Formation of Parental Replicative Forms of ϕ-X174 : Synthesis of the First Complementary Strand

Citation

Zuccarelli, Anthony Joseph (1974) Formation of Parental Replicative Forms of ϕ-X174 : Synthesis of the First Complementary Strand. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/gwm8-pv94. https://resolver.caltech.edu/CaltechTHESIS:07012021-163400628

Abstract

Part I

Mutants of the bacteriophage ϕX174 have been isolated that are less dense than wild-type ϕX phage particles in CsCl. When viral strands from the mutants are hybridized with wild-type complementary strands, the resulting duplex molecules have single-stranded loops characteristic of wild type-deletion heteroduplexes. The mutant phages fail to complement ϕX amber mutants in cistron E, but they do complement mutants in six other cistrons. Based upon contour measurements of phage DNA and duplexes, and the buoyant density of the particles, it is estimated that the mutant viruses have deleted approximately 7% of the ϕX genome in the region of cistron E.

Part II

The formation of circular, double-stranded RF (replicative form) DNA in cells has been observed in the period from 15 seconds to 20 minutes after infection with the SS (single-stranded) DNA bacteriophage ϕXl74. The kinetics of appearance of RF during the first few minutes lead to the conclusion that the new, complementary DNA strand is polymerized in less than 10 seconds (viz. about 600 nucleotides per second).

The structure of RFII (a circular duplex with at least one SS break), RF made after infection with UV damaged phage, and nascent RF (extracted 1 minute after infection) were determined by sedimentation analysis and observations made with the electron microscope. They led to the following generalizations: (a) Normal RFII molecules usually have intact circular viral strands and unit-length linear complementary strands. (b) RF made on UV damaged templates also have circular viral strands, but the complementary strand is shorter than unit-length and regions of SS template are evident. (c) The new complementary strand contains many discontinuities immediately after its synthesis, but these are eventually sealed. (d) The viral strand in nascent RF also appears to be broken. These conclusions are incorporated into a proposed mechanism for the synthesis of the first complementary strand.

Part III

Parental RF molecules were pulse-labeled with [³H]thymidine under conditions expected to label the parts of the new complementary strand which are synthesized last. The RF were analyzed by digestion with a restriction enzyme isolated from Haemophilus influenzae. The pattern of ³H label in the resulting fragments led to the following conclusions: (a) Synthesis of the complementary strand is ordered and begins at one or two specific initiation sites. (b) One initiation is located in or near cistron A. A second initiation near the junction of cistrons G and H may also exist. (c) Synthesis of the new strand is counter-clockwise on the genetic map, in the 5' → 3' direction catalyzed by the known DNA polymerases.

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