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1.
Plasmid ; 42(1): 42-4, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10413664

ABSTRACT

We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.


Subject(s)
DNA Transposable Elements/genetics , Enterococcus/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial/genetics , Enterococcus/isolation & purification , Humans , Molecular Sequence Data , Open Reading Frames , Species Specificity , Streptococcus mutans/genetics
2.
Gene ; 85(1): 205-7, 1989 Dec 21.
Article in English | MEDLINE | ID: mdl-2559874

ABSTRACT

We have developed plasmids with the Tn5 kanamycin-resistance gene (kan) flanked either symmetrically or asymmetrically by several restriction sites. These can be used to provide a selectable genetic marker or to mobilize restriction sites and sense or nonsense codons into genes. The 1.3-kb kan cassette exhibits polarity effects in both directions.


Subject(s)
DNA Transposable Elements , Kanamycin Resistance/genetics , Restriction Mapping , Base Sequence , Codon , Molecular Sequence Data , Oligonucleotide Probes
3.
Plasmid ; 22(3): 275-80, 1989 Nov.
Article in English | MEDLINE | ID: mdl-2561212

ABSTRACT

Two previously characterized mutations in the galOPETK operon of Escherichia coli, galOP-3 and galOPE-490, contain IS2 insertions only 1 bp apart in the gal regulatory region; yet only the former yields Gal+ phenotypic revertants at a detectable frequency. We have shown that the galOPE-490 allele comprises two mutations--an IS2(I) insertion at bp+(2-6) (relative to the gal mRNA start site) plus a C/G to A/T transversion at bp + 59. The latter creates an ochre stop codon and lies within the internal site of the bipartite gal operator; it acts as an operator mutation in an in vivo repressor titration assay. Analysis of a newly isolated allele (galOP-490*) which retains the IS2 of galOPE-490 but is galE+ reveals a reversion frequency approximately 30-fold higher than that of galOP-3. Reversion of galOPE-490 is at least 10,000-fold lower and has not been detectable even under conditions conducive to enhanced double mutations in other systems.


Subject(s)
DNA Transposable Elements , Escherichia coli/genetics , Genes, Bacterial , Genes, Regulator , Mutation , Operon , Alleles , Escherichia coli/cytology , Escherichia coli/metabolism , Galactose/metabolism , Plasmids , Restriction Mapping
4.
Biochimie ; 67(1): 101-8, 1985 Jan.
Article in English | MEDLINE | ID: mdl-3922433

ABSTRACT

The amino acid sequence of thiogalactoside transacetylase, a dimer, has been determined. The monomer contains 202 amino acid residues in a single polypeptide chain and has a molecular weight of 22,671. The analysis was carried out by treatment of the carboxymethylated protein with cyanogen bromide and with trypsin. All seven cyanogen bromide peptides were isolated in pure form and were ordered by peptides isolated from tryptic digests. The sequence analysis was aided by determination of the DNA sequence of the lacA gene. The amino terminus of the protein is heterogenous because the initiator methionine is only partially cleaved. Another rather unusual feature of this cytoplasmic protein is a very hydrophobic segment in the center portion of the chain. Comparison of the amino acid sequence of thiogalactoside transacetylase to those of the lac repressor, beta-galactosidase, and lactose permease did not reveal any marked similarities. Therefore, there is no obvious evolutionary relatedness among proteins of the Lactose Operon.


Subject(s)
Acetyltransferases , Escherichia coli Proteins , Escherichia coli/enzymology , Monosaccharide Transport Proteins , Symporters , Acetyltransferases/genetics , Amino Acid Sequence , Base Sequence , Chromatography, Gel , Cyanogen Bromide , DNA, Bacterial , Lac Operon , Membrane Transport Proteins , Peptide Fragments/isolation & purification , Trypsin , beta-Galactosidase
5.
J Mol Biol ; 169(1): 53-81, 1983 Sep 05.
Article in English | MEDLINE | ID: mdl-6194305

ABSTRACT

The insertion sequence IS2 is a small transposable element of Escherichia coli that lacks any known genetic markers. Insertion of this element in one orientation (I) within bacterial operons blocks expression of downstream genes. In the other orientation (II), IS2 has been associated with the constitutive expression of genes distal to its insertion, suggesting that IS2 might contain promoters directing transcription of IS2(II) into other genes. To test the transcription potential of IS2, we have transcribed in vitro DNA templates from gal3, a Gal- allele in which an IS2(I) is inserted between the gal promoter and the gal genes. We have detected two IS2-specific RNAs which initiate from promoters within IS2 and are transcribed in orientation II (away from the galETK genes). Though the presence and orientation of these promoters suggests that they could be responsible for the constitutive expression of genes adjacent to an IS2(II) element, an alternative role could be for transcription of IS2-encoded genes. Although IS2(I) insertions normally block expression of adjacent genes, certain altered (e.g. mutant) IS2(I) sequences lead to the constitutive expression of downstream genes. We have transcribed DNA templates from galwc5 and galc331, which are Galc alleles that contain altered IS2(I) insertions within the gal operon. For each allele, we have detected two gal-directed transcripts initiating within the IS2 sequence. These RNAs are not detected upon transcription of the unaltered IS2(I) DNA and the promoters arise as a direct consequence of the IS2(I) alterations. This result suggests that these promoters detected in vitro are responsible for the Galc phenotype of these alleles.


Subject(s)
DNA Transposable Elements , Transcription, Genetic , Autoradiography , Base Sequence , DNA, Bacterial/genetics , Escherichia coli/genetics , Gene Expression Regulation , Oligonucleotides/analysis , Operon , RNA, Bacterial/genetics
6.
Nucleic Acids Res ; 10(16): 5015-31, 1982 Aug 25.
Article in English | MEDLINE | ID: mdl-6291000

ABSTRACT

Insertion of the insertion sequence Is2(I) directly before the galE gene of the galactose operon results in a Gal minus phenotype (1, 2). The Gal-constitutive allele galc200 (and its deletion derivative galc200 delta 31) arise from such a Gal minus mutant by the insertion of LS2(II) DNA within the LS2(I) sequence (3). We have transcribed in vitro a DNA template representing the IS2-galE region of galc200 delta 31. Gal-directed transcription initiates at two sites within the IS2(I) sequence, 51 and 52 bp from the IS2-galE junction. The promoter for these transcripts, Pgal200 delta 31, is composed of a novel joint between a -10 region from the IS2(I) DNA and a -35 region contributed by the IS2(II) insertion. No promoters intrinsic to the 121 bp of the IS2(II) sequence also present on the template were detected. The relevance of Pgal200 delta 31 to the Galc phenotype of galc200 and to general mechanisms for the constitutive expression of genes adjacent to IS2 is discussed.


Subject(s)
DNA Transposable Elements , Escherichia coli/genetics , Galactose/genetics , Mutation , Operon , Transcription, Genetic , DNA Restriction Enzymes , DNA, Bacterial/genetics , Nucleic Acid Hybridization , Phenotype , Plasmids , Templates, Genetic
8.
J Biol Chem ; 252(10): 3538-47, 1977 May 25.
Article in English | MEDLINE | ID: mdl-193835

ABSTRACT

We have determined the sequence of 59 base pairs in the DNA preceding the site for initiation of transcription in the galactose operon. DNA from a lambdagal transducing phage was digested with restriction endonucleases to obtain a DNA fragment from the gal regulatory region. This fragment extends from 59 base pairs prior to the transcription initiation site through the 45 base pairs which specify the 5'-terminal sequence of gal mRNA. Analyses of RNA transcripts derived from this fragment and a variety of direct DNA sequence analyses allowed us to deduce the following sequence for the DNA in this fragment: (formula: see text) Position +1 corresponds to the site for initiation of gal mRNA synthesized in the presence of cyclic AMP and its receptor protein, CRP. Transcription experiments indicate, however, that this fragment lacks some element of the gal promoter required for the stimulation of transcription by CRP-cAMP. There are, nonetheless, some similarities between the gal sequence preceding the transcription start site and the sequences of other promoter regions. These include the heptamer sequence T-A-T-G-G-T-T (--12 to --8) and the sequence A-C-A-C-T-T-T (--36 to --30).


Subject(s)
DNA, Bacterial , Escherichia coli , Galactose/metabolism , Genes, Regulator , Operon , Alkylation , Base Sequence , Binding Sites , Coliphages/analysis , DNA Restriction Enzymes , DNA, Bacterial/analysis , DNA, Recombinant/analysis , DNA, Viral/analysis , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/analysis , Phosphoric Diester Hydrolases/metabolism , Pyrimidines/analysis , Receptors, Cyclic AMP/metabolism , Sulfates/pharmacology , Templates, Genetic
9.
Proc Natl Acad Sci U S A ; 71(12): 4940-4, 1974 Dec.
Article in English | MEDLINE | ID: mdl-4612533

ABSTRACT

The 5'-terminal sequence of mRNA from the galactose operon of E. coli has been determined. gal RNA is synthesized in vitro by means of a kinetically controlled purified transcription system and is isolated by a two-step RNA.DNA hybridization scheme. The following sequence of the first 77 nucleotides has been deduced by analysis of oligonucleotides produced by digestion with T(1), pancreatic, and carboxymethylated pancreatic ribonucleases: ppA-U-A-C-C-A-U-A-A-G-C-C-U-A-A-U-G-G-A-G-C-G-A-A-U-U-A-U-G-A-G-A-G-U-U-C-U-G-G-U-U-A-C-C-G-G-U-G-G-U-A-G-C-G-G-U-U-A-C-A-U-U-G-G-A-A-G-U-C-A-U-A-C-C-U-G-U. Residues 27-77 correspond to the amino terminal 17 amino acids of UDPgalactose 4-epimerase (EC 5.1.3.2), the protein specified by the promoter-proximal structural gene of the operon. A self-complementary sequence occurs near the 5' terminus; 12 of 15 nucleotides between residues 4 and 18 are symmetrically located about position 11. The sequence of residues 22-33 closely resembles part of the lactose operator sequence reported previously [Gilbert, W. & Maxam, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3581-3584].


Subject(s)
Escherichia coli/metabolism , Galactose/metabolism , RNA, Bacterial/analysis , RNA, Messenger/analysis , Amino Acid Sequence , Base Sequence , Carbohydrate Epimerases/biosynthesis , Electrophoresis, Paper , Genes , Genetic Code , Oligonucleotides/analysis , RNA, Messenger/biosynthesis , Ribonucleases , Transcription, Genetic
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