Studies of opal mutations in bacteriophage T4B

Hydroxylamine-induced amber and opal mutants are localized on the map of gene 47 of bacteriophage T4B. The matched map of amber and opal mutations showed the presence of four paired sites which seemed to have arisen in the triplet coding for tryptophan.In growth studies o opal mutants in gene 47 in a series of Su⁺ strains the number of strains bearing a gene-suppressor for amber or ochre mutations also had a weak suppressor activity for some opal mutants. This suppressor acitivity is supposedly due to a second mutation in gene Su_uga.A comparative study of the phage yield with amber and opal mutations located in the same (paired) triplet in gene 47 has shown that the suppressor activity depends on the location of the mutant site along the gene.Experiments dealing with the induction of reversions by nitrous acid in amber and opal mutants with mutational sites located in the same triplet of gene 47 (mutant pairs) have shown the essential influence of the nucleotide sequence in the triplet on the frequency of induced reverse mutations at the given site.     

Nonsense mutants in the bacteriophage T4D v gene

Ten UV-sensitive mutants of T4D with the v phenotype were isolated. Of these ten mutants, two are amber and two opal. In UV curves and in photoreactivation and multiplicity reactivation (MR) experiments the nonsense mutants show the v phenotype in su⁻ hosts and almost the T4+ phenotype in su⁺ hosts. The mutations are located between rI and e and are alleles of v₁. In crosses with irradiated and non-irradiated phages the recombinant frequency is not reduced by uvs5.Amber uvs5 propagated in CR63 su⁺ is with B su⁻ just as sensitive to UV as uvs5 propagated in B su⁻, which permits the conclusion that the capsid of T4 phage particles does not contain the v gene product.In addition, four mutants with a relative UV sensitivity equal to that of T4x were isolated. These are discussed in the next paper³³.     

New Late Gene, dar, Involved in DNA Replication of Bacteriophage T4 I. Isolation, Characterization, and Genetic Location

Suppressors of gene 59-defective mutants were isolated by screening spontaneous, temperature-sensitive (ts) revertants of the amber mutant, amC5, in gene 59. Six ts revertants were isolated. No gene 59-defective ts recombinant was obtained by crossing each ts revertant with the wild type, T4D. However, suppressors of gene 59-defective mutants were obtained from two of these ts revertants. These suppressor mutants are referred to as dar (DNA arrested restoration). dar mutants specifically restored the abnormalities, both in DNA synthesis and burst size, caused by gene 59-defective mutants to normal levels. It is unlikely that dar mutants are nonsense suppressors since theý failed to suppress amber mutations in 11 other genes investigated. The genetic expression of dar is controlled by gene 55; therefore, dar is a late gene. The genetic location of dar has been mapped between genes 24 and 25, a region contiguous to late genes. dar appears to be another nonessential gene of T4 since burst sizes of dar were almost identical to those of the wild type. Mutations in dar did not affect genetic recombination and repair of UV-damaged DNA, but caused a sensitivity to hydroxyurea in progeny formation. The effect of the dar mutation on host DNA degradation cannot account for its hydroxyurea sensitivity. dar mutant alleles were recessive to the wild-type allele as judged by restoration of arrested DNA synthesis. The possible mechanisms for the suppression of defects in gene 59 are discussed.     

Mapping of recognition sites for the restriction endonuclease from Escherichia coli K12 on bacteriophage PM2 DNA.

Several mutations in gene B of phage S13 appear to shorten the B protein by elimination of an N-terminal fragment, without destroying the B protein function. The shortened B protein resulting from each of these mutations can block the unique DNA-nicking properties of the S13 gene A protein. Because of the block in gene A function, normal gene B protein may have a function in phage DNA synthesis in addition to its known role in catalyzing capsid assembly.From gel electrophoresis the mutant B protein is estimated to be shorter than the normal S13 B protein by 1720 ± 70 daltons and is therefore believed to be an internal reinitiation fragment. The reinitiated fragments are functional and are made in about twice the amount of the normal B protein.The phage mutants which yield the reinitiation fragments are double mutants, each phage containing the same gene B nonsense mutation and each appearing to contain a different compensating gene B mutation. Various data support the assumption that the compensating mutations are frame-shifts, including the fact that suppression does not restore the normal-sized B protein. The reinitiation is assumed to occur at a pre-existing out-of-phase initiator codon, near the nonsense triplet; the correct reading frame would then be restored by each of the several different compensating mutations.The position of the normal S13 B protein in the gel electrophoresis pattern has been located both by elimination and shifting of the B peak, using appropriate amber mutants. The molecular weight of the S13 B protein is about 17,200, and is 2100 daltons less than the B protein of phage φX174; the S13 B protein can nevertheless substitute for the φX 174 B protein. Thus substantial portions of the B protein can be deleted without destroying its function.     

Host mutant (tabD)-induced inhibition of bacteriophage T4 late transcription: II. Genetic characterization of mutants

In this paper we show that the tabD mutants, selected with ts553 or tsCB53, and described in the accompanying paper (Coppo et al., 1975): (a) are recessive to tab⁺; (b) fail to complement each other, and thus map in the same cistron; (c) by their linkage to rif and their dominance relationships with well characterized amber mutations in the Escherichia coli RNA polymerase operon, probably all map in the gene controlling the synthesis of the β′ subunit of the enzyme. We also describe the isolation of a ts⁺, k^D mutant in phage T4 gene 55, used in the selection of a new tabD mutant (tabD^k292). This tab mutant: (a) generates a defective phenotype which differs somewhat from that of the other tabD mutants; (b) complements the other tabD mutants; (c) by its dominance relationship to amber mutants in the RNA polymerase operon, can be assigned to the structural gene coding for the β subunit of the enzyme.A new type of interaction between T4 genes 55 and 45 is also described. The k^D properties of ts553 (gene 55) are suppressed at 30 °C, by a temperature-sensitive mutation in gene 45. This type of interaction between missense mutations in genes 45 and 55 apparently occurs even in tab⁺ strains, since temperature-sensitive mutations in gene 45 accumulate in lysates of two gene 55 mutants (ts553 and tsA81).     

Electiveness of rapair of plaque-type mutations induced by hydroxylamine in phage kappa of serratia

Phage ? treated extracellularly with hydroxylamine (HA) was preadsorbed to hcr, exr or wild-type (HY) host cells and plated with HY indicator. 5 plaque mutation types were scored. The frequency distributions of the 5 mutation types (mutation spectra) differed with the hosts, the spectrum in exr especially deviating significantly from those in hcr and HY. This indicates electiveness of repair of certain (pre-) mutations within different genomic regions. HA treatment time (dose) influenced the spectra, too, owing to three mutation types giving linear (one-hit) dose curves and two giving parabolic (about two-hit) curves. The host type did not influence these curve shapes. These findings show that the number of HA hits depends on the genomic region where (pre-) mutations occur.Inactivation of phages as well as cells was strongest in exr compared with hcr and HY hosts (factor 1.3). In contrast, induction of all 5 mutation types was lower in exr (factor about 0.5) and hcr (factor about 0.8) than in HY. This indicates that both repair types (probably post- and pre-replicative) are needed for perfecting part of the HA-induced mutations. The part lacking in repair-defective hosts may be caused by lethality within these hosts of certain premutative lesions. The frequency of mixed compared with pure mutant clones was small. Its dose dependence may be due to recessive lethal lesions within the non-premutated DNA strand.     

Mischarging single and double mutants of Escherichia coli sup3 tyrosine transfer RNA

Mischarging mutants of Escherichia coli sup3 tyrosine transfer RNA have been isolated by selecting for suppression of bacterial amber mutations not suppressed by sup3. Five of the mutants have single base changes in the amino acid acceptor stem (A1, A2, U80, U81 and G82). Mutants A1 and A2 are weak thermosensitive suppressors from which thermostable derivatives have been isolated. Some of these derivatives affect the amount of tRNA synthesized but not the sequence (precursor or promoter mutations), and others are double mutants A1U81 and A2U80. The latter mutant does not mischarge. The efficiency of suppression of A1 and A2 can also be increased by recombination events that lead to duplication and triplication of the suppressor gene.The amino acid inserted by some of these mutants at the amber site has been determined. Mutant A1 inserts glutamine, while U81 and A1U81 insert both glutamine and tyrosine.Taken together the results show that the terminal part of the amino acid acceptor stem has an important role in the specificity of aminoacylation by the glutamine and tyrosine synthetase.     

A new type of UV-sensitive mutant of phage T4D

Within a group of more than 20 UV-sensitive mutants of T4D, 4 UV-sensitive mutants with the same sensitivity as T4 x were isolated independently of each other. They were uvs9, uvs21, uvs35, and uvs52. The double mutants with x and y₁₀ were constructed: they are slightly more UV sensitive than T4 v₁. The double mutant with uvs5 was not found. The mutations of uvs9, 21, 35, and 52 are closely linked with v₁. The photoreactivable sector (PRS) is 0.4. One of the mutants, uvs52, has the same sensitivity for methyl methanesulphonate (MMS) as T4⁺, shows a stronger multiplicity reactivation than the wild type, shows the same sensitivity relative to T4⁺ and T4 v₁ in Luria-Latarjet tests and in monocomplex UV inactivation, and raises the recombinant frequency in crosses with irradiated phage. The uvs52⁺ function has the same sensitivity to UV as the v⁺ function. Complementation between uvs52 and v₁, if present is difficult to demonstrate owing to an appreciable MR contribution to increased survival. The possibility that uvs52 is an allele of v₁ is discussed. The observations fit the assumption that uvs52 is an excision-repair mutant with a low excision rate.     

On the mechanism of bromouracil-induced mutagenesis

Bromouracil (BU)-induced mutagenesis of λC17 am o8 phage, in relation to the recombination systems of phage (red) or bacteria (rec), was studied. The mutations investigated were am → am⁺. For efficient BU-induced mutagenesis, red or recA genes as well as bacterial lex gene functions, known to be involved in UV-induced mutagenesis, were required. This suggests a common mechanism or some common step(s) in UV- and BU-induced mutagenesis. Moreover, a several-fold increase was observed in the number of mutants induced by BU in the excision-repair-deficient strain (uvrA), implying that incorporated BU induces some premutational lesions that are recognized and repaired by excision-repair enzymes. A hypothesis on the possible mechanism of BU-induced mutagenesis is proposed, which assumes a common mechanism for UV- and BU-induced mutagenesis, involving recombination repair processes. Incorporation of a tautomeric or ionized form of BU is considered only as a premutational change in DNA activating the dark-repair mechanisms in cells. The observation that BU enhances the frequency of recombination in λ phages also supports teh idea that recombination processes are involved in BU-induced mutagenesis.     

Control of lysogenization by phage P22 I. The P22 cro gene

P22 cro^? mutants were isolated as one class of phage P22 mutants (cly mutants) that have a very high frequeney of lysogeny relative to wild-type P22. These mutants: (1) do not form plaques and over-lysogenize relative to wild-type P22 after infection of a wild-type Salmonella host; (2) are defective in anti-immunity; and (3) fail to turn off high-level synthesis of P22 c2-repressor after infection.P22 cro^? mutations are recessive and map between the P22 c2 and c1 genes. P22 cro^? mutations are suppressed by clear-plaque mutations in the c1 gene, one of which is simultaneously cy^?. They are also suppressed, but incompletely, by mutations in the c2 (repressor) gene, especially those that do not completely abolish c2 gene function.Salmonella host mutants have been isolated that are permissive for the lytic growth of the P22 cro^? mutants.     

An analysis of repressor overproduction by the lambda cIIIs mutant.

Efficient lysogenization of Escherichia coli K12 by bacteriophage λ requires the high level of synthesis of the phage repressor shortly after infection. This high level of synthesis of repressor requires the action of the λ eII and cIII proteins. Certain mutants of λ (λcIIIs) appear to have excess $\text{c}\text{II}\text{c}\text{III}$ activity and can lysogenize more efficiently than λ⁺. The basis for the enhanced lysogenization is that, while two or more infecting phage are necessary for λ⁺ to lysogenize, a single infecting λcIIIs particle is sufficient for lysogenization. Also, repressor levels in cells infected with λcIIIs are higher than in those infected with λ⁺. I report here that repressor overproduction by λcIIIs (1) is due to a much higher rate of repressor synthesis than that of λ⁺; (2) is most marked at low multiplicities of infection, possibly because λcIIIs produces repressor much more efficiently than λ⁺ as a singly infecting phage.     

Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA. I. Identification and mapping of arl mutations

Hyper-rec mutants of Escherichia coli were originally identified as lac-diploid strains whose colonies exhibited unusually high numbers of Lac⁺ papillae during growth on indicator plates (Konrad, 1977). For this work, 38 hyper-rec strains with particularly high frequencies of papillation were selected and screened further, in order to identify those unusually proficient in recombination of bacteriophage λ. The screening procedure, plate-stock growth of λ duplication phages, yielded four strains that exhibited both enhanced recombination of λ and normal (or higher) yields of progeny phage. The mutants displayed the same novel phenotype: phage recombination was normal during the first lytic infection, but was stimulated four- to sixfold if the phages had previously been propagated for several cycles in the mutants. Phages thus appeared to accumulate an enhanced potential for recombination during growth in these four strains. The mutations responsible were designated arl. Enhanced recombination of the phages propagated on arl strains occurred in subsequent test infections of both arl and arl⁺ bacteria, but not in recA cells. Both the high frequency of Lac⁺ papillae and the effects on λ recombination appeared to result from the same mutations. The former phenotype was used for genetic analysis of two arl mutants; their location is near 2 minutes on the E. coli map. Known alleles of two nearby genes, polB and mutT, do not confer a hyper-rec phenotype (by the lac-diploid assay). High-level RecA-constitutive strains do not exhibit enhanced recombination of duplication phages.     

An amber map of Salmonella phage P22

Summary Forty five amber mutants of Salmonella phage P22 were assigned to 12 complementation groups. Two amber mutants could not be classified into any one particular group.Ten of the complementation groups were mapped by two-factor crosses. The map is circular and about 65 units long.The remaining two complementation groups each consist of a single member which form very small plaques. Attempts to map these two mutants by two-factor crosses were unsuccessful.     

An Amber Suppressor of Escherichia coli Strain KO1

The suppression characteristics of Escherichia coli strain KO1 have been investigated. The growth patterns of nonsense mutants of RNA (GA and f2) and DNA (λ and T4) phages suggested that KO1 carried an amber, but not ochre or opal suppressors. The comparison of KO1 with previously identified amber suppressors indicated that KO1 differed from su1, su3 and su6 in its suppression pattern. KO1 and su2 shared some properties in common, for instance, their ability to suppress GA amber mutants with one exception (amN20) and the restriction of suppression capacity by the str^r mutation. However, the suppression efficiency of KO1 (48%) was about three times that of su2 (18%). A possibility that KO1 contained a new amber suppressor is discussed.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

Ethanol-hypersensitive and ethanol-dependent cdc − mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^? mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^? mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^? phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Mutants in the y region of bacteriophage lambda constitutive for repressor synthesis: their isolation and the characterization of the Hyp phenotype

Bacteriophage lambdahyp mutants have been isolated as survivors of Escherichia coli K-12 bacteria lysogenic for lambda Nam7am53cI857. The hyp mutants are characterized by (i) their localization in the y region very close to the imm lambda/imm434 boundary, (ii) polarity on O gene expression, (iii) immediate recovery of lambda immunity at 30 degrees C after prolonged growth of lambda Nam7am53cI857 hyp lysogens at 42 degrees C even in the presence of an active cro gene product, (iv) ability of phage lambda v2v3vs326 but not lambda v1v2v3 to propagate on lambda cI+hyp lysogens, (v) inability to express lambda exonuclease activity after prophage induction, and (vi) inviability at any temperature of phage carrying the hyp mutation. All these properties are referred to collectively as the Hyp phenotype. We show that the Hyp phenotype is due to cII-independent constitutive cI-gene-product synthesis originating in the y region, which results in the synthesis of anti-cro RNA species, and constitutive levels of cro gene product present even in lambda cI+hyp lysogens. A model is presented which is consistent with all the experimental observations.