Cloning and sequence analysis of a cDNA from human ovarian granulosa cells encoding the C-terminal part of human elongation factor 2

A cDNA clone, pHGR81, encoding 358 amino-acid residues of the C-terminal region of human elongation factor 2 (EF-2), was isolated from a human ovarian granulosa cell cDNA library. The deduced amino-acid sequence of pHGR81, when compared with the known identical amino-acid sequences of hamster as well as rat EF-2 revealed a substitution of a glutamine by an alanine residue in the partially determined human sequence. The 15 amino-acid-residue sequence comprising the histidine-715, supposed to be of importance for the biological function of EF-2, is preserved in human EF-2. The coding region of the cDNA insert of pHGR81 displays a homology of 87% to hamster and of 88% to rat EF-2 cDNA. In Northern-transfer analysis, pHGR81 specifically hybridizes with an mRNA species of 3.1 kb.     

Amplification using one-sided polymerase chain reaction, cloning and determination of primary structure of 5'-untranslated cDNA segments coding the somatogenic growth hormone receptor from rat liver]

The method of ligation-mediated, single-sided polymerase chain reaction was applied for the amplification of 5'-untranslated regions of cDNA coding for somatogenic growth hormone receptor from rat liver. The products of polymerase chain reaction were cloned and sequenced. Two variants of 5'-untranslated sequences of growth hormone receptor cDNA corresponding to products of alternative splicing of pre-mRNA were found.     

Heterogeneity of native rat liver elongation factor 2

The high heterogeneity of native rat liver EF-2 prepared from either 105000 x g supernatant or microsome high-salt extract was detected by two-dimensional equilibrium isoelectric focusing-SDS-polyacrylamide gel electrophoresis in the presence of 9.5 M urea. Five spots were always detected, all of Mr 95,000, which were not artefactual for their amount varied when EF-2 was specifically ADP-ribosylated by diphtheria toxin in the presence of NAD+, and/or phosphorylated on a threonine residue by a Ca2+/calmodulin-dependent protein kinase (most likely Ca2+/calmodulin-dependent protein kinase III described by others [(1987) J. Biol. Chem. 262, 17299-17303; (1988) Nature 334, 170-173]). Results of ADP-ribosylation and/or phosphorylation experiments with either unlabeled or labeled reagents ([14C]NAD and [32P]ATP) strongly suggest that our preparation contained native ADP-ribosylated and native phosphorylated forms which could be estimated at about 20% and 40% of the whole EF-2. Phosphorylated and ADP-ribosylated forms of EF-2 could be ADP-ribosylated and phosphorylated, respectively, but a native form both ADP-ribosylated and phosphorylated was not detected. Our results also suggest the existence of a minor native form of EF-2 and of its phosphorylated and ADP-ribosylated derivatives.     

Cloning, primary structure determination and expression of preproinsulin cDNA from human insulinoma in Escherichia coli]

A human insulinoma cDNA library was constructed in expression plasmid vector pUEX1. Clone pUEX1Ins12 was selected from human insulinoma cDNA library by means of hybridization with the insulin probe and a nucleotide sequence of the insertion was determined. It codes for full size amino acid sequence preproinsulin and furthermore, contains the entire 3'-end of noncoding mRNA region and 44 nucleotides from the 5'-untranslated region. The bacterial strain pUEX3Ins8 producing preproinsulin as beta-galactosidase fusion protein was constructed.     

Cloning of metallothionein cDNA from neonatal rat liver

A metallothionein cDNA clone was isolated from a cDNA bank prepared from neonatal r a t liver poly(A)-containing RNA by a colony screening procedure using [³²P]cDNA probes prepared from mRNA of either metal-induced or uninduced rat livers. Nucleotide sequence analysis of this clone showed that it contained the entire 3' untranslated region and 30% of the coding sequence for a rat metallothionein. The sequence is remarkably homologous with the mouse metallothionein-I gene.     

On the nature of cellular ADP-ribosyltransferase from rat liver specific for elongation factor 2

A cellular ADP-ribosyltransferase, specific for elongation factor 2 (EF-2), is found in extracts from rat liver. Co-migrating with EF-2 throughout purification, this activity is, moreover, located in the protein bands corresponding to EF-2 after native or sodium dodecyl sulfate polyacrylamide gel electrophoresis. The observed activity is thus implicated to be an inherent property of EF-2. Preincubation of EF-2 with GuoPPCH2Pox inhibits endogenous, but not diphtheria toxin catalyzed ADP-ribosylation.     

Cloning of several cDNA segments coding for human liver proteins

A human cDNA library was constructed using M13 derivative vectors. The simple and rapid procedures for sequencing single-stranded DNA by the dideoxy chain termination method allowed a screening of individual clones directly by DNA sequence analysis. Some of these clones were identified as coding for: serum albumin, alpha1-antitrypsin, retinol-binding protein, prothrombin, haptoglobin, and metallothionein. Furthermore, a clone coding for aldolase B was tentatively identified on the basis of high sequence homology with rabbit muscle aldolase.     

Cloning and characterization of the chloroplast elongation factor EF-Tu cDNA of Oryza sativa L

From the rice leaf cDNA library, we have cloned a cDNA encoding rice chloroplast translational elongation factor EF-Tu (tufA). The rice tufA cDNA clone contains 1678 nucleotides and codes for a 467 amino acid protein including a putative chloroplast transit peptide of 59 amino acid residues. The predicted molecular mass of the mature protein is approximately 45 kDa. This cDNA clone contains the 61 nucleotides of the 5' untranslated region (UTR) and the 213 nucleotides of 3' UTR. Amino acid sequence identity of the rice tufA with the mature chloroplast EF-Tu proteins of tobacco, pea, arabidopsis, and soybean ranges from 83% to 86%. The deduced polypeptide of the rice tufA cDNA contains GTP binding domains in its N-terminal region and chloroplast EF-Tu signature regions in the C-terminal region. The rice tufA appears to exist as a single copy gene, although its homologues of maize and oat exist as multiple copy genes. The rice tufA gene is located in chromosome 1 and is more highly expressed in the leaf than in root tissue.     

Cloning and structure analysis of the rat apolipoprotein A-I cDNA

Apolipoprotein A-I, the major protein in mammalian high-density lipoprotein, acts as a cofactor for lecithin-cholesterol acyltransferase during the formation of cholesterol ester and as such, is thought to promote cholesterol efflux from peripheral cells to the liver. In this paper, we report the partial purification of rat liver apolipoprotein A-I mRNA by a polysome immunoadsorption technique, and its cDNA cloning. Isolation of two overlapping cDNA clones enabled us to derive the whole rat apolipoprotein A-I cDNA coding sequence. Comparison of the deduced protein sequence with its human counterpart reveals a striking homology between the prepropeptide precursors. Both mature protein amino-terminal regions are very homologous, suggesting that this particular domain could be involved in lipid/protein binding or lecithin-cholesterol acyltransferase activation.     

Cloning and sequence determination of cDNA encoding a second rat liver peroxisomal 3-ketoacyl-CoA thiolase

3-Ketoacyl-coenzyme A thiolase (thiolase) catalyzes the final step of the fatty acid beta-oxidation pathway in peroxisomes. Thiolase is unique among rat liver peroxisomal enzymes in that it is synthesized as a precursor possessing a 26-amino acid (aa) N-terminal extension which is cleaved to generate the mature enzyme. To facilitate further examination of the synthesis, intracellular transport and processing of this enzyme, cDNA clones were selected from a lambda gt11 rat liver library using antiserum raised against peroxisomal thiolase. Upon sequencing several cDNA clones, it was revealed that there are at least two distinct thiolase enzymes localized to rat liver peroxisomes, one identical to the previously published rat liver peroxisomal thiolase (thiolase 1) [Hijikata et al., J. Biol. Chem. 262 (1987) 8151-8158] and a novel thiolase (thiolase 2). The THL2 cDNA possesses a single open reading frame of 1302 nucleotides (nt) encoding a protein of 434 aa (Mr 44790). The coding region of THL2 cDNA exhibits 94.6% nt sequence identity with THL1 and 95.4% identity at the level of aa sequence. Northern-blot analysis indicates that the mRNA encoding thiolase 2 is approx. 1.7 kb in size. The mRNA encoding thiolase 2 is induced approx. twofold upon treatment of rats with the peroxisome-proliferating drug, clofibrate. In contrast, the thiolase 1 mRNA is induced more than tenfold under similar conditions.     

Cloning and expression of a cDNA coding for a rat liver plasma membrane ecto-ATPase. The primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I

The amino acid sequence of the ecto-ATPase from rat liver was deduced from analysis of cDNA clones and a genomic clone. Immunoblots with antibodies raised against a peptide sequence deduced from the cDNA sequence indicated that the determined amino acid sequence is that of the ecto-ATPase. The deduced sequence predicts a 519-amino acid protein with a calculated molecular mass of 57,388 daltons. There are 16 potential asparagine-linked glycosylation sites in the protein. Hydropathy analysis of the deduced amino acid sequence indicates that the protein has two hydrophobic stretches. One is located at the N-terminal and the other is near the C-terminal end. A full-length clone encoding the ecto-ATPase was expressed transiently in mouse L cells and human HeLa cells. The cell lysate from the transfected cells contained immunoreactive ecto-ATPase and Ca2+-stimulated ATPase activities. The expressed protein is glycosylated and has an apparent molecular weight (100,000) similar to that of the rat liver plasma membrane ecto-ATPase.     

Cloning and sequence analysis of cDNA for rat liver uricase

We have isolated cDNA clones for rat liver uricase using an oligonucleotide corresponding to the N-terminal sequence of 8 amino acids. The nucleotide sequences of the cDNAs have been determined, and the amino acid sequence of the protein deduced. A 867-base open reading frame coding for 289 amino acids, corresponding to a molecular mass of 33,274 daltons, was confirmed by matching eight sequences of a total of 53 amino acids from peptide sequence analyses of the fragments generated by lysyl endopeptidase digestion of purified rat liver uricase. The deduced amino acid sequence of rat liver uricase shares 40% homology with that of soybean nodulin-specific uricase and has an N-terminal extension of 7 amino acids. In contrast, soybean uricase has a C-terminal extension of 12 amino acids, which is presumably the result of local gene duplication. Completely different N- and C-terminal structures of the two uricases suggest that the signals for targeting the proteins to the peroxisome are not located on the terminal continuous stretches of amino acids.     

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.     

Molecular cloning and primary structure of cDNA encoding the catalytic domain of rat liver aspartyl-tRNA synthetase

A cDNA clone encoding rat liver aspartyl-tRNA synthetase was isolated by probing a lambda gt11 recombinant cDNA expression library with antibodies directed against the corresponding polypeptide from sheep liver. The 1930-base pairs-long cDNA insert allowed the expression in Escherichia coli of an active enzyme of mammalian origin. The nucleotide sequence of that cDNA, corresponding to the DRS1 gene, was determined. The open reading frame of DRS1 corresponds to a protein of Mr = 57,061, in good agreement with the previously determined molecular weight of the purified enzyme. The deduced amino acid sequence shows extensive homologies with that of yeast cytoplasmic aspartyl-tRNA synthetase, more than 50% of the residues being identical. In rat liver, aspartyl-tRNA synthetase occurs in two distinct forms: a dimeric enzyme and a component of a multienzyme complex comprising the nine aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. The primary structure of the DRS1 gene product is discussed in relation to the occurrence of two distinct forms of that enzyme.     

Complete sequence of the coding region of human elongation factor 2 (EF-2) by enzymatic amplification of cDNA from human ovarian granulosa cells

The use of two primers allowed the specific enzymatic amplification of elongation factor 2 starting with total double-stranded cDNA from human ovarian granulosa cells. The amplified DNA fragment with a length of 1765 bp was restricted and sequenced by the shot gun approach. From the sequences obtained from the amplified fragment and the cDNA insert of pHGR81 [Rapp et al. (1988) Biol. Chem. Hoppe-Seyler 369, 247-250] respectively, the DNA sequence containing the complete coding as well as the 3'-untranslated region was assembled.     

Cloning, nucleotide sequence, and expression of one of two genes coding for yeast elongation factor 1 alpha

One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.     

Protein synthesis elongation factor 1 from rat liver: A zinc metalloenzyme

Highly purified elongation factor 1 (light form, EF1_L) from rat liver contains zinc as determined by atomic absorption spectrophotometry. Analysis has been performed on the most active protein fraction from DEAE-Sephadex chromatography (estimated purity: 90%) and on the main band obtained from this fraction by polyacrylamide gel electrophoresis. The data are consistent with a stoichiometry of approximately one g-atom of zinc per 54,000 daltons of EF1_L protein. A functional role for Zn²⁺ is suggested by the fact that 0.3 mM 1,10-phenanthroline completely abolishes GTP binding by EF1_L (measured by the nitrocellulose filter retention assay), while the isomeric non-chelator 1,7-phenanthroline has no effect. This inhibition can be overcome by the addition of excess zinc ion.