Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have made use of lysogens of a specialized transducing bacteriophage, lambdapyrG+ relA+, to select nonsense (relAnon) and insertion (relAins) mutations in the relA gene. Three independent relAnon mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup6. Three independent relAins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet lightkilled host cells revealed that no stringent factor was coded for by either the relAins or relAnon phages (the latter in a sup+ cell); stringent factor was detected when the relAnon phages were used in a similar experiment with supD or supE host cells. The relAnon and relAins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.
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PMID:Nonsense and insertion mutants in the relA gene of E. coli: cloning relA. 36 54

We have examined the genomes of the temperate bacteriophages Mu and P1 and some of their insertion mutants for hybridization with the prokaryotic transposable elements IS1 and IS2. We used the DNA blotting-hybridization technique in which denatured DNA fragments are transferred to nitrocellulose paper directly from agarose gels and hybridized to 32P-labeled probe DNA. The 800 base pair insertion in an X mutant of Mu was found to hybridize with IS1. The chloramphenicol resistance transposon, Tn9, in Mu X cam mutants was found to be located at or close to the sites of IS1 insertion in X mutants; Tn9 also hybridized with IS1. The restriction endonuclease BalI cleaved IS1 once; it cleaved Tn9 in all Mu X cam mutants twice to release a fragment of about 1700 base pairs. These results support the conclusion that Tn9 contains one copy of IS1 at each end. In the P1cam isolate, from which Tn9 was transposed to Mu, BalI made a third cut in Tn9 giving rise to fragments of about 850 base pairs. The data further suggested that Tn9 is present in tandem copies in the P1cam isolate we examined. P1 itself was found to harbor IS1. The two P1 strains tested had a common fragment containing IS1; one strain had an additional copy of IS1. The IS1 element common to the P1 strains was shown to be the site of the Tn9 insertion in the P1cam isolate examined. No hybridization between IS2 and any of the Mu and P1 strains could be detected.
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PMID:Analysis of transposable elements inserted in the genomes of bacteriophages Mu and P1. 36 86

Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possible other replicons occur more frequently than has hitherto been appreciated. The sequences changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existing in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous, and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.
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PMID:Instability of plasmid DNA sequences: macro and micro evolution of the antibiotic resistance plasmid R6-5. 36 83

TraJ and SfrA are, respectively, plasmid and host (Escherichia coli)-encoded proteins normally required for F plasmid traY promoter function. Beginning with plasmids in which a traY-lacZ fusion gene, designated phi (traY'-'lacZ)hyb, and lacY are expressed from the F plasmid traY promoter, we isolated mutants in which lac gene expression was SfrA or TraJ-independent. A total of 45 of 50 SfrA-independent isolates obtained after 2-aminopurine mutagenesis proved to have chromosomal mutations, whereas four out of four isolates obtained without mutagenesis had plasmid mutations. All of 17 isolates selected for TraJ-independent expression after mutagenesis had plasmid mutations. By restriction endonuclease digestions, 25 of 26 SfrA-independent and TraJ-independent plasmid mutations were insertions. Four of the former and three of the latter were examined further. By sequence analysis, all seven proved to be IS1 or IS2 insertions defining five insertion sites between base-pairs -49 and -82 with respect to the major traY transcription initiation site. In two cases, the same insertion allele was obtained from the two selection schemes. All three of the mutants selected for TraJ-independent gene expression manifested SfrA-independent expression as well, and levels of beta-galactosidase in different plasmid mutant strains lacking TraJ and SfrA were indistinguishable. By primer extension analysis, transcription initiation sites for traY mRNA synthesis were unaltered by the mutations. Replacing the tra sequence upstream from base-pair -78, without genetic selection, increased beta-galactosidase activity in the absence of TraJ and SfrA greater than tenfold. Activity increased two- to threefold more in a traJ+ sfrA mutant strain, and fivefold more in a traJ+ sfrA+ strain. Activity was unaltered in an sfrA+ strain without TraJ. By primer extension analysis, the traY promoter was utilized under all conditions. The data indicate that regulation of traY promoter activity is strongly dependent on sequence context.
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PMID:Regulation of the F plasmid traY promoter in Escherichia coli K12 as a function of sequence context. 190 41

Plasmid pSA5700 from Staphylococcus aureus coding for erythromycin (EmR) and chloramphenicol (CmR) resistance was transformed into Streptococcus pneumoniae. High-copy-number and EmR constitutive mutants of this plasmid were isolated. Transformation frequencies in S. pneumoniae as high as 70% were obtained with a constitutive plasmid as donor DNA, into a recipient cell containing a resident, inducible, high-copy-number plasmid. With the aid of these high frequencies, the site of constitutive mutations could be mapped via a simple marker rescue technique that uses purified restriction endonuclease-generated fragments. One of the EmR constitutive mutants, pFB9, a plasmid originating from a Gram-positive host, was shown to replicate and express EmR and CmR in a Gram-negative organism, Escherichia coli. Four derivatives of pFB9 containing large (0.6-0.9 megadalton) insertion sequences that arose spontaneously in E. coli demonstrated unusual transforming activity, as well as enhanced EmR, in E. coli. The inserted elements mapped to the region in front of the EmR gene. Three of these inserted elements had the size and restriction patterns of insertion sequence IS1, IS2, and IS5. Plasmid pFB9 and derivatives are useful for isolation of new insertion sequences and for comparison of gene expression and illegitimate recombination between Gram-positive and Gram-negative species.
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PMID:Staphylococcal plasmids that replicate and express erythromycin resistance in both Streptococcus pneumoniae and Escherichia coli. 628 51

Several F' plasmids encoding resistance to tetracycline have been derived from a trg::Tn10 Hfr B7 strain of Escherichia coli K-12. One of these plasmids, JGF312, was analyzed by restriction endonuclease digestion and Southern blot hybridization to cloned chromosomal fragments. This analysis revealed that JGF312 was formed by Tn10-promoted deletion from the Tn10 insertion (31.4 min) to within the prophage rac at 30.1 min. Hfr B7 was shown to result from recombination between IS2 of F delta (33-43) and a chromosomal IS2 located within the rac-man region at 30.9 min on the genetic map.
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PMID:Origin of Escherichia coli K-12 Hfr B7. 629 47

The RusA protein of Escherichia coli is an endonuclease that can resolve Holliday intermediates and correct the defects in genetic recombination and DNA repair associated with inactivation of RuvAB or RuvC. The structure of the rusA gene, its organisation in the genome, and its interaction with the Ruv and RecG proteins have been investigated. Recombinant plasmids carrying rusA were identified by their ability to make ruv mutants resistant to UV light. The gene was located to an open reading frame encoding a polypeptide of 120 amino acids. It forms the fifth gene in an operon containing a chain of short, interlinked open reading frames. A similar arrangement was found in the genome of the lambdoid bacteriophage, 82. The two rusA genes show 95% sequence identity. The E. coli operon forms part of the defective lambdoid prophage, DLP12, and is probably derived from a phage related to 82 and PA-2. rusA appears to be very poorly expressed in E. coli, but can be activated by insertion of IS2 or IS10 upstream of the coding sequence to promote transcription. These insertions arise spontaneously in ruv strains as suppressors of the mutant phenotype. Deletion of rusA from the chromosome of either wild-type or ruv mutant strains has no obvious effect on recombination or sensitivity to UV light. Multicopy plasmids expressing RusA alone make ruvA, ruvB, and ruvC mutants resistant to UV light. Suppression depends critically on RecG.
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PMID:Holliday junction resolvases encoded by homologous rusA genes in Escherichia coli K-12 and phage 82. 864 24

The proteasome-like ClpP protease is widely distributed and structurally conserved among bacteria and eukaryotic cell organelles. In Chlamydomonas eugametos, however, the chloroplast clpP gene predicted a much larger ClpP protein containing large insertion sequences (ISs). One insertion sequence, IS2, is 456 amino acid residues long and not similar to known proteins. Here we show that IS2 is an unusual intein, and its protein splicing activity in Escherichia coli cells can be activated by a single amino acid substitution. Analysis of IS2 sequence revealed short sequence motifs that are similar to known intein motifs, including putative LAGLI-DADG endonuclease motifs. But a histidine residue conserved at the C terminus of known inteins is replaced in the IS2 sequence by a glycine residue (Gly455), rendering the IS2 sequence incapable of detectable protein splicing when tested in E. coli cells. Changing Gly455 to histidine activated the ability of IS2 to undergo protein splicing in E. coli cells. The IS2 sequence (intein) was precisely excised from a precursor protein, with the flanking sequences (exteins) joined together by a normal peptide bond.
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PMID:Identification of an unusual intein in chloroplast ClpP protease of Chlamydomonas eugametos. 911 46