Recombination of donor and recipient DNA molecules following conjugation in Escherichia coli K-12
Cooper, Alan Douglas
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A study was made of recombination following conjugation in Escherichia coli K-12 by the isolation of donated DNA fragments. The DNA was isolated after large-scale matings between radioactively-labeled Hfr donor cells and recipient cells density-labeled with 5-iododeoxyuridine in the presence of 5-fluorodeoxyuridine, an inhibitor of thymidylate synthetase. The 30 minute matings were terminated by shearing in a Waring blendor in the presence of azide to poison the males and the males were destroyed by lysis-from-without with a high multiplicity of T6 phage. The merozygotes and female cells were collected by centrifugation removing much of the nonmated male DNA. The remainder of the nonmated male DNA was removed by DNAase and phosphodiesterase digestions followed by differential centrifugations. The "recombinant" DNA - consisting of DNA from the merozygotes and the female cells - was isolated by chloroform-isoamyl alcohol treatment. As a control to establish that the isolated 3H-labeled DNA was the DNA transferred during mating, donor and recipient cells were mixed and the "recombinant" DNA isolated immediately. The specific activity of the "recombinant" DNA from a 30 minute mating was about 10 times greater than the specific activity of the DNA isolated from the 0-time mating used as a control. A mixture of male DNA, radioactively-labeled with 3H-thymidine, and female DNA, density-labeled with 5-iododeoxyuridine, was shown to be separable upon cesium chloride gradient centrifugation. However, when DNA from a 30 minute mating was isolated and centrifuged in cesium chloride the radioactivity banded with the density labeled peak. This indicated that the donated male DNA was associated with the female DNA. The nature of the association of the parental DNAs was determined following the isolation of the "recombinant" DNA immediately after T6 lysis and after further growth of the recombinant cells following a 1:5 dilution into fresh medium. This determination was accomplished by denaturation in 1% formaldehyde and shearing of the "recombinant" DNA isolated from an initial centrifugation in cesium chloride. Mated DNA extracted immediately after T6 lysis banded at a buoyant density of 1.750. The 3H-labeled DNA was associated with this peak. In a control experiment in which the recipient cells were grown in the absence of 5-iododeoxyuridine nearly all the radioactivity was associated with a peak at a buoyant density of 1.711. This indicated that the association of the parental DNAs was not an artifact of the isolation procedure such as the formation of DNA-RNA hybrids or denatured DNA. The "recombinant" DNA from two experiments that banded at a buoyant density of 1.750 was pooled. Upon denaturation of this pooled DNA a peak of radioactivity banded at the density expected for light denatured DNA. Upon shearing of the pooled DNA a peak of radioactivity banded at a buoyant density of 1.711. These results differed from those obtained with DNA extracted after further growth of the female cells following T6 lysis. The DNA from the peak of buoyant density of 1.750 from several gradients was pooled and the aliquots of the DNA taken for shearing and for denaturation. The DNA extracted from the recipient cells 20 minutes after T6 lysis and 140 minutes after T6 lysis exhibited patterns upon shearing and denaturation that were similar to each other. Upon shearing the radioactivity banded at a buoyant density of about 1.750. Upon denaturation the radioactivity banded at a buoyant density expected for density-labeled single-stranded DNA. This indicates that the radioactive nondensity-labeled donor DNA has become incorporated into the density-labeled recipient DNA by the formation of phosphodiester linkages. [TRUNCATED]
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