Abstract
Bacteriophage T5 possesses a double-stranded DNA molecule containing single-strand interruptions and, as a result, denatured T5 DNA consists of a number of single-stranded DNA fragments. The objectives of this investigation were to separate and isolate these single-strand fragments and to map their arrangement in the duplex DNA molecule. To do this I have developed a new gel electrophoretic procedure for separating single-stranded DNA species.
Agarose gel electrophoresis has been used analytically to determine the approximate molecular weights of T5 single-stranded DNA species and under certain conditions to separate the complementary strands of several other bacteriophage DNA molecules. Some characteristics of the gel system and of the electrophoretic behaviour of both double and single-stranded DNA have also been investigated.
Electrophoretic analysis revealed the presence of between forty and fifty single-stranded DNA fragments in denatured T5 DNA, and these have been classified as “major” or “minor” species on the basis of their relative abundances. With wild-type T5+ DNA the electrophoretic separations revealed five major fragments which have molecular weights of 37.0, 14.5, 13.9, 5.1 and 3.8 million. The heat-stable mutant T5st(0) possesses only four major fragments and these have molecular weights of 35.3, 17.2, 14.5 and 3.8 million. The minor fragments occur in much smaller amounts and have molecular weights ranging from as small as 0.16 million up to 28 million. The single-strand fragment patterns of T5 DNA are normally extremely reproducible and most of minor fragments appear to be common to both T5+ and T5st(0) DNA.
I envisage that the major fragments are delineated by “primary” interruptions which are present in every duplex molecule in the population, and that superimposed on this basic structure there must be “secondary” interruptions which occur only at low frequency and at different positions in individual molecules. Therefore, the minor fragments would result from subdivision of major fragments by these occasional secondary interruptions.
For further study of the fragments I have centrifuged denatured T5 DNA in CsC1 density gradients in the presence of poly(G). Gel electrophoretic analysis of fractions from these gradients showed that the 37.0 and 13.9 million major fragments of T5+ DNA and the 35.3 and 17.2 million of T5st(0) DNA are found in the “heavy” buoyant density regions. The other fragments vary in the extent of their interactions with poly(G); and a minor fragment, which has anomalous electrophoretic properties exhibits the strongest poly(g)-interaction.
Studies on the distribution of fragments within the double-strand breakage products of sheared T5 DNA have been performed as well as some hybridisation experiments with isolated major fragments. From the results of these experiments I have proposed a model for the arrangement of major single-strand fragments in the duplex DNA molecules of T5+ and T5st(0). According to this model both molecules contain one intact strand, and 3.8 and 14.5 million fragments at opposite ends of the other strand, but T5+ contains three “primary” interruptions and T5st(0) contains only two. This difference can be explained by a deletion in T5st(0) DNA which eliminates the central T5+ interruption.
The first break by shear in the duplex T5 DNA molecule occurs opposite the interruption separating the 14.5 million fragment from the rest of the “fragmented” strand, but the site of the second preferred point of breakage cannot as yet be established with any certainty.
Finally, the questions of the location of first-step-transfer DNA, of the correlation between the physical and genetic maps, and the possible existence of another major fragment are discussed in terms of the evidence and model presented here.