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Biomedical subjects

S Bron

Publications and source records attributed to S Bron.

At least 109 records · Page 6Linked to original sources
PubMed1983

Transformation in Bacillus subtilis: properties of DNA-binding-deficient mutants.

Transformation-deficient mutants of Bacillus subtilis were selected after replica plating on agar plates containing transforming DNA. Out of 24 mutants tested, 3 showed highly reduced abilities to bind donor DNA; the residual levels of binding were similar to those of noncompetent cells. Transformation and transfection were reduced to nondetectable levels in the mutants. However, transduction with phage SPP1 occurred at normal frequencies. The nuclease activities involved in entry of donor DNA were present in the mutants. Comparison of protein patterns by two-dimensional gel electrophoresis revealed the absence of one major protein in the mutants as compared with the wild-type strain. This protein (molecular weight, approximately 18,000; isoelectric point, 5.0) appeared to be membrane associated. The protein was specific for competent cells, suggesting that it is involved in the binding of donor DNA.

Bacillus subtilis↗
PubMed1983

Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein.

In DNA binding-deficient mutants of Bacillus subtilis a competence-specific protein with a subunit molecular weight of 18,000 was absent. The native protein containing this subunit was purified from B. subtilis membranes by chromatography on hydroxyapatite, DEAE-cellulose, and Sephacryl S-200. This protein appeared to be complexed with a second protein of slightly lower molecular weight (17,000) and a different isoelectric point. The native protein complex (apparent molecular weight, 75,000) contained approximately equal amounts of the two polypeptides and showed a strong DNA-binding activity. Incubation of the complex with plasmid and bacteriophage DNA revealed nuclease activity, specifically directed toward double-stranded DNA. Predominantly single-stranded nicks and a limited number of double-stranded breaks were introduced in the presence of Mg2+ ions. In the presence of Mn2+ ions the complex produced low-molecular-weight breakdown products from the DNA.

Bacillus subtilis↗
PubMed1983

Resistance of bacteriophage H1 to restriction and modification by Bacillus subtilis R.

H1, a 5-hydroxymethyluracil (HMU)-containing Bacillus subtilis bacteriophage, was neither restricted nor modified upon infection of B. subtilis R cells. In vitro, H1 DNA was not restricted by BsuR under standard conditions (200 mM salt), although the expected frequency of -GGCC- cleavage sites was approximately 250. However, four specific sites were cleaved under nonstandard conditions (low salt or high pH) or in the presence of organic solvents, like dimethyl sulfoxide and glycerol. After the substitution of thymine for HMU by DNA cloning in B. subtilis, a BsuR cleavage site was restricted and modified under standard conditions. No additional sites were detected after shotgun-cloning of about 11% of the chromosome. The nucleotide sequence of a cleavage site was found to be 5'. .C-A-Hmu-A-A-C-Hmu-Hmu-Hmu-G-G-C-C-Hmu-A-G-. . .3', which shows the presence of a bona fide BsuR (GGCC) recognition sequence, flanked by (Hmu-A)-rich sequences. The results suggested that the resistance of H1 to restriction and modification by B. subtilis R was due to (i) a strong bias against the GGCC-recognition sequence and (ii) protection of the four remaining GGCC sites as a consequence of HMU-A base pairs flanking the sites.

Bacillus subtilis↗
PubMed1982

Restriction and modification in Bacillus subtilis: effects on transfection under marker rescue conditions.

The role of homology between donor and recipient DNAs in the protection of transfecting DNA against restriction by competent Bacillus subtilis R cells was studied under marker rescue conditions with modified helper phage. By comparing restriction under conditions of preinfection marker rescue and superinfection marker rescue, the significance of DNA homology during the initial stages of DNA processing by competent cells could be studied. The results showed that both in preinfection and in superinfection, complete protection against restriction of transfectants produced via rescue by the modified homologous helper chromosome occurred. Even up to 90 min after entry, DNA entering the helper-mediated pathway of transfection was not affected by restriction. The significance of these findings is discussed in the general context of the role of DNA homology between donor and recipient on the fate of donor DNA in competent B. subtilis, in particular in relation to the effects on restriction.

Bacillus subtilis↗
PubMed1980

Restriction and modification in B. subtilis: effects on transformation and transfection with native and single-stranded DNA.

The effects of restriction in vivo by competent B. subtilis R cells and in vitro by purified endonuclease BsuR on transformation and transfection with native and denatured DNA were investigated. The results show that transformation by either native, or denatured DNA is not affected by restriction, whereas transfection both with native and denatured SPP1 DNA is severely restricted. In contrast to the results obtained in vivo, the biological activity of native and denatured transforming DNA is destroyed by BsuR in vitro, as is the transfecting activity of native and denatured SPP1 DNA. The sensitivity of denatured DNA, either with mixtures of the complementary strands or with separated single strands alone, is significantly lower than that of native DNA. The results are discussed in the context of possible mechanisms underlying the different responses of transforming and transfecting DNA to in vivo restriction by B. subtilis R cells.

Bacillus subtilis↗
PubMed1980

Restriction and modification in B. subtilis: the role of homology between donor and recipient DNA in transformation and transfection.

Non-modified DNAs from phages SPO2 and phi 105, and prophage DNAs extracted from lysogens carrying these phages, were used to transfect isogenic r+m+ B. subtilis recipients which were either non-lysogenic, or had been lysogenized with a homologous or a non-homologous phage. Restriction of transfecting phage and prophage DNA occurred in non-lysogenic recipients and in recipients lysogenic for a non-homologous phage. No effect of restriction was observed when phage or prophage DNA was used to transfect recipients carrying a homologous prophage. This is analogous to the absence of restriction in transformation and indicates that in B. subtilis the distinction between transforming and transforming and transfecting DNA is not made at the initial stages of DNA uptake and processing, but rather at later stages, where recognition of homologous regions in donor and recipient DNA plays an important role.

Bacillus subtilis↗
PubMed1975

Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R.

All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPPI, SPO2 and phi105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo- or endonuclease activity. Only Mg2+ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5 x 10(7)) have an average molecular weight of about 3 x 10(5).

Adenosine Triphosphate↗
PubMed1975

Restriction and modification in B. subtilis. Nucleotide sequence recognised by restriction endonuclease R. Bsu R from strain R.

Restriction endonuclease R from Bacillus subtilis strain R cleaves nonmodified SPP 1 DNA in approximately 80, and lambda DNA in about 200 different sites. DNA digests with this endonuclease and with endonuclease Hae III from Haemophilus aegyptius show identical fragmentation patterns on gel electrophoresis, indicating that the two enzymes recognise the same nucleotide sequence. The polynucleotide kinase reaction was used in conjunction with two-dimensional ionophoretic nucleotide mapping methods to identify the 5'-nucleotide sequences at the sites of cleavage by the B. subtilis restriction endonuclease. The results show that the recognition sequence is (see article) where arrows indicate the points of strand scission. Each of the four possible nucleotides can occur in the positions flanking the recognition site.

Bacillus subtilis↗
PubMed1971

In vitro excision-repair of ultraviolet-irradiated transforming DNA from Bacillus subtilis.

The biological activity of UV-inactivated Bacillus subtilis DNA is partly restored after incubation with a UV-specific endonuclease from Micrococcus lutens in conjunction with DNA polymerase and DNA ligase, both isolated from Escherichia coli. The restored activity is not further increased by photoreactivation. Pyrimidine dimers are specifically liberated when irradiated DNA is exposed to the three enzymes. None of these effects is observed when pancreatic DNase is used instead of UV-specific endonuclease.

Bacillus subtilis↗