RECOMBINATION DURING TRANSFORMATION IN HEMOPHILUS INFLUENZAE

In bacterial transformation, the irreversible uptake by a cell population of DNA extracted from a variant strain is followed by integration and expression of the variant "information" carried by the transforming DNA. Little is known of the details or mechanism of transformation after uptake of the DNA by a cell, i.e., of the process of integration which results in a permanent hereditary change. One must conclude from studies on linked transforming factors, however, that some process of recombination is involved.1 3 The studies reported in this paper were designed to determine the time and some of the conditions for recombination during transformation. Goodgal and Herriott have shown that a population of cells which has irreversibly taken up transforming DNA releases a large part of this activity when lysed shortly after uptake.4 In the experiments to be described, the intracellular fate of transforming DNA has been followed quantitatively by this technique in the case of two linked markers located on the same DNA molecule. Goodgal (3) has shown that in Hemophilus influenzae the two factors responsible for resistance or sensitivity to the antibiotics streptomycin and cathomycin (Novobiocin) are linked. When DNA is extracted from donor cells resistant to both cathomycin and streptomycin and is used to transform cells sensitive to both antibiotics, some 20 per cent of the resistant clones are endowed genetically with resistance to both antibiotics. The remainder of the transformations are to streptomycin resistance alone and to cathomycin resistance alone. The degree of linkage obtained depends to some extent on the method of preparation of the DNA and the conditions of transformation, but for material treated in the same way, the fraction of linked transformations is the same. The failure to retain complete linkage may be attributed to loss or inactivation of one or the other marker in the process of extracting the DNA from the donor cells, in the process of transformation itself, or in both processes. One can transform to streptomycin or cathomycin sensitivity as well as to resistance. The frequency of transformation to sensitivity is the same as to resistance, and the same linkage relationships obtain.3 Resistance and sensitivity thus may be considered as alleles of the same locus, the frequency of genetic incorporation of either or both factors being a function of the spatial relationships between the loci and not of the particular forms utilized in the test.3 When, in the course of transformation, both streptomycin and cathomycin loci are genetically incorporated, linked transformations are obtained. When only one locus is genetically incorporated, transformations result 'which are recombinant with respect to the two markers in question, one marker supplied by the donor population and the other by the recipient population. This is the restricted manner in which the term recombination will be used in this paper, i.e., recombination between two linked loci on a single DNA molecule.