The pyrimidine tracts of bacteriophage øX174

Typescript.Thesis (Ph. D.)--University of Hawaii, 1969.Bibliography: leaves [106]-110.x, 110 l illus., tablesThe bacteriophage ϕX174t containing single stranded, circular DNA as its genetic material, has been studied in some detail with respect to its chemistry and biological function. The frequency of the pyrimidine tracts in the wild type bacteriophage has been determined t and the preponderance of the amount of longer tracts of seven to ten pyrimidine nucleotides, over that which would be expected from a random distribution noted. Further studies of the longer pyrimidine tracts were carried out using the ρ- mutant bacteriophage, which is lysis defective, showing delayed lysis in dilute cultures of the host, E. coli, and lysing only about ten per cent of the cells in dense culture. The DNA of the phage contains a discontinuity, a small region which is not single stranded, and therefore not susceptible to the action of E. coli exonuclease I. This enzyme digests only single stranded DNA and works only on free 5' OH ends. Therefore, digesting ϕX174 chromosomes with pancreatic DNA'ase, an endonuclease, to a limited extent, followed by digestion with E. coli exonuclease I, leaves a population of DNA of varying lengths, all having the same 5' end at the discontinuity. Within this population, then, the frequency of an individual tract's survival, depends on its distance from the discontinuity, and can be used to determine its position on the ϕX174 chromosome. Methods for preparation of pyrimidine tracts, their separation by length into isopliths, and their subfractionation according to their relative cytosine and thymine content have already been developed. When more than one tract of the same length and base composition existed, further separations were not possible. Only the unique tracts could be mapped. This, in the ρ- mutant, consisted of one decanucleotide, two each of nona and octanucleotides and one each of hepta and hexanucleotides. All of these tracts were located in the region of about 30 to 80 per cent of the distance around the chromosome from the discontinuity. The pyrimidine tracts of the ρ- mutant and wild type bacteriophage were compared. There is a decanucleotide of composition C3T7 present in the wild type phage, but not in the ρ- mutant. Some evidence indicates that this mutation, that of a pyrimidine to a purine, occurs at a cytosine residue, leaving an octanucleotide, CIT7 present in the ρ- mutant, but not in the wild type phage. The nature of the ρ- mutation is discussed, along with the possibility that the longer pyrimidine tracts may have a role in initiation of the DNA to messenger RNA transcription process