Conditions Affecting the Isolation from Transformed Cells of Bacillus subtilis of High-Molecular-Weight Single-Stranded Deoxyribonucleic Acid of Donor Origin

Abstract

The deoxyribonucleic acid (DNA) extraction procedure of Piechowska and Fox was evaluated to determine which steps are required for the isolation of high-molecular-weight single-stranded material from transformed cultures of Bacillus subtilis. The results indicate that high-molecular-weight singlestranded DNA can be isolated when certain basic proteins are present at the time of lysis. In the absence of such protective agents as lysozyme or cytC the single-stranded DNA is degraded. The single-stranded DNA can also be protected by being treated with lysozyme at low temperature. The high molecular weight of this single-stranded material and its kinetics of appearance are consistent with its being an intermediate in the transformation process.

Influence of flanking homology and insert size on the transformation frequency ofAcinetobacter baylyiBD413

“…these units are used for all subsequent transformation frequencies given here). Thus there was no significant difference in transformation frequency between the recombination of a point mutation in a coding gene and insertion of the 773-2400 bp gene constructs used, when the DNA sequence was presented as a cell lysate, which is when the homologous flanks were very large (at least greater than 3000 bp each; Davidoff-Abelson and Dubnau, 1973). Although the chromosome of A. baylyi BD413 has seven copies of the 16S rRNA gene (Gralton et al, 1997), the maximum recombination frequency obtained using the 16S rRNA sequence flanks in the donor construct was not significantly different from the maximum obtained for auxotrophic markers or the rifampicin resistance phenotype (around 10 −2 −10 −3 ; Juni and Janik, 1969;Nielsen et al, 1997;Palmen et al, 1993).…”

“…4 Numbers indicate left and right lengths of flanking sequences; * indicates that the lengths of these sequences could not be determined as the donor was lysate DNA (see footnote 5 ). 5 Maximum total donor fragment size for lysates taken up by BD413 was assumed to be >6000 bp (Davidoff-Abelson and Dubnau, 1973).…”

Transfection of Enterobacteriaceae and its applications.

“…coli less efficiently than endolrecBC (183). There are also some preliminary data indicating that pancreatic DNase is inhibited by polyamines (190), and recently a nuclease which is especially active in competent cells of B. subtilis has been shown to be inactive in the presence oflysozyme, spermine, cytochrome c, or protamine (35, 75). Low concentrations of spermine also protect lambda DNA against shear-induced breakage (187), indicating that a more compact configuration of the DNA was achieved.…”

Heteroduplex deoxyribonucleic acid base mismatch repair in bacteria.

“…Naturally occurring DNAmediated transformation, as observed in a limited number of bacteria, provides an example of a recombination process that directly produces heteroduplex DNA. In the case of S. pneumoniae, S. sanguis, and Bacillus subtilis, which are the best studied species among the transformable gram-positive bacteria, double-stranded donor DNA in solution is taken up and rendered single stranded in the entry process (46,143,150,216). This entry process governs subsequent steps by making donor DNA available for recombination only in the form of single-stranded fragments.…”