Genetic recombination : understanding the mechanisms / Harold L.K. Whitehouse.

  • Harold Leslie Keer Whitehouse
Date:
[1982]
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Licence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Credit: Genetic recombination : understanding the mechanisms / Harold L.K. Whitehouse. Source: Wellcome Collection.

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    p-nitrobenzoic acid. The donor bacteria were allowed many generations of growth in a medium containing 32 P as a radioactive label, and deuterium ( 2 H) and 15 N as density labels, before the DNA was isolated. After not more than 15 min incuba tion following cell entry, and hence before DNA replication had occurred, the DNA was extracted from the recipient cells and subjected to density-gradient centrifugation in caesium chloride. It was found that both the streptomycin resistance and the radioactivity of the donor DNA were now associated with DNA only slightly heavier than that of the recipient cells. It was evident that the donor DNA had become associated with the genome of the recipient cells. When the size of the DNA molecules extracted from the recipient cells was reduced, by sonication, from an estimated molecular weight of 2 x 10 7 daltons to about 10 6 daltons, the streptomycin resistance and the radioactivity became associated with heavier molecules, approaching a density halfway between that of the two parents. Denaturation, that is, separation of the strands of the duplex, using heat or alkali, revealed that most of the 32 P was still associated with a large amount of DNA of light density. Denaturation of sonicated extracts, on the other hand, revealed that the bulk of the radioactive label was associated with heavy single strands. Fox and Allen (1964) concluded that the donor material formed part of one strand of the duplex DNA of the recipient genome and was covalently linked to it. They favoured the hypothesis that either strand of the donor DNA could form a hybrid with the complementary strand of the DNA of the recipient. If it were one unique strand, after one bacterial doubling none of the cells receiv ing the other strand would carry donor information. This is contrary to Fox and Hotchkiss’s observation (see above) that the transforming activity multiplies in synchrony with that of the recipient DNA. The steps in the process of recombination in pneumococcal transformation, to which Fox and Allen’s results pointed, were illustrated by Fox (1966) and are shown in Fig. 1. One strand, chosen randomly, of the duplex donor molecule is inserted into the corresponding region of the recipient genome, the other strand of the donor and the displaced strand of the recipient being broken down. Finally, the inserted donor strand is joined covalently to the neighbouring parts of the corresponding recipient strand. Miao and Guild (1970) showed that the donor DNA does not have to be duplex for pneumococcal transformation to take place, but the efficiency of single strands is only about 0.5% of duplex, probably because of inefficient cell entry. Their demonstration depended on the use of the light strands only as donor. The light and heavy strands of the pneumococcal DNA were separated by denaturation, and fractionated by reaction with the ribopolymer poly(G,U). Lacks, Greenberg and Neuberger (1974) obtained mutants of the pneumo coccus lacking a deoxyribonuclease necessary for transformation. They used a strain that was deficient in two major deoxyribonuclease activities known not to affect transformation, and selected mutants defective in transformation, following treatment with the mutagen l-methyl-3-nitro-l-nitrosoguanidine. One group of mutants showed no detectable transformation and failed to bind or take up DNA. Their deoxyribonuclease content was unchanged. A second group of mutants
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