Biomedical subjects

E Luxen

Publications and source records attributed to E Luxen.

8 recordsLinked to original sources

PubMed1988

Instability of recombinant pUB110 plasmids in Bacillus subtilis: plasmid-encoded stability function and effects of DNA inserts.

Two series of pUB110-derived plasmids were constructed to study segregational stability in Bacillus subtilis. pEB plasmids were based on the entire pUB110, whereas pLB plasmids lack the membrane-binding areas BA3 and BA4. Two kinds of stability defects were observed. The first was characterized by a strong size dependency and occurred with different inserts at various positions in pLB and pEB plasmids. Size-dependent reductions in plasmid copy numbers appeared to underly this phenomenon. This may render pUB110 unsuitable for the cloning of inserts larger than about 3 kb, in particular if no selective conditions can be applied. The second defect, observed with pLB plasmids, was caused by the absence of the membrane-binding areas BA3 and BA4. Deletion of BA3 resulted in the accumulation of single-stranded plasmid DNA, suggesting that BA3 contains the initiation signal for complementary strand synthesis. The BA3 region is very rich in hyphenated dyad symmetry which, in single-stranded DNA, could result in several stable alternative secondary structures. It is speculated that the activity of the BA3-associated initiation signal contributes to the segregational stability of pUB110-derived plasmids in B. subtilis. The absence of the BA3 stability function could not account for all stability defects observed. Additional stability functions seemed to be located on the BA4 fragment.

Bacillus subtilis↗

PubMed1987

Stability function in the Bacillus subtilis plasmid pTA 1060.

Plasmid pBB2 (11.3 kb) was constructed by genetically labeling the cryptic Bacillus subtilis plasmid pTA 1060 with the pC194-derived CmR and the pUB110-derived KmR markers. In nonselective media pBB2 was segregationally almost completely stable (loss rates less than or equal to 0.02% per cell generation). In contrast, pBB3, obtained by deleting from pBB2 a region consisting of two ClaI fragments (1.45 and 0.20 kb, respectively), was unstable (loss rates greater than or equal to 0.5% per cell generation). This indicates that a genetic element required for stability is located on one or both of these fragments. In pBB3 cop, a mutant with a two- to threefold increased copy number, the rate of plasmid loss was reduced compared to that of pBB3. The insertion of a 4.2-kb Escherichia coli DNA fragment reduced the stability of pBB2 only slightly, suggesting that this vector may be useful for the cloning of relatively large fragments.

Bacillus subtilis↗

PubMed1985

Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis.

To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability.

Bacillus subtilis↗

PubMed1984

Restriction of hemimethylated DNA by the Bacillus subtilis R system.

The effects of restriction by the BsuR system on hemimethylated SPP1 DNA were investigated. In vitro, single-stranded nicks were introduced in the nonmodified strand of the hemimethylated DNA at the same sites as recognized in nonmodified homoduplex DNA. Transfection with BsuR-treated hemimethylated DNA was severely reduced. In vivo, transfection with hemimethylated DNA was also severely reduced in competent B. subtilis R cells. In contrast, transfection of protoplasts of the R strain with this DNA was not affected. The apparent restriction by competent cells was attributed to the special mode of processing of transfecting 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↗