On the mechanism of integration following transformation with single-stranded DNA of Hemophilus influenzae

Abstract

The biological fate of denatured transforming DNA was examined during transformation of Sr-marked cells with denatured C 25 -marked DNA. Lysates of transforming cells were tested for the presence of the donor transforming activity. It was found that, as a function of time, the denatured C 25 transforming activity decreased in these samples, whereas new native C 25 activity and linked Sr C 25 recombinant activity appeared. Linked recombinant activity indicated integration of the denatured donor DNA. Retention of linked recombinant activity after denaturation provided evidence for the early establishment of covalency between denatured donor and recipient DNA. The physical fate of the denatured donor DNA was determined by cesium chloride density-gradient centrifugation of extracts from transforming cells. The recipient cells were light, labeled with [ 3 H]thymidine and carried the Sr marker; the donor DNA was heavy ( 15 N-D 2 O), labeled with [ 32 P]phosphate and carried the C 25 marker. During uptake of the DNA at low pH, no hybrid density DNA formed and there was no native C 25 or SC recombinant transforming activity. After neutralization, there was a progressive transfer of donor DNA into a hybrid structure concomitant with the appearance of native C 25 and SC recombinant transforming activity. Denaturation of the hybrid DNA increased its density, which indicated that the integrated single-stranded DNA was covalently linked to the recipient DNA. The size of the integrated piece was estimated to be about 6 × 10 6 daltons. Part of the donor DNA banded at its original fully heavy, denatured position, indicating that not all of the donor DNA was covalently linked to the recipient DNA. It is concluded that, aside from the different uptake mechanisms, the processes of integration with single-stranded and double-stranded DNA's are similar.