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PubMed1996

Molecular cloning and expression of the pyrimidine nucleoside phosphorylase gene from Bacillus stearothermophilus TH 6-2.

The pyrimidine nucleoside phosphorylase (Py-NPase) of Bacillus stearothermophilus TH 6-2 is a dimer of 46-kDa subunits and catalyzes the reversible phosphorolysis of uridine and thymidine. The gene encoding this pyrimidine nucleoside phosphorylase (pyn gene) has been cloned and sequenced from B. stearothermophilus TH 6-2. The pyn gene corresponded to an open reading frame of 1299 nucleotides that translates into a putative 433 amino acid protein with a molecular weight of 46,271. The deduced amino terminal sequence of Py-NPase coincided with that previously found for the purified enzyme. The deduced amino acid sequence of Py-NPase shared significant similarity with those of human and Escherichia coli thymidine phosphorylases. The cloned pyn gene was overexpressed in E. coli cells to produce an active enzyme in large quantities that accounted for approximately 20% of the total protein.

Amino Acid Sequence↗
PubMed1985

Cloning and structure of the gene for the subunits of aspartokinase II from Bacillus subtilis.

A library of Bacillus subtilis DNA in lambda Charon 4A (Ferrari, E., Henner, D.J., and Hoch, J.A. (1981) J. Bacteriol. 146, 430-432) was screened by an immunological procedure for DNA sequences encoding aspartokinase II of B. subtilis, an enzyme composed of two nonidentical subunits arranged in an alpha 2 beta 2 structure (Moir, D., and Paulus, H. (1977a) J. Biol. Chem. 252, 4648-4654). A recombinant bacteriophage was identified that harbored an 18-kilobase B. subtilis DNA fragment containing the coding sequences for both aspartokinase subunits. The coding sequence for aspartokinase II was subcloned into bacterial plasmids. In response to transformation with the recombinant plasmids, Escherichia coli produced two polypeptides immunologically related to B. subtilis aspartokinase II with molecular weights (43,000 and 17,000) indistinguishable from those found in enzyme produced in B. subtilis. Peptide mapping by partial proteolysis confirmed the identity of the polypeptides produced by the transformed E. coli cells with the B. subtilis aspartokinase II subunits. The size of the cloned B. subtilis DNA fragment could be reduced to 2.9 kilobases by cleavage with PstI restriction endonuclease without affecting its ability to direct the synthesis of complete aspartokinase II subunits, irrespective of its orientation in the plasmid vector. Further subdivision by cleavage with BamHI restriction endonuclease resulted in the production of truncated aspartokinase subunits, each shortened by the same extent. This suggested that a single DNA sequence encoded both aspartokinase subunits and provided an explanation for the earlier observation that the smaller beta subunit of aspartokinase II was highly homologous or identical with the carboxyl-terminal portion of the alpha subunit (Moir, D., and Paulus, H. (1977b) J. Biol. Chem. 252, 4655-4661). A map of the gene for B. subtilis aspartokinase II is proposed in which the coding sequence for the smaller beta subunit overlaps in the same reading frame the promoter-distal portion of the coding sequence for the alpha subunit.

Antigen-Antibody Complex↗
PubMed1983

Cloning of the Vibrio harveyi luciferase genes: use of a synthetic oligonucleotide probe.

A mixed-sequence synthetic oligonucleotide probe was used to isolate a clone containing the gene encoding the alpha subunit of bacterial luciferase from Vibrio harveyi and part of the gene coding for the beta subunit. DNA sequence analysis has allowed us to determine that the genes are closely linked on the bacterial chromosome and transcribed in the same direction. Comparison of the sequences in the regions preceding the two structural genes has revealed considerable homology and has identified sites that may be involved in the expression of the genes. Identification of a clone from a clone bank of total genomic DNA from this organism shows that mixed probes can be successfully used to isolate a gene of interest from any bacterium provided some protein sequence for the gene product is available.

Amino Acid Sequence↗
PubMed1993

Introduction and expression of the bacterial genes cysE and cysK in eukaryotic cells.

The coding sequences of the cysE and cysK genes from Escherichia coli, which encode the enzymes of the cysteine biosynthetic pathway, namely, serine acetyltransferase (EC 2.3.1.30) and O-acetylserine sulfhydrylase (or cysteine synthase [EC 4.2.99.8]), were modified for expression in eukaryotic cells and introduced into murine L cells. A number of fusion genes comprising the cysE or cysK coding sequences joined to the promoter of the ovine metallothionein-Ia (MT-Ia) gene and various portions of the ovine growth hormone (GH) gene were prepared. Significant differences in the level of transcription were observed, depending on the amount and arrangement of the GH gene sequences used, the highest levels being obtained with the constructs MTCE10 and MTCK7, which contained only the GH 3' untranslated gene sequences. These two constructs were fused to produce the gene MTCEK1. In this single DNA sequence, each bacterial gene is under independent MT-Ia promoter control. Expression of the cysK sequence in this construct (MT-Ia promoter-cysE-3' GH sequence-MT-Ia promoter-cysK-3' GH sequence) was elevated compared with expression of the cysK gene in MTCK7. However, expression of the cysE sequence in MTCEK1 was only 40% of that of the cysE gene cloned into MTCE10. The double-promoter configuration, which enhances the expression of the second gene in MTCEK1, is proposed as a model for the modification of bacterial genes in general.

Acetyltransferases↗
PubMed1990

The meta cleavage operon of TOL degradative plasmid pWW0 comprises 13 genes.

The meta-cleavage operon of TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are xylX(57)-xylY(20)-xylZ(39)-xylL(28)-xylT(1 2)-xylE(36)-xylG(60)-xylF(34)- xylJ(28)-xylQ(42)-xylK(39)-xylI(29)-xylH(4 ). The xylXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xylE, xylG, xylF, xylJ, xylK, xylI, and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxaloccotonate tautomerase, respectively. The functions of xylT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xylX and xylH consists of coding sequences.

Bacterial Proteins↗
PubMed1994

Molecular characterization of the extracellular poly(3-hydroxyoctanoic acid) [P(3HO)] depolymerase gene of Pseudomonas fluorescens GK13 and of its gene product.

phaZPfi, the gene encoding the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, was cloned, sequenced, and characterized. It comprises 837 bp and is transcribed as a monocistronic message of about 950 bp from a putative sigma 70-like promoter 32 bp upstream of the ATG start codon. The deduced protein of 278 amino acids reveals a typical leader peptide at its N terminus. When expressed in Escherichia coli, the mature depolymerase started with Ala-23, whereas the mature enzyme purified from P. fluorescens GK13 started with both Leu-34 and Arg-35 determining proteins of 26,687 and 26,573 Da, respectively. The depolymerase is a strongly hydrophobic protein and includes the lipase consensus sequence Gly-X-Ser-X-Gly, which is known for serine hydrolases. Replacement of the central residue, Ser-172, in the corresponding sequence (Gly-Ile-Ser-Ser-Gly) of PhaZPfl with alanine resulted in complete loss of enzyme activity, indicating that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.

Amino Acid Sequence↗
PubMed1991

Yolk polypeptide gene expression in cultured Drosophila cells.

The transfer of chimaeric plasmids to Drosophila melanogaster cell lines has been examined as a system for investigation of the hormonal regulation of the genes coding for D. melanogaster yolk polypeptide 1 (YP1) and Locusta migratoria vitellogenin B (VgB). Constructs containing promoters and putative 5'-regulatory sequences from these genes, ligated to bacterial chloramphenicol acetyltransferase (CAT) coding DNA, were transfected into Drosophila Kc (Kc-H) and S3 cells, and transient expression of CAT was assayed. Activity was expressed both from the homologous promoter of pYP1CAT and from the heterologous locust promoter of pVgCAT at comparable levels. In S3 cells, with calcium phosphate-mediated transfer of pYP1CAT there was a twofold induction of CAT activity after the addition of 10(-6) M ecdysterone, but no hormonal stimulation was noted when the polycation polybrene was used to achieve transfection. For Kc cells, calcium phosphate was ineffective for transfection, and after transfection with polybrene neither pYP1CAT nor pVgCAT was induced by the juvenile hormone (JH) analog methoprene. It is concluded that S3 cells may be useful for investigating the molecular basis of gene regulation by ecdysteroids, but conditions suitable for the analysis of JH action have not yet been established.

Animals↗
PubMed1989

Regulated expression of foreign genes in mammalian cells under the control of coliphage T3 RNA polymerase and lac repressor.

Systems that stringently regulate the expression of individual genes within a complex genetic background have contributed greatly to the analysis of gene function. In this report the development of a highly regulated expression system in mammalian cells is described in which transcription of a foreign gene is mediated by the bacteriophage T3 RNA polymerase under the control of the Escherichia coli lac repressor. Rabbit kidney cell lines have been established that constitutively express the phage RNA polymerase and lac repressor. The two bacterial proteins regulate the transcription of the coding sequence of the firefly luciferase, which has been placed under the control of a T3 promoter/lac operator fusion. In the presence of the inducer isopropyl beta-D-thiogalactoside, efficient T3 polymerase-dependent transcription is observed, which is tightly repressed in the absence of inducer. Translation of the T3 transcripts can be mediated by vaccinia virus functions. The demonstration that a specific transcription activity can be regulated over a range of several orders of magnitude in higher eukaryotic cells by using a highly specific and nontoxic inducer has broad implications for a variety of studies.

Animals↗
PubMed1990

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium.

The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5' end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.

Acetolactate Synthase↗
PubMed1994

Exploring the microtubule-binding region of bovine microtubule-associated protein-2 (MAP-2): cDNA sequencing, bacterial expression, and site-directed mutagenesis.

A 1.1 kilobase fragment of bovine microtubule-associated protein-2 (MAP-2) cDNA coding for bovine MAP-2 microtubule-binding region (MTBR) was sequenced. Relative to mouse, rat, and human MAP-2, we observed striking preservation of primary structure, even beyond the sequence and spacing of the three nonidentical peptide repeats responsible for microtubule-binding interactions. For further analysis of microtubule-MAP interactions using site-directed mutagenesis, we developed a bacterial expression system coding for the MT-binding fragment of MAP-2 starting at the thrombin cleavage site (position 1629) and continuing to the C-terminus. This MT-binding fragment was purified to homogeneity by taking advantage of the unusual heat-stability and isoelectric properties of this cytomatrix component. We found that the MT-binding domain readily promoted tubulin polymerization, and the critical tubulin concentration was reduced in the presence of this recombinant protein. Because a second repeated sequence analogue can promote tubulin polymerization as well as displace the MT-binding region of MAP-2, this study was designed to learn more about the importance of each repeated sequence in MT binding. Accordingly, we mutated the first and third sequences to resemble the second repeated sequence, thereby generating the mutants designed m12-m2-m3, m1-m32, and m12-m2-m32. These recombinant proteins bound with an affinity comparable to or slightly better than equal concentrations of wild-type MT-binding fragment. Likewise, when the first or third sequence was replaced by an exact copy of the second octadecapeptide repeat, there was little, if any, increase in binding affinity, as reflected in the ability of mutant MT-binding fragments to promote tubulin polymerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Selenocysteine: the 21st amino acid.

Great excitement was elicited in the field of selenium biochemistry in 1986 by the parallel discoveries that the genes encoding the selenoproteins glutathione peroxidase and bacterial formate dehydrogenase each contain an in-frame TGA codon within their coding sequence. We now know that this codon directs the incorporation of selenium, in the form of selenocysteine, into these proteins. Working with the bacterial system has led to a rapid increase in our knowledge of selenocysteine biosynthesis and to the exciting discovery that this system can now be regarded as an expansion of the genetic code. The prerequisites for such a definition are co-translational insertion into the polypeptide chain and the occurrence of a tRNA molecule which carries selenocysteine. Both of these criteria are fulfilled and, moreover, tRNASec even has its own special translation factor which delivers it to the translating ribosome. It is the aim of this article to review the events leading to the elucidation of selenocysteine as being the 21st amino acid.

Bacterial Proteins↗
PubMed1993

The Streptomyces coelicolor glnR gene encodes a protein similar to other bacterial response regulators.

The Streptomyces coelicolor glnR gene positively regulates the transcription of the glutamine synthetase-encoding glnA gene. The nucleotide sequence of a 1682-bp DNA segment containing glnR was determined. The deduced amino acid sequence of the GlnR protein was found to be similar to the sequence of several bacterial response regulators that are known to function as transcriptional activators. Primer extension analysis of glnR mRNA identified three transcriptional start points (tsp) upstream from the glnR coding sequence.

Amino Acid Sequence↗
PubMed1986

The alpha 2 cDNA sequence of human haptoglobin carries a bacterial promoter functional in vivo.

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete alpha 2FS beta precursor protein or only for the beta subunit have been placed under the control of the lambda PR promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the PR promoter, the complete alpha 2FS beta constructions constitutively express a smaller polypeptide of approximately 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (alpha 2 PF and alpha 2 PS) located in the duplicated alpha 2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp alpha 2 cDNA sequence, when fused upstream to the cDNA coding for alpha 1-antitrypsin, constitutively promotes in vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against alpha 1-antitrypsin or haptoglobin.

Cloning, Molecular↗
PubMed1994

Enhancement of serine-sensitivity by a gene encoding rhodanese-like protein in Escherichia coli.

When cells of Escherichia coli are grown on lactate (or other carbon sources), an addition of serine to the medium causes growth inhibition. This growth inhibition is caused by inhibition by serine of homoserine dehydrogenase I, which is involved in threonine-isoleucine biosynthesis [Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T. (1990) Biochem. Biophys. Res. Commun. 168, 1211-1216]. We have cloned and sequenced genes which enhance the serine-sensitivity. Two open reading frames were found and designated as sseA and sseB. Introduction of either sseA or sseB gene, or both, into E. coli cells enhanced the serine-sensitivity. The sseA gene elicited stronger enhancement than sseB. The deduced amino acid sequence of SseA showed considerable similarity with that of bovine liver rhodanese, which catalyzes sulfur transfer from thiosulfate. We observed a twofold increase in rhodanese activity in E. coli cells harboring a plasmid carrying the sseA gene. The position of sseA in the genetic map is around 52'. However, sseA is different from cysM, which codes for O-acetylserine sulfhydrylase-B, an enzyme catalyzing sulfur transfer from thiosulfate to O-acetylserine, the map position of which is also around 52'.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence and molecular evolution of the gene coding for glyceraldehyde-3-phosphate dehydrogenase in the thermoacidophilic archaebacterium Sulfolobus solfataricus.

A Sulfolobus solfataricus genomic library cloned in the EMBL3 phage was screened using as probes synthetic oligonucleotides designed from the known amino acid sequence of a peptide obtained from the purified glyceraldehyde-3-phosphate dehydrogenase (aGAPD) protein. The screening led to the isolation of six recombinant phages (lambda G1-lambda G6) and one of them (lambda G4) contained the entire GAPD gene. The deduced amino acid sequence accounts for a protein made of 341 amino acids and the initial methionine is encoded by a GTG triplet. Alignment of the S. solfataricus aGAPD sequence versus GAPD from archaea, eukarya, and bacteria showed that aGAPD is very similar to other archaebacterial but not to eukaryotic or eubacterial GAPD. For known archaebacterial GAPD sequences, the rate of nucleotide substitutions per site per year showed that these sequences are homologous not only at the amino acid but also at the nucleotide level. The evolutionary rates are nearly similar to those reported for other eukaryotic genes.

Amino Acid Sequence↗
PubMed1989

Gene UL2 of herpes simplex virus type 1 encodes a uracil-DNA glycosylase.

An insertion mutant of herpes simplex virus type 1 has been constructed which carries the lacZ gene from Escherichia coli within the coding sequence of gene UL2, which is in the long unique region of the genome. In a one-step growth curve experiment this recombinant (called in 1601) grew as well as the wild-type (wt) parent virus, indicating that the UL2 gene is dispensable for growth in tissue culture. Analysis of in 1601 DNA with restriction endonucleases showed no detectable changes from the wt apart from the insertion. Extracts of cells infected with in 1601 possessed levels of viral DNA polymerase and alkaline exonuclease activities similar to those infected with the wt, but unlike the wt had negligible uracil-DNA glycosylase activity, suggesting strongly that the product of the UL2 gene is the uracil-DNA glycosylase. The sequence of the uracil-DNA glycosylase gene of E. coli was recently published, and the encoded amino acid sequence of this shows clear similarity to that of UL2, confirming our results.

Amino Acid Sequence↗
PubMed1990

A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy.

Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol. At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3. At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.

Bacteriophages↗