Nonsense Mutants in the rII A Cistron of Bacteriophage T4

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of Escherichia coli, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC → AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.     

Characterization of T4 Mutants That Partially Suppress the Inability of T4rII to Grow in Lambda Lysogens

In the course of isolating viable T4 deletions that affect plaque morphology (Homyk and Weil 1974), two closely linked point mutants, sip1 and sip2, were obtained. They map between genes t and 52, cause a reduction in plaque size and burst size, and partially suppress the lethality of rII mutants for growth in lambda lysogens. These characteristics demonstrate that sip1 and sip2 are similar to mutants previously reported by Freedman and Brenner (1972). In addition, D. Hall (personal communication) has shown that sip1 and sip2 are similar to the mutant farP85, which affects the regulation of a number of early genes ( Chace and Hall 1975).——Sip suppression of rII mutants can be demonstrated in one-step growth experiments, even when both rII genes are completely deleted. This indicates that sip mutants do not simply reduce the level of rII gene products required for growth in a lambda lysogen. Instead, they alter the growth cycle so as to partially circumvent the need for any rII products.——Mutations at two other sites, designated L₁ and L₂, reverse the poor phage growth caused by sip and, in the one case tested, reverse the rII-suppressing ability of sip.     

Streptomycin-Induced Phenotypic Suppression of Hydroxylamine-Induced Nonsense Mutants in the rII A Cistron of Bacteriophage T4

Twenty-one hydroxylamine-induced rII A cistron nonsense mutants were tested for streptomycin (SM)-induced phenotypic suppression by exposing Escherichia coli SBO (nonpermissive host) to phage in the presence and absence of SM. All nine amber, four of six ochre, and five of six opal mutants were phenotypically suppressible by SM. For suppressible mutants, the ratio of the average burst size in the presence of SM to size in the absence of SM ranged from 12 to 242 for the ambers, 3 to 33 for the ochres, and 4 to 14 for the opals. Increased susceptibility of the amber mutants to SM-induced phenotypic suppression relative to the susceptibility of the opal and ochre mutants may reflect a neighboring base effect, such that a 3′-terminal adenine inhibits misreading of a 5′-terminal uracil.     

The structure of rII diploids of phage T4

Summary Crosses between the rII deletion 1589 and an overlapping deletion such as 638 which lies entirely within the rIIB cistron generate a few T4 phage particles, the so-called rII diploids, which contain two copies of the rII region, one derived from each parent in the cross. A specific model is proposed to account for the properties of these rII diploids. This model postulates: 1) the rII diploids contain a tandem duplication, 2) the duplicated region extends both to the left and right of the rII region itself, and 3) during phage multiplication recombination occurs between homologous regions of the duplication. These assumptions lead to precise predictions on the following points: 1) the frequency at which haploid 1589 and 638 phage particles are generated during multiplication, 2) the ratio of 1589 and 638 phage amongst the segregants, 3) the relative lengths of terminal redundancy to be found in the rII diploids, the 1589 and 638 segregants and wild-type T4, and 4) the formation and properties of homozygous diploids containing two copies either of the 1589 or 638 region.Experiments are reported which validate the model on these points and also indicate how the homozygous diploids can be utilized to generate new rII diploids with structures which would otherwise be unobtainable.     

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.     

Mutants of phage HK239 defective in excluding phages lambda T4rII, P1, and P2

Summary Escherichia coli cells lysogenic for temperate phage HK239 exclude phages , HK022, P1 vir, P2, and rII mutants of phage T4. After mutagenic treatment, four isolates were obtained for their inability to exclude T4rII. It is shown that this mutation, designated exc, is located in the prophage HK239, and that, it also abolishes the exclusion of phages , HK022, P1 vir, and P2.     

Replication and Recombination in Ligase-Deficient rII Bacteriophage T4D

Deoxyribonucleic acid replication and genetic recombination were investigated after infection of Escherichia coli with ligase-deficient rII bacteriophage T4D. The major observations are: (i) deoxyribonucleic acid synthesis is discontinuous, (ii) the discontinuities are more slowly repaired than in wild-type infection, (iii) host ligase is required for viability, and (iv) genetic recombination is increased.     

Mutagen specificity and position effects on mutation in T4rII nonsense sites.

In 14 sites in the T4rII region, spontaneous and induced interconversions of ochre (UAA) and opal (UGA) alleles, as well as the reversion of the nonsense sites to r+, were studied. The mutagens employed were 2-aminopurine (2AP), 5-bromouracil (5BU), N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and hydroxylamine (HA). With the test system employed, mutagen specificity (i.e., the preferential induction of A: T leads to G: C or G: C leads to A: T mutation at a given site) can be studied. Simultaneously, the response of similar base pairs at various locations in the same or in different nucleotide triplets throughout the rII region, to a given mutagen, can be compared. 2-Aminopurine can induce transitions of both A: T and G: C base pairs at high rates. This mutagen shows no preference for either direction. Furthermore, there is a correlation between the response to 2AP of an A: T pair occupying a given site, and that of a G: C pair occupying the same site. NTG and HA induce G: C leads to A: T transitions almost exclusively. However, there is a correlation between the low rates of A: T leads to G: C transition induced in each of the various sites by these mutagens and those of G: C leads to A: T. 5-Bromouracil induces transitions from G: C to A: T more readily than from A: T to G: C. With 5BU-induced mutation, there is no correlation between the rates of G: C leads to A: T transitions and those of A: T leads to G: C. In UAA sites, all three adenine:thymidine paris respond to 2AP mutagenesis in a similar pattern, In each position in the triplet, response to 2 AP is correlated with that to 5BU. In UGA sites, there are correlations among the spontaneous as well as the 2AP-, HA- and NTG-induced transition rates. 5BU-induced transition rates are usually not correlated with those induced by other mutagens or with the sponatneous ones.     

The DNA-Delay Mutants of Bacteriophage T4

Mutants of phage T4 defective in genes 39, 52, 58-61, and 60 (the DNA delay or DD genes) are characterized by a delay in phage DNA synthesis during infection of a nonpermissive Escherichia coli host. Amber (am) mutants defective in these genes yield burst sizes varying from 30 to 110 at 37 C in E. coli lacking an am suppressor. It was found that when DD am mutants are grown on a non-permissive host at 25 C, rather than at 37 C, phage yield is reduced on the average 61-fold. At 25 C incorporation of labeled thymidine into phage DNA is also reduced to 3 to 10% of wild-type levels. Mutants defective in the DD genes were found to promote increased recombination as well as increased base substitution and addition-deletion mutation. These observations indicate that the products of the DD genes are necessary for normal DNA synthesis. The multiplication of the DD am mutants on an Su⁻ host at 37 C is about 50-fold inhibited if prior to infection the host cells were grown at 25 C. This suggests that a compensating host function allows multiplication of DD am mutants at 37 C in the Su⁻ host, and that this function is active in cells grown at 37 C prior to infection, but is inactive when the prior growth is at 25 C. Further results are described which suggest that the products of genes 52, 60, and 39 as well as a host product interact with each other.