Mapping of prophage and mature deoxyribonucleic acid from temperate Bacillus bacteriophage phi 105 by marker rescue.

Abstract

By using temperature-sensitive (ts) and suppressor-sensitive (sus) mutants, 11 essential genes have been identified in phage phi105. The order of the genes has been established in two- and three-factor crosses. The genes can be arranged in a linear order; this order is identical in the vegetative phage and in the prophage. One gene essential for phage deoxyribonucleic acid (DNA) synthesis has been found. Marker rescue from prophage and mature DNA, taken up by competent bacteria, was studied by superinfection with phage carrying one sus and one ts mutation. In prophage DNA, all single markers studied are rescued at similar frequencies. The frequency of co-rescue of two markers is proportional to the recombinational distance between the markers. Thus, colinearity between the genetic map and the position on the DNA molecule of those mutations used to establish the map is demonstrated. The results indicate that the recombination frequencies observed in vegetative crosses are a relative measure of the physical distance between markers. All single markers are not rescued at equal frequencies from mature DNA. The frequency of co-rescue of two markers is related to the recombinational distance only over a distance about one-fourth or less of the genetic map. Markers separated by 10% recombination, or more, are co-rescued at 5 to 10% of the frequency of rescue of single markers. Shearing of mature DNA into half-sized molecules reduces the efficiency by which single markers are rescued by a factor of 5 to 10. The results of experiments on co-rescue of two markers from half-sized mature DNA indicate a preferred break-point near the middle of the genetic map; the results are compatible with a nonpermuted sequence in mature DNA. It is pointed out and discussed that mature DNA exhibits several anomalies in marker rescue experiments.