Formation of the parental replicative form of bacteriophage φX174

Abstract

The formation of parental replicative forms was observed in the period from 15 seconds to 20 minutes after infection with the single-stranded bacteriophage φX174. The rapid appearance of the first double-stranded molecules indicates that the new DNA strand is polymerized in less than 10 seconds. The distribution of pulse-label in parental RF† suggests that synthesis of the new complementary strand is most often initiated in or near cistron A and proceeds in the direction A-H-G-F-E-D-C-B-A. A second initiation site may also exist near cistron G. Parental RF DNA isolated at various times after infection was characterized by centrifugal analysis. The following observations were made. 1.(i) At one minute after infection an appreciable fraction of the parental RF molecules appear to be in intermediate stages of synthesis; they have complementary strands which are substantially incomplete. 2.(ii) Nascent parental RF molecules which appear to be completely double-stranded have complementary strands with several discontinuities. 3.(iii) A portion of the viral strands in nascent parental RF sediment more slowly than unit-length strands. 4.(iv) Complete parental RFII molecules consist of intact circular viral strands and unit-length complementary strands. 5.(v) Parental RF made from phage exposed to ultraviolet light also have intact circular viral strands, but the complementary strands are shorter than unit-length. 6.(vi) The amount of complementary DNA made after infection with phage exposed to various doses of ultraviolet light suggests that the new strand has only one initiation site.