993

An improved method for generating single-chain antibodies from hybridomas.

Cloning the correct VL kappa gene from hybridomas derived from MOPC-21 can be problematic because such cell lines variably express a transcript which is aberrantly rearranged at the VJ recombination site. Cellular levels of the aberrant transcript can exceed that of productive light chain RNA, so a large proportion of the VL gene-derived products obtained on PCR amplification of hybridoma cDNA may not encode a functional protein. We have developed a method in which antibody variable region genes are recovered from hybridoma cDNA using a unique set of V gene family-specific primers; the V region genes are then spliced by PCR, in the form 5'-VL-LINKER-VH-3' (where the linker encodes [GlyGlyGlyGlySer]3), and cloned into an expression vector under control of T7 RNA polymerase. Plasmid DNA is isolated from colonies, and the insert is expressed in an in vitro rabbit reticulocyte lysate-based coupled transcription/translation system, in a microtiter plate format. Since aberrantly rearranged VL kappa genes contain a translation termination codon at amino acid position 105, only constructs containing the correctly rearranged gene produce a protein of the predicted size. We demonstrate the method by producing the single-chain form of OKT9, a murine IgG1 which binds to the human transferrin receptor, and extend the results to show that the protein generated by the in vitro expression system retains the antigen binding properties of the parent antibody. Our method will be generally useful for screening single-chain antibodies for function prior to large scale production in vivo.

Amino Acid Sequence↗