Mapping of Prophage and Mature Deoxyribonuleic Acid from Temperate Bacillus Bacteriophage φ105 by Marker Rescue

Abstract

By using temperature-sensitive (ts) and suppressor-sensitive (sus) mutants, 11 essential genes have been identified in phage 4105. 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.

Transfection of Enterobacteriaceae and its applications.

“…Since only lambda DNA fragments carrying a cohesive end were active in their transfection system, internal fragments of the chromosome could not be detected. Because internal fragments of B. subtilis phage to coliphage T4 DNAs are active in helped transfection and coliphage T4 assays, the colinearity of the genetic and physical maps of these phages could be documented even more completely (4,9,141,150,415).…”

Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule

“…The phage mutants used in these experiments carried either a susl4 or sus9 mutation. sus14 maps in gene B, and sus9 maps in gene L; the former is near the left end of the genetic map, and the latter is at the right end (1,24). The results of these experiments ( 23,1977 from the right and fragment D from the left end of the genome.…”

“…Marker rescue. The procedure followed was that described by Armentrout and Rutberg (1). The 4105 DNA preparations used were unfractionated EcoRIdigested DNA and separated EcoRI-generated fragments that were recovered from the agarose gels by the "freeze-squeeze" method of Thuring et al (32).…”

Transfection ofBacillus subtiliswith phage φ105 DNA: Effect of φ105EcoRI restriction endonuclease fragments

“…The su +3 gene was introduced into 3G18 by congression with primary selection for ade + and using SR135 DNA. Phage ~105 wild type, q~105 sus-mutants and the methods used for preparing phage and phage DNA have been described [8,9]. The methods for transfection have recently been described [3,4].…”