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At least 595 records · Page 33Linked to original sources
PubMed1985

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30.

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].

Amino Acid Sequence↗
PubMed1985

Cloning and sequence analysis of porcine myoglobin cDNA.

Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heart-myoglobin mRNA. Sequence analysis revealed 59 nucleotides (nt) in the 5'-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3'-untranslated regions. The myoglobin cDNA showed a high G + C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-249], a high degree of homology is observed in the 5'-untranslated region and in parts of the 3'-untranslated region, as well as in the coding region.

Amino Acid Sequence↗
PubMed1985

Properties of rat and mouse beta-glucuronidase mRNA and cDNA, including evidence for sequence polymorphism and genetic regulation of mRNA levels.

cDNA clones containing partial sequences for beta-glucuronidase (beta G) were constructed from rat preputial gland RNA and identified by their ability to selectively hybridize beta G mRNA. One such rat clone was used to isolate several cross-hybridizing clones from a mouse-cDNA library prepared from kidney RNA from androgen-treated animals. Together, the set of mouse clones spans about 2.0 kb of the 2.6-kb beta G mRNA. Using these cDNA clones as probes, a genomic polymorphism for DNA restriction fragment size was found that proved to be genetically linked to the beta G gene complex. A fragment of beta G cDNA was subcloned into a vector carrying an SP6 polymerase promoter to provide a template for the in vitro synthesis of single-stranded RNA complementary to beta G mRNA. This provided an extremely sensitive probe for the assay of beta G mRNA sequences. Using either nick-translated cDNA or transcribed RNA as a hybridization probe, we found that mouse beta G RNA levels are strongly induced by testosterone, and that induction by testosterone is pituitary-dependent. During the lag period preceding induction, during the induction period itself, and during deinduction following removal of testosterone, beta G mRNA levels paralleled rates of beta G synthesis previously measured by in vivo pulse-labelling experiments. Genetic variation in the extent of induction affected either the level of beta G mRNA or its efficiency of translation depending on the strain of mice tested.

Amino Acid Sequence↗
PubMed1986

Cosmid vectors for high efficiency DNA-mediated transformation and gene amplification in mammalian cells: studies with the human growth hormone gene.

We have constructed two new recombinant cosmid vectors that can be used for direct expression and amplification of genomic DNA in mammalian cells. The vectors allow cloning of DNA fragments up to 40 kb in size. Each carries two dominant selectable markers: the bacterial neo gene and the mouse DHFR gene. In the first vector, pCV001, the neo and DHFR genes are regulated by the SV40 early promoter, and in the second, pAVCV007, by the avian sarcoma virus LTR promoter. The neo gene served as a dominant marker for the selection of transformants in all mammalian cell types, and we demonstrate here that the LTR promoter significantly improved the efficiency of DNA-mediated transformation of a human cell line. We isolated the human growth hormone genes from genomic libraries prepared in these cosmid vectors and used these recombinant cosmids for direct transfections of cultured cells. Selection of transformants in increasing concentrations of methotrexate led to the outgrowth of resistant cell populations carrying amplified copies of the DHFR marker. A 40-1000-fold coamplification of the hGH genes was observed in the different transfected cell lines, along with a corresponding increase in transcription and translation activity of the hGH gene. Gene amplification could be achieved in both DHFR deficient or normal cell lines. High level expression of a cloned gene mediated by gene amplification should facilitate characterization of DNA sequences, as well as isolation of specific gene products for biochemical, functional, and pharmacological studies.

Animals↗
PubMed1988

Complete sequence of the mouse type-II keratin EndoA: its amino-terminal region resembles mitochondrial signal peptides.

We have isolated a clone, pKA56, from a cDNA library prepared from poly(A)/RNA of F9ACc19 cells. Northern-blot analysis showed that this clone recognizes a 1.9-kb mRNA which is expressed strongly in F9 differentiated cells but only faintly detected in F9 stem cells. Sequence determination revealed that this mRNA codes for EndoA, the murine homologue of the human type-II keratin No. 8. This is the first report of the complete coding sequence of a mammalian keratin No. 8. Comparison of mouse EndoA with keratin No. 8 of humans, cows and frogs indicated a strong evolutionary conservation. The first 16 amino acid residues of the N-terminal domain of EndoA are also homologous to other type-II keratins and, to a lesser extent, to other intermediate filament (IF) proteins. Furthermore, this region is predicted to adopt an amphiphilic alpha-helical conformation similar to that of mitochondrial signal peptides. Conservation of that sequence and of other segments of the end domains of EndoA supports the idea that those regions are implicated in the specific organization of the IF network in the cell and in the interactions of IF with other cell constituents.

Amino Acid Sequence↗
PubMed1988

cDNA cloning and sequence analysis of a chicken gene expressed during the gonadal development and homologous to mammalian cytochrome P-450c17.

A cDNA clone, pLOA0511, was isolated from the cDNA library prepared from the left ovaries of one- to three-day-old chickens. The transcript corresponding to this cDNA clone is approx. 1.9 kb in length and is present in steroidogenic tissues (ovary, testis and adrenal gland) but is undetectable in non-steroidogenic tissues. Relative abundance of the transcript in the ovary and testis is high and developmentally regulated but is much lower in the adrenal gland. Relative levels of this transcript in the developing left ovary and the regressing right ovary of the one- to three-day-old chicken are similar. The nucleotide sequence of this cDNA clone shows significant homology to some mammalian cytochromes P-450. Out of the 508 amino acids (aa) coded, 244 and 243 aa residues are identical with those of the bovine and human cytochrome P-450c17 (steroid 17 alpha-hydroxylase/17,20 lyase), respectively. There are three highly conserved regions among the bovine, human and chicken sequences, one of which is unique to P-450c17. These data suggest strongly that this cDNA corresponds to the mRNA of a chicken cytochrome P-450c17.

Amino Acid Sequence↗
PubMed1988

Molecular cloning and expression in Escherichia coli of the cDNA coding for rat lipocortin I (calpactin II).

Lipocortins (LC) are a family of proteins that were initially described to be induced by glucocorticosteroids and to inhibit phospholipase A2 (PLA2). Using oligodeoxynucleotide probes corresponding to partial amino acid (aa) sequences of rat lipocortin I (LCI), we have isolated a cDNA clone for rat LCI from a cDNA library prepared from poly(A)+RNA of peritoneal cells of dexamethasone-treated rat. The cDNA insert (1355 bp) had an open reading frame of 1038 bp that encoded a 346-aa polypeptide (Mr 38,784). The nucleotide sequence and the amino acid sequence deduced from it showed high homology with the reported sequences of human LCI. A plasmid containing the trc promoter and cDNA sequence for 346 aa residues of the rat LCI was constructed and expressed in Escherichia coli. Antibody to human LCI crossreacted with the recombinant rat LCI, and the recombinant protein had characteristics of natural rat LCI including PLA2 inhibitory activity in vitro.

Amino Acid Sequence↗
PubMed1988

Isolation of a human cDNA of urokinase and its expression in COS-1 cells.

The cDNA encoding human urokinase (UK) has been isolated from a cDNA library prepared from human normal fibroblast (WI38) cells, which had been stimulated by endothelial cell growth factor and heparin. This cDNA was sequenced and found to contain a few silent substitutions, thus encoding the same amino acids as deduced from the published genomic sequence of UK. After modification, the cDNA of UK was inserted into a transient expression vector and used to transfect COS-1 cells. The recombinant UK protein (rUK) in the serum-free medium of transfected COS-1 cells was characterized by biochemical and functional assays. These studies indicated that rUK from COS-1 cells is glycosylated, enzymatically active, and very similar to native single-chain plasminogen activator (scuPA). Therefore, such rUK can be a convenient source of scuPA for any further studies.

Amino Acid Sequence↗
PubMed1988

Cloning and expression of the MspI restriction and modification genes.

The genes for the MspI restriction (R) and modification enzymes (recognition sequence CCGG) have been cloned into Escherichia coli using the vector pBR322. Clones carrying both genes have been isolated from libraries prepared with EcoRI, HindIII and BamHI. The smallest fragment that encodes both activities is a 3.6-kb HindIII fragment. Plasmids purified from the clones are fully resistant to digestion by MspI, indicating that the modification gene is functional in E. coli. The clones remain sensitive to phage infection, however, indicating that the endonuclease is dysfunctional. When the R gene is brought under the control of the inducible leftward promoter from phage lambda, the level of endonuclease increases and the level of methylase decreases, suggesting that the genes are transcribed in opposite directions.

Cloning, Molecular↗
PubMed1988

Molecular cloning of eel growth hormone cDNA and its expression in Escherichia coli.

cDNA clones coding for growth hormone (eGH) of Japanese eel (Anguilla japonica) have been isolated from a cDNA library prepared from pituitary gland poly(A)+ RNA. The nucleotide sequence of the eGH cDNA was determined. It codes for the prehormone of 209 amino acids (aa) including a putative signal peptide of 19 aa. The deduced amino acid sequence was identical with that determined for eGH protein. The primary structure of eGH was compared with those of other species growth hormones (chum salmon, chicken, rat, and human). Mature eGH was expressed in Escherichia coli harboring a plasmid in which the eGH cDNA was under control of the phage lambda pL promoter. Recombinant eGH polypeptide was immunoreactive to rabbit antiserum against natural eGH. Furthermore, eGH derivative with amino-terminal deletion (delta 1-3 eGH) was produced in E. coli reaching up to 5% of total cellular proteins.

Amino Acid Sequence↗
PubMed1989

Carp growth hormone: molecular cloning and sequencing of cDNA.

cDNA clones of the fish Cyprinus carpio growth hormone (GH) mRNA have been isolated from a cDNA library prepared from carp pituitary gland poly(A)+RNA. The nucleotide sequence of one of the carp GH cDNA clones containing an insert of 1164 nucleotides (nt) was determined. The cDNA sequence was found to encode a polypeptide of 210 amino acids (aa) including a signal peptide of 22 aa and to contain 5' and 3' untranslated regions of the mRNA of 36 and 498 nt, respectively. The carp GH presents a 63% amino acid sequence homology with the salmon GH, has structural features common with other GH polypeptides of mammalian or avian origin and contains domains of conserved sequence near the N- and C-terminal regions. Southern blot hybridization of carp genomic DNA with GH cDNA probes shows the presence of at least two GH-coding sequences in the fish genome.

Amino Acid Sequence↗
PubMed1989

The nucleotide sequence of a microvitellogenin encoding gene from the tobacco hornworm, Manduca sexta.

A microvitellogenin (mVg)-coding gene (mvg) has been isolated from a lambda phage library prepared from the tobacco hornworm, Manduca sexta. One of the lambda clones had a 15-kb insert and contained the entire mvg gene. A DNA fragment (3.0 kb) containing this mvg gene has been sequenced. Southern blot analysis showed that there may be more than one mvg gene in M. sexta. The putative transcriptional start point (tsp) for the cloned mvg was determined by primer extension analysis. This gene contains a single intron in the 5'-noncoding region. The 5'-flanking sequence was compared to the 5'-conserved regions of yolk polypeptide-encoding genes (yp) of Drosophila melanogaster. Two regions were found in the 5'-flanking sequence of the mvg gene that have 66% similarity to the D. melanogaster yp consensus sequence that is believed to be involved in gene expression controlled by ecdysteroids. Furthermore, the sequences flanking these two regions are also similar to the ecdysone-responsive elements found in several genes of D. melanogaster. In fact, preliminary experiments showed that mVg mRNA synthesis is induced by the 20-hydroxyecdysone. Four regions of the mvg gene resemble the upstream conserved regions of the two vitellogenin-encoding genes of the locust, Locusta migratoria. The nucleotide sequence of mvg has 70% similarity to the sequence of one or more of the 30-kDa hemolymph proteins of Bombyx mori. This indicates a very close evolutionary relationship between these proteins.

Amino Acid Sequence↗
PubMed1990

Cloning, expression and characterization of the Sau3AI restriction and modification genes in Staphylococcus carnosus TM300.

The genes encoding the restriction enzyme (ENase) and modification enzyme (MTase) of Staphylococcus aureus 3A (recognition sequence 5'-GATC-3') have been cloned in Staphylococcus carnosus TM300 using the vector pCA44. Clones carrying both genes were isolated from DNA libraries prepared with MboI + BamHI. The DNA region encoding M.Sau3AI was subcloned on a 3.66-kb EcoRV fragment in vector pT181mcs. Plasmids purified from the clones were resistant to digestion with Sau3AI, indicating that the sau3AIM gene was expressed and the product was functional in S. carnosus. Cell lysates of clones with both activities encoded on plasmid pSEM7, cut DNA with the same pattern as Sau3AI, showing that the sau3AIR gene was also expressed and the ENase was functional in S. carnosus. Sequence analysis shows that both genes are transcribed in the same direction and encode polypeptides with calculated Mrs of 56,477 for R.Sau3AI and 47,300 for M.Sau3AI. Efforts to clone one or both genes in Escherichia coli have so far failed.

Amino Acid Sequence↗
PubMed1991

Cloning of trichosanthin cDNA and its expression in Escherichia coli.

Several cDNA clones coding for trichosanthin (TCS) have been isolated from a cDNA library prepared from the poly(A)+RNA of the root tuber of Trichosanthes kirilowii Maximowicz. The nucleotide sequence codes for a protein of 289 amino acids (aa) including a putative signal peptide of 23 aa and an extra 19 aa at the C terminus; the latter two have not been found in TCS obtained from the natural product [Collins et al., J. Biol. Chem. 265 (1990) 8665-8669]. Recombinant TCS (reTCS) was synthesized in Escherichia coli, in which the cDNA without the signal sequence was expressed under the control of the trc promoter; reTCS was detected by a rabbit anti-TCS antiserum.

Amino Acid Sequence↗
PubMed1991

The gene encoding squalene epoxidase from Saccharomyces cerevisiae: cloning and characterization.

The gene (ERG1) encoding squalene epoxidase (ERG) from Saccharomyces cerevisiae was cloned. It was isolated from a gene library, prepared from an allylamine-resistant (AlR) S. cerevisiae mutant, by screening transformants in a sensitive strain for AlR colonies. The ERG tested in a cell-free extract from one of these transformants proved to be resistant to the Al derivative, terbinafine. From this result, we concluded that the recombinant plasmid in the transformant carried an allelic form of the ERG1 gene. The nucleotide sequence showed the presence of one open reading frame coding for a 55,190-Da peptide of 496 amino acids. Southern hybridization experiments allowed us to localize the ERG1 gene on yeast chromosome 15.

Allylamine↗
PubMed1991

Cloning and sequencing a cDNA encoding human ribosomal protein S25.

A full-length cDNA clone has been isolated from a cDNA library prepared from mRNA of adriamycin-resistant human leukemia HL60 cells. The nucleotide sequence of this cDNA has been determined and the protein coded for by the gene identified. The cDNA encodes a polypeptide of 125 amino acids (aa) with a deduced Mr of 13750. The deduced aa sequence of this protein has 56% homology to yeast ribosomal protein S31. Western-blot analysis using antibodies directed against a synthetic peptide based on the deduced aa sequence identifies the gene product as the human ribosomal protein S25.

Amino Acid Sequence↗
PubMed1991

Cloning and sequencing of a jack bean urease-encoding cDNA.

A cDNA which encodes the entire amino acid (aa) sequence of the mature jack bean urease has been cloned in Escherichia coli from a library prepared from the mRNA of developing jack beans. It was necessary to use reverse transcriptase in the cDNA was obtained in the form of two contiguous DNA fragments, each of which was completely sequenced. The conceptual translation of the nt sequence gave an 840-aa sequence which was identical to the directly determined sequence except for one conservative aa substitution (Takashima et al., Eur. J. Biochem. 175 (1988) 151-165). These data constitute the first report on the cloning and sequence of the cDNA encoding a urease from any higher plant.

Amino Acid Sequence↗
PubMed1992

Cloning and molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata.

The glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) has been identified from a genomic DNA library prepared from the plant pathogenic fungus Glomerella cingulata. Nucleotide sequence data revealed that this gene codes for a putative 338-amino-acid protein encoded by two exons of 129 and 885 bp, separated by an intron 216 bp long. The 5' leader sequence is also spliced by an intron of 156 bp. A cDNA clone was prepared using the polymerase chain reaction, the sequence of which was used to confirm the presence of the intron in the coding sequence and the splicing of the 5' leader sequence. The transcriptional start point (tsp) was mapped at -253 nt from the site of the initiation of translation by primer extension and is adjacent to a 42-bp pyrimidine-rich region. The general structure of the 5' flanking region shows similarities to gpdA from Aspergillus nidulans. The putative protein product is 71-86% identical at the aa level to GPDs from Aspergillus nidulans, Cryphonectria parasitica, Curvularia lunata, Podospora anserina and Ustilago maydis.

Amino Acid Sequence↗