The structure of the bacterophage alpha DNA molecule : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University

Abstract

Selective precipitation with polyethylene glycol is an efficient method for concentrating and purifying bacteriophage α and other phages. The phage α DNA molecule has a molecular weight of 33 million. When prepared by phenol extraction of crude phage suspensions, it contains many single-strand breaks. When prepared by phenol extraction of purified phage, it contains approximately one randomly-located single-strand break per molecule. The number of single-strand breaks can be further reduced by changing the conditions of the phenol extraction. The complementary single strands of α DNA can be separated by MAK chromatography followed by self-annealing and hydroxylapatite chromatography, but this procedure results in extensive breakage of the strands. An alternative procedure has been developed using CsCl gradient centrifuging in the presence of polyguanylic acid (polyG) to give an efficient separation of the intact strands in 100 µg. quantities. Both the L strand and the H strand of α DNA form complexes with polyG, although to different extents. The PolyG binding sites in the L strand appear to be confined to a small segment having a similar buoyant density to the H strand. Sequences of consecutive pyrimidine nucleotides of all lengths up to 13 have been detected in diphenylamine-formic acid digests of α DNA. There is a slight general tendency towards clustering of the pyrimidine nucleotides, sequences of lengths 1-4 being present at below random frequencies, and longer sequences being present at above random frequencies. These same general features are found in diphenylamine digests of the separated H and L strands. The distribution of pyrimidine nucleotide sequence lengths in a DNA does not appear to follow a rhythmic code of the type found in RNA phages. Preliminary analyses have been made of the longest pyrimidine nucleotide sequences in α DNA, and of the distribution of various sequences between the two strands. The dialysis of pyrimidine deoxyoligonucleotides was investigated, and found to be strongly influenced by cytosine content. This may reflect an unusual conformation of cytosine-rich oligonucleotides at low ionic strength. Gel filtration was found to provide a satisfactory method for the preliminary fractionation of diphenylamine digests on the basis of chain length.