Cloning and nucleotide sequence of the chlD locus

The nucleotide sequence of a Sau3A1 restriction nuclease fragment that complemented an Escherichia coli chlD::Mu cts mutant strain was determined. DNA and deduced amino acid sequence analysis revealed two open reading frames (ORFs) that potentially codes for proteins with amino acid sequence homology with binding protein-dependent transport systems. One of the ORFs showed a sequence that encoded a protein with properties that were characteristic of a hydrophobic inner membrane protein. The other ORF, which was responsible for complementing a chlD mutant, encoded a protein with conserved sequences in nucleotide-binding proteins and hydrophilic inner membrane proteins in active transport systems. A proposal that the chlD locus is the molybdate transport operon is discussed in terms of the chlD phenotype.     

Nucleotide sequence of the R721 shufflon.

The shufflon is a DNA region that undergoes complex rearrangement mediated by the product of a putative site-specific recombinase gene, rci. The DNA sequences of the shufflon region and the rci gene of IncI2 plasmid R721 were determined. The R721 shufflon consists of three invertible DNA segments that are homologous to the shufflon segments found in IncI1 plasmid R64. Structural analysis of open reading frames indicated that the R721 shufflon possibly functions as a biological switch for selecting one of the six pilV genes in which the N-terminal region is constant and the C-terminal region is variable. The R721 rci gene was shown to encode a basic protein of 374 amino acid residues.     

Cloning and nucleotide sequence of the chimpanzee c-myc gene

A DNA fragment covering the chimpanzee c-myc locus was cloned from the DNA of peripheral blood lymphocytes, sequenced, and compared to its human c-myc counterpart. The two nucleotide sequences were found to be highly homologous (99%). The divergence rate between the two species was 0.4% in exons and 1.7% in introns. The different TATA-boxes described in the human myc gene were also identified in the chimpanzee sequence and an open reading frame (ORF) was observed which overlaps the chimpanzee c-myc first exon. This latter ORF contained three silent mutations with regard to the human region, whereas the chimpanzee Myc oncoprotein coded by exons 2 and 3 differed by two amino acids from the human one.     

Cloning and nucleotide sequence of placental hGH-V cDNA

We have previously demonstrated the presence in human placenta and maternal serum of a GH variant, called human placental growth hormone (hPGH). We have also shown that the hGH-V gene is expressed at the placental level thus possibly coding for hPGH. The hGH-V cDNA has now been isolated from a lambda gt 11 human placenta cDNA library. Its sequence has been determined which firmly establishes the GH-V gene mode of splicing as well as the GH-V protein structure. Our data give final evidence of placental hGH-V gene expression and reinforce the hypothesis of identity between the hGH-V protein and hPGH.     

Cloning and nucleotide sequence of ovine prolactin cDNA

A cDNA expression library was constructed in the lambda gt 11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5' region of this cDNA. Translation initiation could occur at the 5' proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283-292)], with its substitution of the A at -3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3'-untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.     

Cloning and nucleotide sequence of the murine interleukin-3 gene

Southern hybridization analysis using a probe derived from a murine interleukin-3 (IL-3) cDNA clone revealed the presence of a single IL-3 gene in the haploid murine genome. An 8600-base-pair (8.6-kb) murine genomic EcoRI fragment containing the IL-3 gene was isolated by screening a library of size-fractionated genomic EcoRI fragments cloned in lambda gtWES X lambda B. The nucleotide sequence of a 3.5-kb region of the cloned DNA encompassing the IL-3 gene was determined. The gene contains four introns of 96, 993, 135 and 122 base pairs (bp), located within the coding region. The large intron contains 12 copies of a 14-15-bp tandem repeating sequence which resembles a human cellular homologue of a BKV enhancer sequence. The nucleotide sequence of the exons agrees exactly with that of an IL-3 cDNA cloned from WEHI-3, a tumorigenic cell line which over-produces IL-3, establishing that the unprocessed primary structure of IL-3 is identical in WEHI-3 and in BALB/c mice. Southern hybridization has revealed genomic alteration in the vicinity of the IL-3 gene in WEHI-3 cells.     

Cloning and nucleotide sequence analysis of the human beta-microseminoprotein gene

Beta-microseminoprotein (MSP) is a small protein (94 amino acids) synthesized by the epithelial cells of the prostate gland and secreted into the seminal plasma. Restriction endonuclease mapping of human genomic DNA with a human MSP cDNA probe identified a 19 kilobase (Kb) hybridizing band in both EcoRI and BamHI digestions. Subsequently, the 19 Kb EcoRI fragment of human genomic DNA containing the MSP gene was isolated and cloned into an EMBL4 phage vector. Screening of the recombinant phages resulted in the isolation of one clone containing the MSP gene. Restriction endonuclease mapping and sequence analysis of this clone revealed the human MSP gene of approximately 15 Kb in length. The gene contains four exons and three large introns of approximately 6, 1, and 7 Kb.     

Cloning and nucleotide sequence of rat ornithine decarboxylase cDNA

The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17) catalyses the first and rate-limiting step in polyamine biosynthesis. Its activity is markedly increased in rapidly growing or regenerating tissue and is subject to regulation by a variety of trophic and mitogenic stimuli. ODC is therefore believed to play an essential role in the onset of cellular proliferation. In a molecular-biological approach to investigate ODC regulation upon induction by tumor promoters in rat liver we isolated an almost full-length rat ODC cDNA clone of 2.4 kb (designated pODC.E10) from a cDNA library of testosterone-induced rat kidney poly(A)+ RNA. Characterization by restriction-endonuclease mapping and sequence analysis showed strong homology to mouse ODC cDNA sequences previously published [Gupta and Coffino, J. Biol. Chem. 260 (1985) 2941-2944; Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673-1677; Hickok et al., Proc. Natl. Acad. Sci. USA 83 (1986) 594-598]. This homology is most pronounced in the 461-aa-spanning coding region, amounting to 94% and 97% at the DNA and protein levels, respectively. In the 423-nt 5' leader the rat-mouse homology (approx. 75%) is most pronounced in a region of about 175 nt directly upstream from the translational start site. The leader sequence also contains a perfect inverted repeat of 54 nt and ten additional upstream ATG triplets, which are all followed by nonsense codons before the initiating ATG. In the 633-nt 3' trailer region of pODC.E10 an additional polyadenylation signal is observed more than 300 nt upstream from the 3' end. Rat-mouse homology is about 80% up to this first polyadenylation signal and is considerably less thereafter. The presence of two alternate polyadenylation sites most likely accounts for the 3' size heterogeneity observed in the two ODC mRNAs of 2.1 and 2.6 kb, respectively. In rat liver both mRNAs are coordinately induced by different tumor promoters. Finally, Southern blot analysis of normal rat liver and rat hepatoma DNA revealed that rat ODC, as in other rodents, belongs to a multigene family.     

Cloning and nucleotide sequence of Bacillus stearothermophilus pyruvate carboxylase

A gene for prokaryotic pyruvate carboxylase (PC) was cloned from Bacillus stearothermophilus. It has an open reading frame of 3441 base pairs which can code for a protein of 128 353 Da. Not only the molecular size and domain organization but also the deduced amino acid sequence of B. stearothermophilus PC are similar to those of eukaryotic PCs.     

Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens

The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.     

Cloning and nucleotide sequence of the Brucella abortus groE operon.

The cloning and sequencing of the Brucella abortus groES and groEL genes are reported. The genes are adjacent on the Brucella chromosome, and presumably comprise a functional operon. Putative promoter and terminator sequences are also identified. The groES gene exhibits 60%, and the groEl gene 69%, sequence identity with the corresponding Escherichia coli genes.     

Cloning and nucleotide sequence of the Vibrio proteolyticus aminopeptidase gene.

The gene encoding the Vibrio proteolyticus aminopeptidase was cloned and sequenced and its amino acid sequence was deduced. The gene encodes a 54 kDa protein, larger than the previously reported size of 30 kDa for the purified aminopeptidase. Sequence alignments revealed a 43-45% homology with two other Vibrio sp. extracellular proteinases.     

Cloning and characterization of a highly repetitive fish nucleotide sequence

We have cloned and sequenced a highly repetitive HindIII fragment of DNA from the common carp Cyprinus carpio. It represents a tandemly repeated sequence with a monomeric unit of 245 bp and comprises 8% of the fish genome. Higher units of this monomer appear as a ladder in Southern blots. The monomeric unit has been sequenced; it is A + T-rich with some direct and some inverse-repeat nucleotide clusters.     

Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.