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PubMed1983

Characterization of the yeast mitochondrial locus necessary for tRNA biosynthesis: DNA sequence analysis and identification of a new transcript.

Most components necessary for the biosynthesis of mitochondrial tRNAs are coded by nuclear genes, but one mitochondrial locus other than the tRNA genes themselves is required to make functional tRNAs in the yeast Saccharomyces cerevisiae. DNA sequence analysis of this yeast mitochondrial tRNA synthesis locus is reported here. This region of mitochondrial DNA is almost exclusively A+T-rich DNA with one G+C-rich element. Despite the unusual structure of the DNA in this region, we have demonstrated that it codes for a heretofore unidentified mitochondrial transcript about 450 bases in length. Since this RNA is the only RNA encoded by the tRNA synthesis locus, it must be the active agent of the locus. This RNA could either act autonomously through RNA-RNA interactions or as part of an RNA-protein complex to effect tRNA biosynthesis.

Base Sequence↗
PubMed1989

Two precursors of melanin-concentrating hormone: DNA sequence analysis and in situ immunochemical localization.

Two precursors to Chinook salmon (Oncorhynchus tshawytscha) melanin-concentrating hormone, an important factor in teleosts involved in the control of skin pigmentation and stress responsiveness, have been identified from DNA sequence analysis. Both precursors encode proteins of 132 amino acids and they share 107/132 amino acid identities. The biologically active 17-residue peptide is located at the C terminus of both precursors and can be liberated by proteolytic cleavage following two adjacent arginine residues. Additional putative proteolytic processing sites are located within the two precursors. Northern analysis demonstrated an intense hybridization signal of 750 nucleotides in the hypothalamus. Immunocytochemical studies as well as in situ hybridization analyses identify intensely staining cell bodies in the hypothalamus in the area of the lateral tuberal nucleus.

Amino Acid Sequence↗
PubMed1988

DNA sequence analysis of the Olir2-76 and Ossr1-92 alleles of the Oli-2 region of the yeast Saccharomyces cerevisiae. Analysis of related amino-acid substitutions and protein-antibiotic interaction.

Petite deletion mapping helped to generate a fine-structure genetic map of the Oli-2 region of the mitochondrial genome of Saccharomyces cerevisiae. Here we report the DNA sequence analysis of the Oli-2 region from two drug-resistant alleles (Olir2-76 and Ossr1-92) which are located in the gene for subunit-6 of mitochondrial ATPase, in agreement with their genetic locations on the mitochondrial genome. An analysis of the corresponding amino-acid substitutions is also presented in the context of protein-antibiotic interactions.

Adenosine Triphosphatases↗
PubMed1989

DNA sequence analysis of the HLA-DRw12 allele.

The complete DNA sequence of a DR beta chain cDNA encoding the DRw12 allele has been determined. The sequence of this DRB1 allele reveals a structural relationship to the group of other DRB1 genes found on DRw52 haplotypes, such as DR3, -w11, -w13, -w14, and -w8. The structural similarities among this group of alleles are particularly evident in the first hypervariable as well as in the 3' untranslated region. The second hypervariable region contains a unique sequence not identified in any other DRB1 allele. The third hypervariable region appears to have arisen by gene conversion events involving two DRB1 chain genes, DR7 and DR1 or DR2/Dw21.

Alleles↗
PubMed1992

Cloning and DNA sequence analysis of a Serpulina (Treponema) hyodysenteriae gene encoding a periplasmic flagellar sheath protein.

A Serpulina (Treponema) hyodysenteriae expression library was constructed in vector lambda ZAP and screened with a polyclonal antiserum raised against S. hyodysenteriae periplasmic flagella. A single immunoreactive plaque was chosen for further analysis. The recombinant phage from this plaque contained a gene encoding the 44-kDa protein that is on the outer layer (or sheath) of the periplasmic flagella. DNA sequence analysis showed that the gene encodes a protein of 320 amino acids. The protein is homologous to the flagellar sheath proteins of Treponema pallidum and Spirochaeta aurantia but not to any other flagellar proteins. We designated the cloned S. hyodysenteriae flagellar sheath protein gene flaA and the encoded protein FlaA. The 19 N-terminal amino acid residues of FlaA constitute a signal peptide that is cleaved from the protein before assembly onto the flagella in the periplasm. Amino acid residues 20 to 38 correspond to the N-terminal amino acid sequence of the native protein. Upstream from the gene, DNA motifs that are similar to the consensus Escherichia coli -35 and -10 promoter sequences and a ribosome binding site were identified. Downstream from the gene, two inverted repeat sequences that may serve as a rho-independent transcription termination signal are present.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and DNA sequence analysis of cDNA encoding chicken homologue of the Bcl-2 oncoprotein.

We have isolated a 2228 bp cDNA clone encoding a chicken homologue of the human Bcl-2 oncoprotein by low-stringency hybridization screening of a lambda gt10 cDNA library derived from a chicken B-cell lymphoma. DNA sequence analysis of this cDNA revealed an open reading frame predicting a polypeptide of 232 amino acids and an M(r) of 25,839. The predicted protein is highly homologous to the human (73%) and mouse (70%) Bcl-2 proteins, and contains a hydrophobic stretch of amino acids within its carboxyl-end (213-229) consistent with an integral membrane protein. Areas of very high sequence homology shared by all three Bcl-2 proteins at the NH2-terminus (amino acids 1-33) and within the last 150 amino acids of these proteins suggest the presence of at least two evolutionarily conserved domains within the family of Bcl-2 proteins that may be important either for their targeting to mitochondria or their ability to block programmed cell death.

Amino Acid Sequence↗
PubMed1986

DNA sequence analysis of diuron-resistant mutations in the mitochondrial cytochrome b gene of Saccharomyces cerevisiae.

Diuron (3-[3,4-dichlorophenyl]-1,1-dimethylurea), an inhibitor of mitochondrial respiration, blocks the yeast respiratory chain between cytochrome b and c1. Diuron-resistant mutants of Saccharomyces cerevisiae have been selected and several mutations localized to the mitochondrial cytochrome b gene. The present paper identifies specific DNA base changes within the cytochrome b gene conferring diuron-resistance. DNA sequence analysis was done utilizing primer extension of crude mitochondrial RNA preparations in the presence of reverse transcriptase. Five independent diuron-resistant mutations have been sequenced.

Amino Acid Sequence↗
PubMed1984

DNA sequence analysis of the Serratia marcescens ompA gene: implications for the organisation of an enterobacterial outer membrane protein.

The cloned ompA gene from Serratia marcescens was fully expressed in Escherichia coli and its product correctly assembled into the outer membrane. The S. marcescens polypeptide was not functionally equivalent to the E. coli OmpA protein, which serves as a phage receptor and as a component of several colicin uptake systems. DNA sequence analysis of the gene showed that three regions of the protein likely to be exposed on the cell surface not only differed extensively from the corresponding regions of the E. coli polypeptide but also from all other sequenced OmpA proteins. It is suggested that this sequence polymorphism represents a safety mechanism by which the various enterobacterial species can avoid cross-infection by noxious agents such as phages or colicins.

Bacterial Outer Membrane Proteins↗
PubMed1990

DNA sequence analysis of in vivo hprt mutation in human T lymphocytes.

We have determined the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (hprt) mutations that arose in vivo in the T lymphocytes of a normal male subject. In previous studies approximately 16% (23/141) of the mutants from this individual analyzed by Southern blot displayed large structural alterations in hprt. Thirty-two mutants without these large hprt structural alterations produced sufficient hprt cDNA for polymerase chain reaction amplification and DNA sequence analysis. Base substitutions in hprt cDNA resulting in missense mutations and one mRNA splicing aberration (inclusion of intron sequences) were observed in 18/32 of these these mutants; substitutions occurred at both AT and GC base pairs. Small deletions (3/32), a tandem change and a single base insertion were also observed among the hprt cDNAs. Exon skipping and inclusion of hprt intron sequences in the hprt cDNA were observed in an additional 9/32 of the mutants. Analysis of T cell receptor (TCR) gene rearrangements revealed that six of eight mutants with an identical hprt T----A transversion displayed the same TCR rearrangement pattern, indicating that they were clonally related and arose from a single in vivo mutational event.

Base Sequence↗
PubMed1986

Cloning and DNA sequence analysis of the wild-type and mutant cyclic AMP receptor protein genes from Salmonella typhimurium.

The crp gene from Salmonella typhimurium, as well as two mutant adenylate cyclase regulation genes designated crpacr-3 and crpacr-4, were cloned into the EcoRI site of plasmid pUC8. Initially cloned on 5.6-kilobase fragments isolated from EcoRI digests of chromosomal DNA, these genes were further subcloned into the BamHI-EcoRI site of plasmid pBR322. When tested, Escherichia coli crp deletion strains harboring the clones regained their ability to pleiotropically ferment catabolite-repressible sugars. Also, the crpacr-containing strains displayed sensitivity to exogenous cyclic AMP (cAMP) when grown on eosin-methylene blue medium with xylose as the carbon source. The proteins encoded by the S. typhimurium wild-type and mutant crp genes were found to have similar molecular weights when compared with the wild-type cAMP receptor protein (CRP) from E. coli. DNA sequence analysis of the wild-type crp gene showed only a three-nucleotide difference from the E. coli sequence, suggesting little divergence of the crp gene between these organisms. The crpacr sequences, however, each contained single nucleotide changes resulting in amino acid substitutions at position 130 of the CRP. Based on the site at which these substitutions occur, the crpacr mutations are believed to affect CRP-cAMP interactions.

Amino Acid Sequence↗
PubMed1989

DNA sequence analysis, gene product identification, and localization of flagellar motor components of Escherichia coli.

The Escherichia coli operon designated flaA contains seven flagellar genes; among them are two switch protein genes whose products are believed to interface with the motility and chemotaxis machinery of the cell. Complementation analysis using several plasmids carrying different portions of the flaA operon and analysis of expression of these plasmids in minicells allowed the identification of two flagellar gene products. The MotD (now called FliN) protein, a flagellar switch protein, was determined to have an apparent molecular weight of 16,000, and the FlaAI (FliL) protein, encoded by a previously unidentified gene, had an apparent molecular weight of 17,000. DNA sequence analysis of the motD gene revealed an open reading frame of 414 base pairs. There were two possible initiation codons (ATG) for motD translation, the first of which overlapped with the termination codon of the upstream gene, flaAII (fliN). The wild-type flaAI gene on the chromosome was replaced with a flaAI gene mutated in vitro. Loss of the flaAI gene product resulted in a nonmotile and nonflagellated phenotype. The subcellular location for both the MotD and FlaAI proteins was determined; the FlaAI protein partitioned exclusively in the insoluble fraction of a whole minicell sonic extract, whereas the MotD protein remained in both the soluble and insoluble fractions. In addition, we subcloned a 2.2-kilobase-pair DNA fragment capable of complementing the remaining four genes of the flaA operon (flbD [fliO], flaR [fliP], flaQ [fliQ], and flaP [fliR]).

Amino Acid Sequence↗
PubMed1990

Comparative DNA sequence analysis of the host shutoff genes of different strains of herpes simplex virus: type 2 strain HG52 encodes a truncated UL41 product.

Herpes simplex virus (HSV) particles contain a factor which can shut off host protein synthesis during the earliest stages of infection. The efficiency of shutoff varies between different strains of virus, type 2 strains being generally more active than type 1 strains. However, HSV-2 strain HG52 is deficient in host cell shutoff. We have investigated the basis of the different shutoff phenotypes of a strong shutoff strain (HSV-2 strain G), a weak shutoff virus (HSV-1 strain 17 syn+) and HG52 by comparative DNA sequence analysis of gene UL41, which encodes the virion-associated host shutoff factor. The results show that the UL41 genes of strains G and 17 are 86% homologous and that the lack of shutoff by HG52 is likely to be explained by a frameshift mutation within its UL41 coding sequence.

Amino Acid Sequence↗
PubMed1990

DNA sequence analysis of five genes; tnsA, B, C, D and E, required for Tn7 transposition.

A region of DNA sequence of the bacterial transposon Tn7, which is required for transposition, has been determined. This DNA sequence completes an 8351 base pair (bp) region containing five long open reading frames (ORF's) that correspond to the genetically defined genes, tnsA, B, C, D and E, required for Tn7 transposition. All of the ORF's are oriented in the same direction, ie. inward from the element's right end. The genes are in a very compact arrangement with the presumed initiation codons never more than two bases beyond the preceding termination codon. Domains with similarity to the helix-turn-helix genre of Cro-like, sequence specific DNA binding sites occur within the deduced amino acid (a.a.) sequence of the TnsA, TnsB, TnsD and TnsE proteins. Translation of the tnsC ORF reveals strong homology to a consensus sequence for nucleotide binding sites as well as a region of similarity to a transcriptional activator (MalT). No striking a.a. sequence similarity to other DNA recombinases is observed. The possible roles of these proteins in Tn7 transposition is discussed in light of the analysis presented.

Amino Acid Sequence↗
PubMed1991

DNA sequence analysis and restriction fragment length polymorphism (RFLP) typing of the HLA-DQw2 alleles associated with dermatitis herpetiformis.

Dermatitis herpetiformis (DH) is a blistering autoimmune skin disease associated with a 95-100% incidence of the HLA class II antigen HLA-DQw2. Although the precise role of this antigen in the pathogenesis of DH is unclear, one theory proposes that patients with DH possess a molecularly unique subtype of the HLA-DQw2 antigen that causes immune abnormalities eventuating in the clinical manifestations of DH. To test this hypothesis, we performed DNA sequence analysis on the highly polymorphic HLA-DQB1 and HLA-DQA1 loci of eight patients with dermatitis herpetiformis. All DQB1 alleles sequenced were identical to the previously described HLA-DQB*0201 allele from HLA-DQw2 normal subjects. In addition, DQA1 alleles sequenced were identical to those alleles previously associated with HLA-DQw2 (DQA*0201, DQA*0501). These data document that although HLA-DQw2 appears to be a necessary element in the pathogenesis of DH, the development of DH is not dependent on the presence of a unique HLA-DQw2 antigen. HLA-DQ allelic typing by restriction fragment length polymorphism analysis of PCR-amplified HLA-DQA1 and HLA-DQB1 fragments was also performed in ten patients with DH to determine the allelic distribution among both HLA-DR3 (eight patients) and non-DR3 (two patients) DH patients. At the HLA-DQ beta chain locus, all patients possessed the DQB1*0201 allele. At the HLA-DQ alpha chain locus, all HLA-DR3 patients and one non-DR3 patient displayed a pattern consistent with the DQA1*0501 allele, whereas one non-DR3 patient displayed a pattern consistent with the DQA1*0201 allele. These data document that patients with DH do not express a unique HLA-DQw2 heterodimer, that the HLA-DQw2 molecules present in patients with DH have no DNA sequence differences from those found in normal HLA-DQw2 subjects and therefore that susceptibility to DH is not due to a unique HLA-DQw2 molecule.

Alleles↗
PubMed1990

[DNA sequence analysis of HLA-DQB genes associated with insulin-dependent diabetes mellitus in Japanese].

Recently it has been reported that 57th amino acid of DQ beta antigen was a non-aspartic acid in the most Caucasian patients with insulin-dependent diabetes mellitus (IDDM). Using serological analysis, HLA-DQ antigens show a strong association with IDDM rather than the HLA-DR antigens. In the Caucasian population, IDDM is associated with DQw2 and DQw3 antigens. However, in the Japanese population, DQw4 and DQw9 antigens are strongly associated with IDDM. In order to determine the possible significance of the 57th amino acid of DQ beta chain for the susceptibility to IDDM in Japanese, we performed DNA Sequence analysis of HLA-DQB from Japanese IDDM patient, its same HLA genotyped healthy sibling and B cell line with Japanese IDDM associated haplotype. The results revealed that all of the 57th amino acid are aspartic acid which is thought to contribute to develop IDDM resistance in Caucasian. Furthermore we could not find any other amino acid sequence of DQ beta chain which might contribute to the susceptibility of the Japanese IDDM.

Amino Acid Sequence↗
PubMed1991

The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis.

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.

Amino Acid Sequence↗
PubMed1990

Genetic and DNA sequence analysis of the Salmonella typhimurium virulence plasmid gene encoding the 28,000-molecular-weight protein.

We have confirmed that the 28,000-molecular-weight (28K) protein encoded by the virA gene of the 90-kilobase Salmonella typhimurium virulence plasmid is a virulence factor. It was previously shown that a Tn5 insertion, vir-22::Tn5, located in the virulence plasmid greatly attenuated virulence for mice and inhibited the production of a 28K protein (P.A. Gulig and R. Curtiss III, Infect. Immun. 56:3262-3271, 1988). Plasmid pYA426 fully complemented vir-22::Tn5 to virulence by increasing splenic infection after oral inoculation and encoded the 28K protein. To identify the virulence gene(s) of pYA426 mutated by vir-22::Tn5, we constructed nested deletions in pYA426 and examined deletion derivatives for their abilities to complement vir-22::Tn5. Only derivatives still producing the 28K protein complemented vir-22::Tn5. Furthermore, the smallest complementing derivative encoded only the 28K protein, as determined by DNA sequence analysis. Therefore, the 28K protein is sufficient for complementation of the attenuating mutation vir-22::Tn5 and must be the virulence factor inhibited by the insertion. We determined the nucleotide sequence of the 1.2-kilobase BamHI-EcoRI fragment encoding the 28K protein and identified the structural gene, virA. A 723-base-pair open reading frame which encodes a peptide with a molecular weight of 27,572 was found.

Amino Acid Sequence↗
PubMed1986

Rat liver glutathione S-transferases. DNA sequence analysis of a Yb2 cDNA clone and regulation of the Yb1 and Yb2 mRNAs by phenobarbital.

We have constructed a cDNA clone, pGTA/C48, which is complementary to the rat liver glutathione S-transferase Yb2 mRNA. Recombinant clone pGTA/C48 contains a cDNA insert of 845 base pairs which overlaps nucleotides 108-952 of the Yb1 cDNA clone, pGTA/C44, described previously by our laboratory (Ding, G. J.-F., Lu, A. Y. H., and Pickett, C. B. (1985) J. Biol. Chem. 260, 13268-13271). Over the protein coding region of the Yb1 and Yb2 cDNA clones there is an 84% nucleotide sequence homology, whereas the 3' untranslated regions are only 32% homologous. The complete amino acid sequence of the Yb2 subunit has been determined from a combination of DNA sequence analysis of pGTA/C48 and conventional protein sequence analysis of the glutathione S-transferase Yb1 Yb2 heterodimer. The Yb2 subunit is comprised of 218 amino acids with a molecular weight of 25,705 and has an amino acid sequence which is 79% homologous to the sequence of the Yb1 subunit. We have utilized the divergent 3' untranslated regions of three rat liver glutathione S-transferase cDNA clones as specific probes to determine the effect of phenobarbital on the level of Yb1, Yb2, and Yc mRNAs. Our results clearly show that the Yb1 and Yb2 mRNAs are elevated approximately 5-6-fold by phenobarbital administration; whereas the Yc mRNA is only modestly elevated by this xenobiotic. Finally, our data suggest that the Yb2 subunit is encoded by a gene(s) which is distinct from the Yb1 gene(s) and provides direct evidence for the existence of multiple glutathione S-transferase Yb genes in the rat.

Amino Acid Sequence↗