Abstract
Normal preparations of B. subtilis DNA have weight average native molecular weights of 10 to 30 x 10(6). For any given preparation the upper and lower 95% size limits may differ by a factor of ten or more. Single-stranded molecular weights indicate an average of 1 to 4 breaks per single strand of the native DNA. The reduction in transforming activity and viscosity following DNAase I digestion can be accounted for by a direct relationship between the transforming activity of a DNA and its single-stranded molecular weight. Uptake studies with DNAase I treated heavy ((2)H(15)N (3)H) DNA show that single strand breaks inhibit integration less than transformation. A provisional estimate of the size of the integrated region based on correlating the single strand size of the donor-recipient complex with the donor-recipient density differences following alkali denaturation came to 1530 nucleotides. Using a competent, nonleaky thymine-requiring strain of B. subtilis grown in 5-BU medium before and after transformation, it was shown that (a) No detectable amount of DNA synthesis is necessary for the initial stages of integration, (b) Cells which have recently been replicating DNA are not competent. (c) Cells containing donor DNA show a lag in DNA replication following transformation, (d) When donor DNA is replicated it initially appears in a density region between light and hybrid. This indicates that it includes the transition point formed at the time of reinitiation of DNA synthesis in the presence of 5-BU following transformation. A model is proposed in which donor DNA is integrated at the stationary growing point of the competent cell, which is in a state of suspended DNA synthesis.
Highlights
There are two major reasons for a general interest in the elucidation of the molecular mechanisms of integration and recombination in DNA-mediated bacterial transformation
The first problem is that observed frequencies of transformation cannot be accounted for unless a competent cell has a high probability of integrating a donor molecule appropriate for the region in the neighborhood of its replicating point
Those molecules homologous for the appropriate segment of the recipient genome will be permanently integrated. This molecular traffic might be responsible for the transient uptake reported for pneumococcus by Lerman and Tolmach [20]. It might account for the excess of native DNA found "inside" the cell in some of the experiments reported by Bodmer and Ganesan [4]
Summary
There are two major reasons for a general interest in the elucidation of the molecular mechanisms of integration and recombination in DNA-mediated bacterial transformation. There is the hope that these mechanisms will suggest studies directed toward an understanding of the molecular basis for recombination in higher organisms. The unique opportunity a transforming system provides for the study of the physical and chemical properties of DNA molecules in relation to their biological activity cannot fully be exploited without an understanding of the molecular mechanisms involved
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