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At least 1,747 records · Page 97Linked to original sources
PubMed1993

A chloroplast DNA deletion located in RNA polymerase gene rpoC2 in CMS lines of sorghum.

Fertile lines of sorghum (Sorghum bicolor) were shown to differ from cytoplasmic male sterile (CMS) lines by the presence of a 3.8 kb HindIII chloroplast DNA fragment in the former and a smaller (3.7 kb) fragment in the latter. DNA/DNA hybridization studies showed that these two fragments are homologous. Fertile plants from S. versicolor, S. almum, S. halepense, and Sorghastrum nutans (Yellow Indiangrass) also have the 3.8 kb fragment, and CMS lines studied containing A1, A2 and A3 cytoplasms have the 3.7 kb fragment. The size difference between the two fragments was localized to a 1.0 kb SacI-HindIII fragment by restriction mapping. A 165 bp deletion, which is flanked by a 51 bp tandem repeat, was identified in the CMS lines by sequencing the clones. Comparison of the two sequences with those from maize, rice, tobacco, spinach, pea, and liverwort revealed that the deleted sequence is located in the middle of the RNA polymerase beta" subunit encoded by the gene rpoC2. The amino acid sequence deleted in the CMS lines is in a monocot-specific region which contains two protein motifs that are characteristic of several transcriptional activation factors, namely, a leucine zipper motif and an acidic domain capable of forming an amphipathic alpha-helix. Further studies designed to determine whether or not the deletion is involved in CMS of sorghum are underway.

Amino Acid Sequence↗
PubMed1996

Genomic structure and polymorphism of the human thromboxane synthase-encoding gene.

Thromboxane synthase (TS) is a cytochrome P-450 (CYP450) enzyme catalyzing the conversion of prostaglandin endoperoxide (PGH2) into thromboxane A2 (TxA2) which plays a crucial role in hemostasis and cardiovascular diseases. Twelve genomic clones containing the DNA encoding the human TS gene (hTS) were isolated and characterized to determine the exon/intron boundaries and restriction maps of the nearly contiguous structure of the gene. The hTS contains 13 exons spanning more than 150 kb. Its first five exons, divided by relatively large introns, spread over 100 kb, but encode less than one third of the full-length TS transcript. Southern analysis indicates that the human haploid genome contains a single copy of the TS gene. Although multiple transcription start points (tsp) are utilized, transcription of hTS is primarily TATA-independent, as determined by promoter-directed reporter gene expression in transfected cells. A dinucleotide (CA) repetitive sequence identified in the ninth intron of the gene exhibits allelic polymorphism. At least four distinctive alleles, containing from 13 to 20 copies of the CA repeats, have been detected.

Base Sequence↗
PubMed1991

Discrete regions of the avian beta-globin gene cluster have tissue-specific hypersensitivity to cleavage by sonication in nuclei.

We have analyzed the DNA released by sonication from avian nuclei, crosslinked with formaldehyde, by restriction mapping and equilibrium density gradient centrifugation. Our results indicate that regions flanking the adult beta-globin gene in adult reticulocytes have increased sensitivity to the mechanical shearing caused by sonication. These regions are near, and may overlap, the known nuclease hypersensitive regions that flank this gene. Like the hypersensitivity of these regions to nuclease digestion, the increased sensitivity to sonication is tissue-specific and appears to be due chiefly to the absence of nucleosome structures in these regions. Analysis of crosslinked chromatin fractionated by density gradient sedimentation suggests that DNA sequences near the matrix attachment region/enhancer element located 3' to the adult beta-globin gene are associated with transcriptionally-engaged chromatin fractions from both adult reticulocytes and day 5 chick embryo primitive erythroid cells. Sonication appears to be a useful tool for the study of chromatin structure.

Animals↗
PubMed1988

Characteristics of RP4 tellurite-resistance transposon Tn521.

A restriction map of the tellurite-resistance (Ter) transposon Tn521 (parent plasmid RP4Ter) was prepared. Five sites from RP4Ter, including the EcoRI origin, were found in pIN25::Tn521. Tn521 was inserted into a transferable 27.5 kb vector (pCU109) to make three different insertion mutants, in which the size of Tn521 was measured accurately at 4.5 kb. Unlike the Ter of IncHI2 plasmids, that of Tn521 in RP4Ter was non-inducible. Ter was expressed in five widely differing bacterial species to which RP4Ter was transferred from Escherichia coli. Electron micrographs of bacteria expressing the Ter of RP4Ter, H complex plasmids, and chromosomal mutants, all revealed similar tellurium metal crystallites when the bacteria were grown in potassium tellurite medium. No other Ter determinants were found amongst 54 plasmids representing most incompatibility groups (excluding the H complex).

DNA Transposable Elements↗
PubMed1993

Characterization of the baiH gene encoding a bile acid-inducible NADH:flavin oxidoreductase from Eubacterium sp. strain VPI 12708.

A cholate-inducible, NADH-dependent flavin oxidoreductase from the intestinal bacterium Eubacterium sp. strain VPI 12708 was purified 372-fold to apparent electrophoretic homogeneity. The subunit and native molecular weights were estimated to be 72,000 and 210,000, respectively, suggesting a homotrimeric organization. Three peaks of NADH:flavin oxidoreductase activity (forms I, II, and III) eluted from a DEAE-high-performance liquid chromatography column. Absorption spectra revealed that purified form III, but not form I, contained bound flavin, which dissociated during purification to generate form I. Enzyme activity was inhibited by sulfhydryl-reactive compounds, acriflavine, o-phenanthroline, and EDTA. Activity assays and Western blot (immunoblot) analysis confirmed that expression of the enzyme was cholate inducible. The first 25 N-terminal amino acid residues of purified NADH:flavin oxidoreductase were determined, and a corresponding oligonucleotide probe was synthesized for use in cloning of the associated gene, baiH. Restriction mapping, sequence data, and RNA blot analysis suggested that the baiH gene was located on a previously described, cholate-inducible operon > or = 10 kb long. The baiH gene encoded a 72,006-Da polypeptide containing 661 amino acids. The deduced amino acid sequence of the baiH gene was homologous to that of NADH oxidase from Thermoanaerobium brockii, trimethylamine dehydrogenase from methylotrophic bacterium W3A1, Old Yellow Enzyme from Saccharomyces carlsbergensis, and the product of the baiC gene of Eubacterium sp. strain VPI 12708, located upstream from the baiH gene in the cholate-inducible operon. Alignment of these five sequences revealed potential ligands for an iron-sulfur cluster, a putative flavin adenine dinucleotide-binding domain, and two other well-conserved domains of unknown function.

Amino Acid Sequence↗
PubMed1988

Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli.

The genomic locus responsible for production of the globin portion of Vitreoscilla hemoglobin (VtHb), the only well-characterized bacterial hemoglobin (Hb), has been cloned and expressed in Escherichia coli. A 17-mer oligodeoxynucleotide, corresponding to a region of the VtHb amino acid sequence was used as a hybridization probe to screen a Vitreoscilla genomic library constructed in broad-host-range cosmid vector pVK102. E. coli, carrying recombinant pVK102:H5 which contained a 16.5-kb insert of Vitreoscilla genomic DNA, produced three to four times more Hb than Vitreoscilla. Restriction mapping and subcloning revealed that the globin-coding gene (vgb) was completely localized on a 1.4-kb HindIII-SalI fragment of the 16.5-kb insert. Production of VtHb still occurred when this 1.4-kb fragment was cloned in plasmids pUC8 and pUC9 in opposite orientations, suggesting the presence of a Vitreoscilla promoter on this fragment. A single copy of this gene on the chromosome was indicated by Southern-blot analysis, and a 450-500-nt RNA transcript specific for the globin gene was detected after Northern hybridization. A partially purified Hb preparation from E. coli harboring the recombinant plasmid had identical spectral properties and subunit molecular size as authentic VtHb. The Hb in respiring cells of E. coli was in the physiologically functional oxyHb form.

Blotting, Northern↗
PubMed1989

Structure of the rat alpha 2-macroglobulin-coding gene.

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein, i.e., produced upon tissue inflammation. Genomic DNA clones covering the entire sequence of the alpha 2M gene were isolated and characterized by restriction mapping. Southern blotting and (partial) DNA sequencing. The rat alpha 2M gene is approx. 50 kb in length and consists of 36 exons ranging in size from 21 to 229 bp. Two functional domains, a bait region and a thiol ester site, are encoded by the exon 18 and 24, respectively. Several possible regulatory signals such as a TPA-inducible enhancer core, an identifier sequence, purine-pyrimidine alternative stretches and viral enhancer core sequences were identified. Several genomic DNA clones which cross-hybridized with the alpha 2M cDNA probe were also identified. Sequence analysis showed that they possessed sequences identical to a part of the rat alpha 1-inhibitor III cDNA and that they had a strikingly similar exon organization to the alpha 2M gene.

Animals↗
PubMed1993

Thyrotropin regulates thyroglobulin mRNA splicing and differential processing.

Rat thyroid tissue and cultured rat thyrocyte lines contain two thyroglobulin (Tg) mRNAs: a 9 kb rTg-1 mRNA encoding the 330, kDa Tg monomer and a recently described 0.95 kb rTg-2 mRNA. These transcripts have identical 5' coding sequences (641 nucleotides); however, the 3' end of rTg-2 is comprised of coding and non-coding sequences not present in rTg-1. To determine if a single Tg gene encoded both mRNA species, a genomic clone was isolated which spanned the full-length rTg-2 cDNA sequence. The promoter sequence and restriction map were the same as for the previously characterized rTg-1 gene, indicating that rTg-1 and rTg-2 mRNAs are splicing variants derived from the same Tg gene. The unique 3' end of rTg-2 mRNA comprised a single exon which was intronic with respect to rTg-1 mRNA formation. The level of rTg-2 in cultured rat thyrocytes was more sensitive to thyrotropin (TSH) regulation than was rTg-1. rTg-2 mRNA was rapidly (and reversibly) depleted to nearly undetectable levels after TSH removal, unlike rTg-1. Conversely, TSH rapidly restored control levels of rTg-2 mRNA in such depleted cells. The data thus support a model of TSH-induced splicing and regulation of the two Tg mRNAs in the rat.

Amino Acid Sequence↗
PubMed1991

Structural analysis of the gene encoding rat uricase.

The uricase gene was isolated from rat genomic DNA libraries. The gene spans 40 kb and consists of eight exons. All the exon-intron junctional sequences conform to the canonical GT/AG rule. The restriction map of the isolated clones and Southern blot analysis revealed that the enzyme is encoded by a single-copy gene. Analysis of the transcription initiation site of rat uricase mRNA indicated the differential use of consecutive nucleotides; the principal repeat is located 55 nucleotides upstream from the first methionine codon. Nucleotide sequence analysis of the 5'-flanking region showed the presence of a TATA (ATAAAA) sequence at nucleotides 30 to 25 and of a CAAT (GGTCAAT) sequence at nucleotides 63 to 57 upstream of the principal transcription initiation site. The 5'-flanking region contains another possible regulatory sequence (TGTCGACA) homologous to the cAMP-regulatory element. The palindromic sequence is located 158 to 151 nucleotides upstream from the transcription initiation site, surrounded by a direct repeat (TCAGCAA).

Amino Acid Sequence↗
PubMed1992

The telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p.

A telomere YAC clone containing the most distal 115 kb of chromosome arm 4p has been previously isolated. This clone is of particular interest as it spans a potential candidate region for the Huntington disease gene. The YAC was subcloned into a phage vector, and a high-resolution restriction map extending to within 13 kb of the telomere was constructed. In situ hybridization of the YAC to human metaphase spreads gives a peak of hybridization on 4pter but also an increase in the number of signals close to several other telomeres. Where possible, these results were investigated further by the hybridization of probes from the YAC to somatic cell hybrids containing single human chromosomes. This analysis indicates that the most telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p. The extent of this homology makes it less likely that the mutation for Huntington's disease is located within the telomere YAC clone.

Animals↗
PubMed1990

Molecular cloning of a gene encoding an ARS binding factor from the yeast Saccharomyces cerevisiae.

We report the isolation of the gene for origin binding factor 1 (OBF1) from the yeast Saccharomyces cerevisiae by screening a yeast genomic DNA library in lambda gt11 with an ARS-specific oligonucleotide probe. One recombinant encoded a fusion protein of approximately 180 kDa that bound ARS-specific oligonucleotide probes in vitro. The restriction map of this gene was determined after isolation of the complete gene by screening a yeast genomic DNA library in YEp24. Characterization of the gene for OBF1 by pulsed-field gel electrophoresis and Northern and Southern blot analyses demonstrated that (i) the gene is located in chromosome IV, (ii) the gene is a single-copy gene, (iii) the mRNA is approximately 3.8 kilobases, which could code for an approximately 130-kDa polypeptide, consistent with the reported size of OBF1. An antibody, affinity-purified using the lysogen-encoded fusion protein, specifically detected an approximately 130-kDa polypeptide in yeast extract. The isolation of the gene for OBF1 should allow further analysis of the mechanism of action of this protein in vitro and in vivo.

Base Sequence↗
PubMed2002

Antioxidant enzymes in blood of patients with Friedreich's ataxia.

BACKGROUND AND AIMS: Increased generation of reactive oxygen species and mitochondrial dysfunction may underlie the pathophysiology of Friedreich's ataxia, the most common inherited ataxia, due to GAA expansion in a gene coding for a mitochondrial protein (frataxin), implicated in the regulation of iron metabolism. Because iron overload would cause oxidative stress in Friedreich's ataxia, we investigated the enzyme antioxidant system in the blood of 14 patients by determining superoxide dismutase, glutathione peroxidase, and glutathione transferase catalytic activities. We also studied the glutathione S-transferase genotype polymorphism in order to evaluate its possible influence on enzyme activity. METHODS: Blood samples were obtained from 14 unrelated patients with Friedreich's ataxia and 21 age matched healthy subjects. Antioxidant enzyme determinations were spectrophotometrically assayed using specific substrates; the glutathione S-transferase genotype polymorphism was analysed by endonuclease restriction mapping of exon 5 and 6 amplification products. RESULTS: There was a significant elevation of the superoxide dismutase/glutathione peroxidase activity ratio (0.037 (0.01) v 0.025 (0.008) of controls) and an 83% rise of glutathione transferase specific activity (0.22 (0.1) v 0.12 (0.03) nmol/min/mg protein) in blood of patients with Friedreich's ataxia than in the controls. The genotype polymorphism of glutathione S-transferase enzyme did not show any relevant differences when compared to that of healthy subjects. CONCLUSIONS: Data show an impairment in vivo of antioxidant enzymes in patients with Friedreich's ataxia and provide evidence of an increased sensitivity to oxidative stress, supporting a consistent role of free radical cytotoxicity in the pathophysiology of the disease.

Adolescent↗
PubMed1991

Cloning and expression of a Clostridium thermocellum DNA fragment that encodes a protein related to cellulosome component SL.

Antibodies raised against the SL subunit of the Clostridium thermocellum cellulosome were used to screen a library of C. thermocellum chromosomal DNA fragments constructed in the vector lambda gt11. A DNA fragment that encoded a polypeptide that crossreacted with the anti-SL antibodies was isolated and its restriction map elucidated. No similarity with other previously cloned DNA fragments has been found. The anti-SL crossreacting polypeptide was isolated from recombinant Escherichia coli and found to have a mol mass of 37,000 Da and to possess low levels of CMCase and Avicelase activity. Using CMC as the substrate, a temperature optimum of 55 degrees C and a pH optimum of 6.6 were observed. These properties were compared to those of C. thermocellum SL isolated by electroelution from an SDS gel, which was also found to possess low levels of CMCase and Avicelase activities. In addition, the SL proteins produced in C. thermocellum and E. coli were able to interact positively against Avicel with an endoglucanase (Ss) purified from the C. thermocellum crude cellulase preparation, and with a recombinant protein that crossreacted with anti-Ss antibodies.

Cellulase↗
PubMed1990

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli.

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.

Aminobutyrates↗
PubMed1991

Molecular cloning of a novel isolate of feline immunodeficiency virus biologically and genetically different from the original U.S. isolate.

The Japanese isolate (TM1 strain) of feline immunodeficiency virus (FIV) which replicates in a feline CD4 (fCD4)-positive lymphoblastoid cell line (MYA-1 cells) was molecularly cloned from extrachromosomal closed circular DNA. The restriction map of the clone, termed pFTM 191 complete genome (CG), showed a considerable difference from that of the U.S. isolate (Petaluma strain) of FIV. The sequence homology in the long terminal repeat between the TM1 and Petaluma strain was 82%. The pFTM 191 CG was biologically active after transfection into Crandell feline kidney cells which were permissive for replication of FIV Petaluma. However, the progeny virions could not reinfect fCD4-negative Crandell feline kidney cells but could infect fCD4-positive MYA-1 cells. When a specific-pathogen-free cat was inoculated with the virus derived from the pFTM 191 CG, the cat seroconverted within 8 weeks postinoculation and FIV was reisolated at 4, 8, and 20 weeks postinoculation. These results indicate the infectivity of the pFTM 191 CG in vivo.

Animals↗
PubMed1997

In vivo recombination between two strains of the genus Nucleopolyhedrovirus in its natural host, Spodoptera exigua.

The DNA restriction map for the enzymes BamHI, BglII, PstI, and XbaI of SeMNPV-US (Se-US), the best-studied Nucleopolyhedrovirus strain isolated from Spodoptera exigua in California, was used as a reference to construct that of SeMNPV-SP2 (Se-SP2), a closely related Spanish strain of the same virus. After coinfection of S. exigua with both the Se-US and Se-SP2 strains per os, a recombinant virus (SeMNPV-SUR1 [Se-SUR1]) was detected after one passage which quickly replaced the parental viruses. A physical map of Se-SUR1 DNA was constructed for BamHI, BglII, PstI, and XbaI and compared to that of the parental viruses, Se-US and Se-SP2. Se-SUR1 is the result of at least four crossover events between Se-US and Se-SP2 and not of selection of a minor variant in one of the parental viruses. Bioassays of the recombinant and parental strains against L2 beet armyworm larvae demonstrated that their 50% lethal dose values were not significantly different. The differences in median lethal time values are too small to explain the replacement of the parental viruses by the recombinant virus upon successive passage in vivo, although it cannot be ruled out as an explanation for the selective advantage of the recombinant strain, Se-SUR1. The consequences of the release of nonindigenous or recombinant baculovirus strains in agro-ecosystems are discussed.

Animals↗
PubMed1991

Allelic exclusion of membrane but not secreted immunoglobulin in a mature B cell line.

Although many B lymphocytes contain two completely rearranged immunoglobulin (Ig) heavy (H) chain genes, only one of the alleles specifies a protein product. This phenomenon is termed allelic exclusion and is controlled in part by the membrane portion of Ig H chains. We have identified a mature B cell line derived from murine bone marrow that expresses two H chain proteins, gamma 3 and gamma 2b. Proteins of appropriate size for the membrane and secreted forms of both H chains are produced and both IgG3 and IgG2b are secreted from the cells. However, only IgG3 is expressed on the membrane. Detailed restriction mapping of the H chain genes indicates that both are rearranged, one containing a VHJ558 gene linked to the gamma 3 constant region and the other a VHS107 gene linked to the gamma 2b constant region. Primer extension sequencing of the RNA reveals that the gamma 2b RNA is transcribed from the allele containing VHS107 sequences while the gamma 3 RNA is transcribed from the allele containing VHJ558 sequences. Thus, this mature B cell line secretes two distinct antibody molecules but is allelically excluded at the level of surface Ig expression. This cell line provides a model system to study factors, in addition to aberrant rearrangement, that influence allelic exclusion.

Alleles↗
PubMed1991

Polymorphism within the nuclear and 2 micron genomes of Saccharomyces cerevisiae.

Seven strains of bakers' yeast were obtained as a representative sample of the Spanish baking industry. The nuclear genome was monitored for polymorphism by transverse alternating field electrophoresis (TAFE) and restriction maps of 2 micron DNA were produced. All seven strains were uniquely different when evaluated by their total chromosomal lengths whereas only two 2 micron variants were defined. There was no apparent correlation between chromosomal and plasmid polymorphism. The extensive chromosomal polymorphism within one 2 micron DNA type indicates the rapid and relatively recent evolution of the nuclear genome. The hybrid origin (S. cerevisiae-S. monacensis) of lager yeast was critically evaluated by TAFE analysis of S. cerevisiae and S. carlsbergensis chromosomes. The absence of corresponding S. cerevisiae chromosomes III and XIII in S. carlsbergensis argued against the hybrid origin of lager strains. We discuss limitations of the hybrid origin hypothesis of industrial yeasts and propose that the molecular coevolution observed in 2 micron DNA serves as a useful additional mechanism for rationalization of some of the structural polymorphism of the nuclear genome.

Chromosomes, Fungal↗