Isolation and characterization of eft-1, an elongation factor 2-like gene on chromosome III of Caenorhabditis elegans.

A gene (eft-1) encoding an elongation factor 2-like protein was isolated from a region adjacent to the polyubiquitin gene, ubq-1, of Caenorhabditis elegans. Sequence analysis of genomic and cDNA clones revealed that the deduced amino acid sequence of the protein (EFT-1) is 38% identical to that of mammalian and Drosophila elongation factor 2 (EF-2). The entire eft-1 gene is approximately 3.8 kb in length and contains 5 exons separated by short introns of 46-75 bp. The 2,547-bp open reading frame predicts a protein of 849 amino acid residues (calculated Mr, 96,151). Conserved sequences shared among a variety of GTP-binding proteins including EF-2 are found in the amino-terminal third of EFT-1. The carboxy-terminal half contains regions with 40-57% similarity (including conservative changes) with segments characteristic of EF-2 and its prokaryotic homolog, EF-G. However, the histidyl residue target for ADP-ribosylation of EF-2 by diphtheria toxin is replaced by tyrosine in EFT-1. Southern and Northern blot analyses indicate that eft-1 is a single-copy gene that is expressed at all stages of nematode development. Amplification of fragments encoding highly conserved regions of EF-2 using the polymerase chain reaction led to the isolation of a fragment encoding the modifiable histidyl residue and which likely represents part of the C. elegans EF-2 gene (eft-2). This suggests that EFT-1 is not the C. elegans homolog of EF-2, but a closely related protein.     

Structure and expression of elongation factor 2 gene during development of Dictyostelium discoideum

A cDNA library constructed from poly(A)+ RNA isolated from Dictyostelium discoideum cells at 12 h of development was screened with the hamster elongation factor 2 (EF-2) cDNA. Several different cDNA clones which hybridized were isolated after a second screening. A cDNA clone representing the 5'-end of the mRNA was obtained by primer extension. By comparing the amino acid sequence deduced from the nucleotide sequences of these clones with that of hamster EF-2, we found enough homology between them to conclude that the isolated clones were complementary to the mRNA of D. discoideum EF-2. The N terminus which is the GTP-binding domain and the C-terminal half where it interacts with a ribosome showed a high degree of homology. The amino acid sequence of the carboxyl half includes that it contain a site of ADP-ribosylation by diphtheria toxin. From the Northern blotting analysis, the size of the mRNA was estimated to be 2.6 kilobases. The expression of the mRNA was high in vegetative cells, became maximal at the aggregation stage, and decreased thereafter through development. Upon differentiation of prespore and prestalk cells, the mRNA was highly enriched in the former over the latter. ADP-ribosylation assay of EF-2 protein by diphtheria toxin showed nearly the same developmental changes for the protein as the mRNA. However, prestalk cells were found to contain the same amount of the protein as prespore cells. The Southern blot analyses indicated that the gene encoding EF-2 is unique.     

Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF-7.

We present the complete sequence of an mRNA which is induced by estrogen in the human breast cancer cell line MCF-7 [pS2 mRNA, Masiakowski et al., Nucleic Acids Res. 10, 7895-7903 (1982)]. Primer extension and cloning of double-stranded cDNA (ds-cDNA) into a vector designed to make full-length cDNA were used to determine the sequence of the fifteen 5'-terminal nucleotides which were not present in the original pS2 ds-cDNA clone. The mRNA sequence has a major open reading frame encoding 84 amino-acids, flanked by a 40 nucleotide 5'-untranslated region and a 198 nucleotide 3'-untranslated region preceding the polyA tail. The 3'-untranslated region contains a polyadenylation signal, AUUAAA, 14 nucleotides upstream from the polyA tail. The derived protein sequence contains a putative signal peptide region suggesting that the protein may be secreted. The nucleotide and derived amino-acid sequences were compared to previously determined sequences, particularly to those of hormone-regulated proteins and growth factors, and no obvious similarities were observed.     

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.     

Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence

Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA lambda gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two positive clones. The library was rescreened with a radiolabeled cDNA probe made from one of these clones, thus identifying an additional 13 positive clones. Sequencing of the largest two of these overlapping clones resulted in 2672 bp of cDNA sequence containing partial 5'- and 3'-untranslated sequences of 286 and 1159 nucleotides, respectively, and a complete open reading frame of 1227 bp encoding a polypeptide of 409 amino acids (46 kDa), consistent with the 48 +/- 3 kDa cell-free translation product of rat smooth muscle cell RNA that was immunoprecipitated by anti-bovine lysyl oxidase. The rat aorta cDNA-derived amino acid sequence contains the sequence of each of the six peptides isolated and sequenced from the 32-kDa bovine aorta enzyme, including the C-terminal peptide with sequence identity of 96%. Northern blots screened with lysyl oxidase cDNA probes identified hybridizing species of 5.8 and 4.5 kb in mRNA of rat aorta and lung, while dot blot analyses were negative for lysyl oxidase mRNA in preparations of rat brain, liver, kidney, and heart. A 258-bp segment of the 3'-untranslated region of lysyl oxidase cDNA is 93% identical with a highly conserved region of the 3'-untranslated sequence of rat elastin cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and expression analysis of a new member of the cytochrome P450, CYP2A15 from the Chinese hamster, encoding testosterone 7alpha-hydroxylase.

We cloned a new cytochrome P450 cDNA encoding testosterone 7alpha-hydroxylase in the Chinese hamster, designated CYP2A15 which shares significant amino acid sequence homology with members of the CYP2A subfamily. The CYP2A15 cDNA was isolated by screening a liver cDNA library and the sequence contains an open reading frame of 1482 nucleotides encoding a polypeptide of 493 amino acids with a calculated molecular mass of 56,295 Da. This is flanked by a 5'-untranslated region of 2 bp and a 3' untranslated region of 191 bp including the poly(A) tail. We determined the catalytic activity of CYP2A15 using microsomes obtained by transient expression of its cDNA in transfected COS-7 cells. The heterologously expressed CYP2A15 was found to hydroxylate testosterone at position 7alpha in a reconstituted system. RT-PCR experiments revealed that the mRNA of CYP2A15 was expressed in liver, but not detected in kidney, lung, or small intestine. The expression of CYP2A15 mRNA was slightly induced by treatment with either rifampicin or 3-methylcholanthrene.     

Vimentin cDNA clones covering the complete intermediate-filament protein are found in an EHS tumor cDNA library

Intermediate filaments are part of the cytoskeleton of most cells. To analyze changes in intermediate filament synthesis, we have isolated two cDNA clones (pV-C25, pV-C877) that cover the complete coding sequence of the murine intermediate filament protein vimentin. The cDNA clones were isolated from a murine Engelbreth-Holm-Swan (EHS) tumor cDNA library by screening under (i) non-stringent conditions with a synthetic oligodeoxynucleotide (oligo), LW-36, which is specific for type-IV collagen, and (ii) stringent conditions with oligo LW75, which was derived from the vimentin clone pV-C25. The cDNA clones contain 38 nucleotides (nt) of the 5'-untranslated region, 1398 nt of the coding region and 7 nt of the 3'-untranslated region. Comparing the mouse sequence with the published sequence for vimentin from hamster, human and chicken, we find shared identities of 99, 97 and 87%, respectively. Since the cDNA clones have been isolated from a basement membrane tumor (EHS) cDNA library, we measured the vimentin mRNA production in EHS tumor cells in culture, and found that this mRNA is half as abundant as mRNA for type-IV mRNA.     

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.     

Purification, characterization, and cDNA cloning of a novel metallothionein-like, cadmium-binding protein from Caenorhabditis elegans

Caenorhabditis elegans adapted for survival in high concentrations of Cd(II) express a heavy metal binding protein designated C. elegans metallothionein-like protein or MT-Ce. This protein was purified to homogeneity and characterized. MT-Ce binds 6 mol of Cd(II)/mol protein. The sequence of 39 amino-terminal residues in MT-Ce was determined. A radiolabeled 41-mer oligonucleotide, designed from the partial MT-Ce sequence, was used in conjunction with sucrose gradient centrifugation to obtain size-fractionated poly(A+) RNA enriched in MT-Ce sequences. Subsequently, cloned cDNAs, corresponding to MT-Ce mRNA sequences, were isolated from a lambda ZapII cDNA library prepared from the enriched template mRNA. cDNA and protein sequence analysis revealed that MT-Ce comprises 62 amino acid residues and has a predicted Mr of 6462. Seventeen of the 18 Cys residues in the nematode cadmium-binding protein are included in Cys-X-Cys and X-Cys-Cys-X motifs that are characteristic of mammalian metallothioneins (MTs). However, the resemblance of MT-Ce to mammalian MTs is superficial. The amino acid sequence of MT-Ce is unique, and neither its putative alpha and beta domains nor its Cys residues can be readily aligned with the corresponding regions of other eukaryotic MTs. This suggests that MT-Ce is an example of convergent evolution. The MT-Ce mRNA level in nematodes that were selected and grown with Cd(II) concentrations that are lethal for wild-type worms, was 55-fold higher than the level of MT-Ce mRNA in wild-type C. elegans. Comparison of the sequences of MT-Ce cDNAs revealed the occurrence of two types of MT-Ce mRNA. Each contains an identical coding region, but the cDNAs diverge markedly in their 5'-untranslated regions. This suggests the possibilities of regulation by alternative splicing and/or the presence of multiple MT-Ce genes encoding a single protein, but controlled by different regulatory elements.     

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.     

Nucleotide sequence of rat alpha 1-acid glycoprotein messenger RNA

The complete nucleotide sequence of rat alpha 1-acid glycoprotein (alpha 1-AGP) mRNA has been determined from cloned double-stranded cDNA. The coding portion of the mRNA was bounded at the ends by a 5'-untranslated region of 35 nucleotides in length and a 3'-untranslated region of 119 nucleotides in length. The 3'-untranslated region contains the characteristic AAUAAA sequence ending 18 nucleotides from the 3'-terminal poly(A) segment. The 5'-region of the mRNA contains two in-phase AUG codons separated by 12 nucleotides. Comparison with the known NH2-terminal amino acid sequence of serum rat alpha 1-AGP suggests that the primary translation product of the mRNA contains an additional 14 or 18 amino acids that are not present in the mature form of the protein, which contains 187 amino acids. The inferred amino acid sequence of rat alpha 1-AGP and the known amino acid sequence of human alpha 1-AGP have several regions of identity clustered in the NH2-terminal portion of the proteins. The carboxyl-terminal regions show significantly less homology. Six potential asparagine glycosylation sites are found in the rat sequence, and four of these sites are in positions similar to known glycosylation sites in the human protein. Furthermore, three of these potential glycosylation sites are in a region that exhibits extensive amino acid sequence conservation, suggesting that this region may be important for the biological function of alpha 1-AGP.     

Molecular cloning and primary structure of rat thyroxine-binding globulin

Rat thyroxine-binding globulin (TBG) cDNAs were isolated from a rat liver cDNA library by using a human TBG cDNA as a probe. From two overlapping cDNA inserts, an aligned cDNA sequence of 1714 nucleotides was obtained. There was 70% homology with human TBG cDNA over the span of 1526 nucleotides. In order to confirm that the cloned cDNA encodes rat TBG and to localize the NH2-terminal amino acid of the mature molecule, the protein was purified by affinity chromatography and subjected to direct protein microsequencing. The NH2-terminal amino acid sequence was identical with that deduced from the nucleotide sequence. The rat TBG cDNA sequenced consisted of a truncated leader sequence (35 nucleotides), the complete sequence encoding the mature protein (1194 nucleotides) and the 3'-untranslated region (485 nucleotides), containing two polyadenylation signals. It was deduced that rat TBG consists of 398 amino acids (Mr = 44,607), three NH2-terminal residues more than human TBG, with which it shares 76% homology in primary structure. Of the six potential N-glycosylation sites, four are located in conserved positions compared to human TBG. Northern blot analysis of rat liver revealed an approximately 1.8-kilobase TBG mRNA. Its amount increased markedly following thyroidectomy and decreased with thyroxine treatment in a dose-dependent manner.