Effects of Uvsx, Uvsy and DNA Topoisomerase on the Formation of Tandem Duplications of the Rii Gene in Bacteriophage T4

We have characterized tandem duplications in the rII regions of phage T4. The rII deletion r1589 blocks only the function of the rIIA cistron, although it extends into the B cistron. Another rII deletion, r1236, blocks the function of the rIIB cistron and overlaps r1589. When a cross is made between r1589 and r1236, true rII(+) progeny cannot form. Instead, anomalous phenotypically rII(+) phages are detected carrying an rII region from each parent. Analyses of nucleotide sequences of the recombination junctions indicate that recombination takes place between short regions of homology (from 2 to 10 bp). Open reading frames of the recombinants deduced from the nucleotide sequences reveal that they contain a normal rIIA cistron and one of a variety of fused, duplicated rIIB cistrons. The T4 uvsX and uvsY genes, which participate in homologous recombination, are involved in this duplication formation. T4 DNA topoisomerase is encoded by genes 39, 52 and 60. Mutations in 52 and 60 reduced the frequency of such duplications, but mutations in gene 39 and some in gene 52 did not. Hence, the effects of topoisomerase mutations are allele-specific. Models are proposed in which these proteins are involved in tandem duplication.     

The isolation and characterization of a T4 mutant partially defective in recombination

Summary Starting with an rII diploid isolate of T4 a procedure is reported for isolating diploid variants which generate segregants at a reduced frequency. The properties of one such variant, which exhibits a four-fold reduction in segregation frequency, is discussed in detail. It is shown: 1) this variant has acquired at least two extra mutations outside the rII region 2) these mutations confer a four to seven-fold reduction in recombination frequency as measured in standard phage crosses 3) the mutations also cause a 20% increase in UV-sensitivity and a marked effect both on multiplicity-reactivation and on the intracellular development of radiation-resistance.     

Novel rII duplications in bacteriophage T4.

The properties of two rII complementation heterozygotes (D5B and D7A) of bacteriophage T4 are described. These strains are characterized by their stability, each forming less than 10-3 r segregants among their viable progeny, and by their segregation of only one of the two parental types. No increase in r progeny was found on crossing D7A or D5B with T4r+, indicating that the duplications in these strains are not separated by an essential region of the phage genome. Both D5B and D7A from h-2+/h-4+ heterozygotes at frequencies similar to T4r+, suggesting that the duplicated regions in these strains are short. The progeny of these h-2+/h-4+ heterozygotes retain heterozygosity for rII but not for h: therefore, D5B and D7A are not stabilized terminal redundancy complementation heterozygotes. We conclude that D5B and D7A contain very short tandem duplications and we present structures consistent with the observed characteristics of these phages.     

The Genetic Constitution of Tandem Duplications of the rII of Bacteriophage T4d

Methods for genetically mapping the end points of tandem duplications of the rII region of bacteriophage T4D are presented. Analysis of ten duplications indicates (1) that the position of duplication end points and therefore the length of the duplicated segment differ in strains of independent origin; (2) that there is a direct relationship between segregation frequency and length; (3) that segregation is more frequent than expected on the basis of standard genetic mapping; and (4) that while duplications frequently include non-rII genetic material, frequently they do not include the entire rII region. The duplications studied range from less than two to about five cistrons in length.     

Properties of Bacteriophage T4 Mutants Defective in Gene 30 (Deoxyribonucleic Acid Ligase) and the rII Gene

In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.     

Analysis of expression of the rII gene function of bacteriophage T4.

Temperature-sensitive (ts) mutants of the T4 phage rII gene were islated and used in temperature shift experiments that revelaed two different expressions for the normal rII (rII+) gene function in vivo: (i) an early expression (0 to 12 min postinfection at 30 C) that prevents restriction of T4 growth in Escherichia coli hosts lysogenic for gamma phage, and (ii) a later expression (12 to 18 min postinfection at 30 C) that results in restriction of T4 growth when the phage DNA ligase (gene 30) is missing. The earlier expression appeared to coincide with the period of synthesis of the protein product of the T4 rIIA cistron, whereas the later expression occurred after rIIA protein synthesis had stopped. The synthesis of the protein product of the rIIB cistron continues for several minutes after rIIA protein synthesis ceases (O'Farrell and Gold, 1973). The two rII+ gene expressions might require different molar ratios of the rIIA and rIIB proteins. It is possible that the separate expressions of rII+ gene function are manifestations of different associations between the two rII proteins and other T4-induced proteins that are synthesized or activated at different times after phage infection.     

Selective Allele Loss in Mixed Infections with T4 Bacteriophage

Evidence is presented that when E. coli B is mixedly infected with T4D wild type and rII deletion mutants, the excess DNA of the wild type allele is lost. No loss is seen in mixed infections with rII point mutants and wild type. In similar experiments with lysozyme addition mutants, the mutant allele is lost. We believe these results demonstrate a repair system which removes "loops" in heteroduplex DNA molecules. A number of phage and host functions have been tested for involvement in the repair of the excess DNA, and T4 genes x and v have been implicated in this process.     

Synthesis of Bacteriophage and Host DNA in Toluene-Treated Cells Prepared from T4-Infected Escherichia coli: Role of Bacteriophage Gene D2a

We investigated the synthesis of DNA in toluene-treated cells prepared from Escherichia coli infected with bacteriophage T4. If the phage carry certain rII deletion mutations, those which extend into the nearby D2a region, the following results are obtained: (i) phage DNA synthesis occurs unless the phage carries certain DNA-negative mutations; and (ii) host DNA synthesis occurs even though the phage infection has already resulted in the cessation of host DNA synthesis in vivo. The latter result indicates that the phage-induced cessation of host DNA synthesis is not due to an irreversible inactivation of an essential component of the replication apparatus. If the phage are D2a(+), host DNA synthesis in toluene-treated infected cells is markedly reduced; phage DNA synthesis is probably also reduced somewhat. These D2a effects, considered along with our earlier work, suggest that a D2a-controlled nuclease, specific for cytosine-containing DNA, is active in toluene-treated cells.     

In vivo study of fidelity of DNA double-strand break repair in bacteriophage T4

A method for in vivo studying the fidelity of DNA double-strand break (DSB) repair in bacteriophage T4 has been developed. The frequency of reversion of rII mutations to the wild phenotype was measured in i segC+ x i ets 1 segCDelta crosses, where ets 1 is an insertion in the initial part of the rII gene carrying a sequence recognized by SegC endonuclease; i designates a rIIB or rIIA mutation located at some distance from ets 1, and segCDelta is a deletion in the segC gene. In such cross, a DSB occurs in the site of ets 1. Their repair involves genetic recombination and DNA replication in the neighborhood of ets 1. In parallel, the frequency of reversion of the same i mutant in the absence of DSBs is measured in i x i self-crosses. Reversions of different types (base substitutions, deletions, insertions) can be studied with the use of structurally different i mutations located at varying distances from ets 1. The reversion frequencies were determined for three rIIB mutations and one rIIA mutation. The results obtained suggest that DSB repair in bacteriophage T4 is a process of high fidelity with the rate of errors that does not essentially exceed that in the case of usual phage multiplication.     

Induction of mutation in phage T4 by extracellular treatment with methylene blue and visible light

Summary Photodynamic inactivation with methylene blue and light (MB+Li) of maximally sensitized phages T4 and T4rII nearly followed single-hit kinetics. Not so mutation induction, which in one phage (T4rII N₁₇) tested showed multi-hit kinetics. This difference and the fact that T4rII phages with a GC base pair at the site of the rII-mutation showed no enhanced backmutation when compared to rII phages with an AT base pair or to sign mutants suggests that there is no guanine specificity for MB+Li-induced mutation in phage T4.     

Genetic mapping by duplication segregation in Salmonella enterica

MudP and MudQ elements were used to induce duplications in Salmonella enterica by formation of a triple crossover between two transduced fragments and the host chromosome. The large size (36 kb) of MudP and MudQ is a favorable trait for duplication formation, probably because homology length is a limiting factor for the central crossover. Additional requirements are a multiplicity of infection of 2 or higher in the infecting phage suspensions (which reflects the need of two transduced fragments) and an exponentially growing recipient (which reflects the need of a chromosome replication fork). We describe a set of 11 strains of S. enterica, each carrying a chromosomal duplication with known endpoints. The collection covers all the Salmonella chromosome except the terminus. For mapping, a dominant marker (e.g., a transposon insertion in or near the locus to be mapped) is transduced into the 11-strain set. Several transductants from each cross are grown nonselectively, and haploid segregants are scored for the presence of the marker. If all the segregants contain the transduced marker, it maps outside the duplication interval. If the marker is found only in a fraction of the segregants, it maps within the duplicated region.     

The pathway of recombination in phage T4

Summary A study has been made of the effect of modifying the products of the early T4 genes on the frequency with which haploid segregants are generated by recombination from a phage harbouring a standard genetic duplication. Alterations in the products of genes 32, 44, 46, 47 and 59 have been found to significantly decrease the segregation frequency and are, therefore, considered to be involved in the T4 recombination pathway.     

9-Aminoacridine mutagenesis of bacteriophage T4 intracellular DNA.

Most of the intracellular T4 DNA made in the presence of 9-aminoacridine is of lower molecular weight than mature T4 DNA and does not get packaged into phage particles. Using a T4 DNA transformation assay, we have examined this intracellular T4 DNA for its content of 9-aminoacridine-induced revertants of certain rII gene frameshift mutations. The proportion of acridine-induced revertants in the intracellular DNA population is close to that found in the phage progency made in the presence of 9-aminoacridine. Thus, the generation of low molecular weight T4 DNA in the presence of 9-aminoacridine is not, in itself, also a mutagenic process.     

Nonreplicated DNA and DNA Fragments in T4 r? Bacteriophage Particles: Phenotypic Mixing of a Phage Protein

"Conservative phage" containing a genome derived from an infecting phage particle which has not undergone replication in the cell but nevertheless has become encapsulated and released in a normal phage particle, are found after infection of Escherichia coli with rII(-) or rI(-) mutants under conditions which result in rapid lysis. If such conservative phage are derived from a mixed infection with v(+) and v(1) phage, they display phenotypic mixing of the v gene product (an endonuclease carried in the phage particle). Populations of rI and rII mutant phage grown under conditions of rapid lysis include particles containing short DNA fragments. It is suggested that a "maturation defect", common to rI and rII mutants, but absent in rIII mutants, may account for the encapsulation of nonreplicated DNA as well as that of the DNA fragments.     

The isolation and characterization of TabR bacteria: Hosts that restrict bacteriophage T4 rII mutants

Summary We have isolated mutants of Escherichia coli B (called TabR) that restrict the growth of bacteriophage T4 rII mutants at high temperature. TabR strains lysed very rapidly after infection with rII mutants, and no progeny phage were produced. T4⁺-infected TabR cells also lysed quickly, but the cells remained intact long enough to give a small burst. We have selected pseudorevertants of rII deletion mutants that grow on TabR at high temperature; tk (thymidine kinase) is a component of one class of these pseudorevertants.T4 strains harboring mutations in genes 12, 16, 25, 34, 36, 45 and 63 were also specifically restricted on TabR strains at high temperature. Bacteriophages T2, T4, T5, T6, and T7 grew normally on TabR, while , 80, and P1 failed to grow at any temperature. The most restrictive TabR strains were auxotrophic for methionine at high temperature, and most spontaneous Met⁺ revertants had also lost the ability to restrict rII mutants, suggesting that the TabR phenotype and methionine auxotrophy result from the same mutation.Although the mechanism by which TabR strains exert their restriction has not been determined, one model is described. The potential uses of these and similar strains is discussed.     

Isolation of Plaque-Forming, Galactose-Transducing Strains of Phage Lambda

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.—A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.     

Bacteriophage-induced functions in Escherichia coli K (lambda) infected with rII mutants of bacteriophage T4

Rutberg, Blanka (Karolinska Institutet, Stockholm, Sweden), and Lars Rutberg. Bacteriophage-induced functions in Escherichia coli K(lambda) infected with rII mutants of bacteriophage T4. J. Bacteriol. 91:76-80. 1966.-When Escherichia coli K(lambda) was infected with rII mutants of phage T4, deoxycytidine triphosphatase, one of the phage-induced early enzymes, was produced at initially the same rate as in r(+)-infected cells. Deoxyribonuclease activity was one-third to one-half of that of r(+)-infected cells. This lower deoxyribonuclease activity was observed also in other hosts or when infection was made with rI or rIII mutants. Presence of chloramphenicol did not allow a continued synthesis of phage deoxyribonucleic acid in rII-infected K(lambda). No phage lysozyme was detected nor was any antiphage serum-blocking antigen found in rII-infected K(lambda). It is suggested that the rII gene is of significance for the expression of phage-induced late functions in the host K(lambda).     

Ultraviolet mutagenesis in bacteriophage T-4. I. Irradiation of extracellular phage particles

Drake, John W. (University of Illinois, Urbana). Ultraviolet mutagenesis in bacteriophage T4. I. Irradiation of extracellular phage particles. J. Bacteriol. 91:1775-1780. 1966.-Ultraviolet (UV) irradiation of extracellular T4 phage particles induces about 2 x 10(-4)r mutations per lethal hit. The mutants largely escape detection unless the irradiated phages are plated with very soft overlay agar. Multiplicity reactivation is not a prerequisite for mutagenesis. A much higher frequency of base pair substitution-type mutants is induced than is found in the spontaneous background, but sign mutants are also induced. Nearly half of the mutants map into previously identified UV hot spots. The rII mutants induced extracellularly are very similar to those induced intracellularly. The mutants also appear to result from direct radiation effects upon the bacteriophage deoxyribonucleic acid.