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.     

Nanovariant RNAs: nucleotide sequence and interaction with bacteriophage Qbeta replicase

Gene 2 amber mutants of bacteriophage T4 grown on su^? hosts produce whole particles of which less than 0.5% are infective on su⁺ hosts. Although the DNA of such particles is full-sized and un-nicked, it is degraded to acid-soluble fragments after infection of exo V⁺ hosts. This breakdown does not occur on exo V^? deficient hosts, and such hosts are fully permissive for gene 2-defective particles. We have now determined that giant-headed, gene 2-defective particles containing several genome lengths of DNA per head are fully infective on exo V⁺ hosts even though part of the parental DNA is degraded to acid-soluble fragments early after infection. Restriction of gene 2-defective particles must therefore be due to exonucleolytic degradation of the incoming DNA. If the parental DNA is of sufficient length to enable a complete genome to survive this degradation before production of anti-exoV, such particles are now infective.     

Characterization of an amber suppressor in Pneumococcus.

Summary Partial revertant has been isolated, with resistance to aminopretin intermediate between wild type and mutant. This phenotype is the result of a mutation at a gene unlinked to the amiA locus. This suppressor mutation (su⁺) has no phenotypic characteristics by itself except a slow growth. 9 amiA mutants (belonging to 6 sites) are affected by su⁺ out of the 30 investigated mutants (i.e. 22 sites). The efficiency of suppression is site dependent. Two sites out of 14 mutants belonging to the thymidilate synthetase gene are suppressible. Thymidilate synthetase activity is partially restored by su⁺. Optochin mutants can also be suppressed. Thus su⁺ is not gene specific but site specific. Moreover when the str-41 allele conferring resistance to streptomycine is introduced by transformation, the suppression effect is restricted. All these properties are characteristic of an informational suppressor.The t-RNA extracted from the suppressor strain su⁺ but not the wild type restored the synthesis of coat protein coded by RNA from an amber mutant of bacteriophage f2. Attempts to detect ochre suppression activity gave negative results. It is suggested that the su⁺ gene is amber specific.Thus su⁺ can provide insight into the nature of suppressible mutations which should be point mutations. Both low efficiency and high efficiency mutants are affected by su⁺; this is additional evidence that both categories contain point mutations.     

Participation of bacteriophage T4 gene 41 in replication repair

The location of the non-essential T4 mutant uvs79, with defective replication repair, is described. After crosses with double mutants dispersed over the early region of T4, a linkage was observed with the double mutant am41 : am42. For more accurate location, crosses were made with single mutants. Uvs79 proved to be located between mutants amC23 and amN81 in gene 41, as shown by 3-point crosses. No genetic complementation with respect to multiplicity reactivation was found between amN81 and uvs79 after a co-infection of an su^? host. Apparently, mutant amN81 is disturbed as to replication repair and, owing to its lack of DNA synthesis, also in replication-dependent recombination repair. Consequently, the product of gene 41 has a function additional to its RNA-primer induction during replication of undamaged DNA. Presumably, the product of gene 41 induces RNA primers opposite DNA regions containing lesions. This capability is believed to be specifically affected by the uvs79 mutation.     

Nucleotide alterations in the bacteriophage T4 glutamine transfer RNA that affect ochre suppressor activity

We have determined the nucleotide sequences of the glutamine transfer RNAs that are coded by wild-type and psu₂⁺ ochre-suppressor strains of bacteriophage T4. The two transfer RNAs have the same sequence except for their anticodons, where NUG in the wild-type species is mutated to NUA in the psu₂⁺ species (N is a modified residue of U). This mutation is believed to confer suppressor activity on the psu₂⁺ glutamine tRNA. Three mutants derived from psu₂⁺ by loss of suppressor activity have been characterized with respect to their sequence alterations. Each mutant specifies a transfer RNA differing from the psu₂⁺ species by a nucleotide substitution that occupies a base-paired region in the cloverleaf arrangement of the molecule. The mutants synthesize a reduced amount of tRNA that is defective in nucleotide modifications and processing at the 5′ and 3′ termini.     

Ultraviolet light sensitive mutants of Coprinus lagopus. I. Isolation and characterization

4UV-sensitive mutants have been isolated from the wild type strain BC9/66 of Coprinus lagopus by following a new replica plating technique. Complementation and recombination studies between these mutants suggest 3 gene loci uvs1, uvs2 and uvs3, two of the mutants being allelic (uvs3-1 and uvs3-2). The mutants uvs2, uvs3-1 and uvs3-2 show photoreactivation whereas the mutant uvs1 appears to be deficient in this respect. None of the mutants of the wild type showed significant recovery after dark holding.     

The psu1+ amber suppressor gene of bacteriophage T4: identification of its amino acid and transfer RNA

Previous work identified the psu⁺₁ amber suppressor gene of bacteriophage T4. Analysis of protein arising from suppression now shows that psu⁺₁ specifies the insertion of serine in response to the amber triplet. The efficiency of suppression is 70%.The psu₁⁺ gene affects a serine transfer RNA coded by bacteriophage T4. Comparative ribonuclease T₁ fingerprint analysis of the serine transfer RNAs made by wild type T4 and psu⁺₁ strains shows a specific alteration in the patterns, presumably reflecting a mutational alteration in the anticodon of the transfer RNA. Mutations which result in the loss of suppressor activity define two genes: one is apparently the structural gene for the serine transfer RNA; the function of the second gene, M1, is less clear. Mutational inactivation of either gene prevents the appearance of the serine transfer RNA and a second transfer RNA, which has not yet been associated with its cognate amino acid. M1 mutants are also deficient in the production of several additional transfer RNA species, as well as several larger RNAs. The significance of these results in relation to transfer RNA biosynthesis is discussed.     

Mutator activity in uvs mutants of Aspergillus nidulans

Summary The frequency of selenate-resistant spontaneous mutants was determined among the conidia of two uvs ⁺, two allelic uvsB, one uvsD, three allelic uvsC and three allelic uvsE strains of Aspergillus nidulans. In the uvsB, uvsD, uvsC and uvsE mutants the median frequencies of mutation were respectively 1.7, 1.8, 8.7 and 4.0 times as high as in the uvs ⁺ strains. The selenate resistance resulted from mutation at the chromosomal loci sB or sC. It is concluded that the uvs alleles enhance spontaneous mutation in chromosomal genes.     

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.     

Analysis of specific misreading in Escherichia coli

The pattern of specific misreading by nonsense suppressors has been investigated using nonsense mutants in the rIIB gene of phage T4 and in the lacZ gene of Escherichia coli. It is shown that a su⁺ transfer RNA which reads UAG also misreads UAA but not UGA, a su⁺ tRNA which reads UAA (while it also reads UAG by wobble) misreads UGA and a su⁺ tRNA which reads UGA also probably misreads UAA but not UAG.These specific types of errors in translation occur in the absence of streptomycin. The addition of the drug raises their level without altering the pattern described. A ribosomal mutation str A reduces the level of specific misreading; by contrast, a ram mutation strongly increases this level. In all cases the specific pattern is not affected.The rate of specific misreading of nonsense codons in different cases ranges from less than 0.001% to more than 3%. Since the frequency of misreading is sitespecific (unpublished observations), the rates obtained cannot be extrapolated to any other codon at any other site.     

Suppression of temperature sensitive mutants of bacteriophage T4 by bacterial opal suppressors

Summary Some of the partial revertants from opal (UGA) mutants of bacteriophage T4 are temperature sensitive in su ^– host cells but are still temperature resistant in su ⁺ cells. Hence these revertants are missense mutants suppressible by bacterial opal suppressors. Such a suppression may be explained in terms of codon-anticodon interactions by the wobble hypothesis.     

Identification of transfer RNA suppressors in Escherichia coli. I. Amber suppressor su+2, an anticodon mutant of tRNA2Gln.

In order to isolate the gene for amber suppressor su⁺2 (SupE) in Escherichia coli, a non-defective su⁺2-transducing phage lambda was isolated in three steps: first, deletion derivatives of F′su⁺2 gal (λ) were selected, linking su⁺2 to the right-hand prophage attachment site, att^λPB′; second, these F′-factors were relysogenized by λ and defective transducing phages, λdsu⁺2, were produced by induction; and third, non-defective λpsu⁺2 transducing phages were produced by recombination of λdsu⁺2 isolates with λ. Upon infection by λpsu⁺2, the production of transferRNAs accepting glutamine and methionine was markedly stimulated. Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNA₂^Gln, mutant tRNA₂^Gln and tRNA_m^Met. The mutant tRNA₂^Gln carried a singlebase alteration from G to A at the 3′-end of the anticodon. The production of tRNA₁^Gln was not stimulated by the infection of λpsu⁺2. We conclude that the wild-type allele of su⁺2 (SupE) is the structural gene for tRNA₂^Gln, and the su⁺2 amber suppressor was derived by a single base mutation, changing the anticodon from CUG to CUA, in one of the multi-copy genes for tRNA₂^Gln. The fact that λpsu⁺2 also induces the production of tRNA_m^Met suggests that this tRNA is encoded in the same chromosomal region of E. coli as is tRNA₂^Gln.     

Cold-sensitive ompB mutants affecting expression of ompC in Escherichia coli K12

The regulatory locus ompB, consisting of 2 genes, ompR and envZ, is required for the expression of ompC and ompF genes encoding the major outer membrane porin proteins OmpC and OmpF in Escherichia coli K12. We utilized localized mutagenesis to isolate cold-sensitive mutants in the ompB operon. The isolated mutants exhibited a cold-sensitive OmpC⁻ phenotype, but remained OmpF⁺. Furthermore, ompC expression was still regulated by medium osmolarity. The cold-sensitive OmpC⁻ phenotype was complemented by plasmids carrying the wild-type ompB operon, but not by plasmids containing either envZ or ompR genes alone. This suggests that the mutations are in the ompB promotor. We show that the mutations can be used to control expression vectors based on the ompC promotor.     

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.     

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.     

Detection of long eukaryote-specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos.

Precursor molecules for Escherichia coli tRNAs that accumulated in a temperature-sensitive mutant defective in tRNA synthesis (TS709) were investigated. More than 20 precursors were purified by two-dimensional polyacrylamide gel electrophoresis. The purified molecules were analyzed by RNA fingerprint analysis and/or in vitro processing after treatment with E. coli cell-free extracts. The molecular sizes of most of the precursors identified were in the range of 4 to 5 S RNAs, although several larger ones were also detected. Fingerprint analysis revealed that the precursors generally differ from the corresponding mature tRNAs in the 5′ termini, having extra nucleotides. Thus, the genetic block in TS709 was shown to affect the trimming of the 5′ side of tRNA by impairing the function of RNAase P. Although this mutant had been isolated as a conditional mutant defective in the synthesis of su⁺ 3 tRNA₁^Tyr, the synthesis of many tRNA species was affected at high temperature. On the basis of their mode of maturation in vivo, the precursor molecules were discussed as intermediates in tRNA biosynthesis in E. coli. Accumulation of these intermediates was accounted for as a common feature of E. coli mutants defective in RNAase P function.     

MUTANT GENES REGULATING THE INDUCIBILITY OF KYNURENINE SYNTHESIS

Alterations in the cellular synthesis of kynurenine in the larval fatbody of Drosophila melanogaster may be obtained by feeding the precursor tryptophan or by changing the genotype. In the wild type Ore-R strain, autofluorescent kynurenine globules normally occur in the cells in the anterior regions of the fatbody designated as regions 1, 2, and 3. When tryptophan is included in the larval diet, kynurenine will develop throughout the entire fatbody, thus extending to the cells in regions 4, 5, and 6. In the fatbodies of both the sepia mutant strain and the mutant combinations of the suppressible vermilion alleles with the suppressor gene (su²-s, v¹ and su²-s, v²), kynurenine is found in the cells from region 1 through region 4. This involvement of additional cells in the synthesis of kynurenine occurs under the usual culture conditions for Drosophila. When sepia larvae are fed tryptophan, kynurenine appears in all of the cells of the fatbody. However, dietary tryptophan does not induce kynurenine production in cells in regions 5 and 6 in the mutant combination su²-s, v¹ or su²-s, v². In the latter strains, an increase in the quantity of kynurenine in the fatbody is detected, but this increase remains limited to the same cells in which kynurenine production is found under normal feeding conditions. When the v^36f allele is combined with the su²-s allele, an extremely faint autofluorescence characteristic of kynurenine is found in some of the anteriormost fat cells of regions 1 and 2. This autofluorescence becomes intensified when tryptophan is fed to su²-s, v^36f larvae. The genetic control of kynurenine synthesis in the cells of the fatbody of Drosophila melanogaster has been previously demonstrated. The present observations establish genetic regulation of the ability to induce kynurenine production within a cell through the administration of the inducer tryptophan. Kynurenine production has been considered as a unit function of the cell as a whole rather than of the enzyme alone, and it has been concluded that even though cells in different parts of the body perform this same function (kynurenine production), the gene loci regulating this function may be different for cells in different regions of the body. A phenomenon of overlapping domains of gene actions at the cellular level offers a genetic and cellular basis for developmental and physiological homeostasis.