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A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2-35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region.

T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon. These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells. They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein. Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth. Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity. The mutation at amino acid 64 of 4A' appears to have little effect on 4A function. The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth.

The capsid of bacteriophage T4 is composed of two essential structural proteins, gp23, the major constituent of the capsid, and gp24, a less prevalent protein that is located in the pentameric vertices of the capsid. gp24 is required both to stabilize the capsid and to allow it to be further matured. This requirement can be eliminated by bypass-24 (byp24) mutations within g23. We have isolated, cloned and sequenced several new byp24 mutations. These mutations are cold-sensitive in the absence of gp24, and are located in regions of g23 not known to contain any other mutations affecting capsid assembly. The cold-sensitivity of the byp24 mutations can be reduced by further mutations within g23 (trb mutations). Cloning and sequencing of these trb mutations has revealed that they lie in regions of g23 that contain clusters of mutations that cause the production of high levels of petite and giant phage (ptg mutations). Despite the proximity of the trb mutations to the ptg mutations, none of the ptg mutations has a Trb phenotype. The mutation ptE920g, which is also located near one of the ptg clusters, and which produces only petite and wild-type phage, has been shown to confer a Trb but not a Byp24 phenotype. The relevance of these observations to our understanding of capsid assembly is discussed.

We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants. Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP. The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.

In terms of evolution and fitness, the most significant spontaneous mutation rate is likely to be that for the entire genome (or its nonfrivolous fraction). Information is now available to calculate this rate for several DNA-based haploid microbes, including bacteriophages with single- or double-stranded DNA, a bacterium, a yeast, and a filamentous fungus. Their genome sizes vary by approximately 6500-fold. Their average mutation rates per base pair vary by approximately 16,000-fold, whereas their mutation rates per genome vary by only approximately 2.5-fold, apparently randomly, around a mean value of 0.0033 per DNA replication. The average mutation rate per base pair is inversely proportional to genome size. Therefore, a nearly invariant microbial mutation rate appears to have evolved. Because this rate is uniform in such diverse organisms, it is likely to be determined by deep general forces, perhaps by a balance between the usually deleterious effects of mutation and the physiological costs of further reducing mutation rates.

Deinococcus radiodurans has 2 endonucleases that incise UV-irradiated DNA. UV endonuclease-alpha and UV endonuclease-beta, that are believed to functionally overlap. Both endonucleases must be mutationally inactivated to yield an incisionless, markedly UV-sensitive phenotype. denV, the bacteriophage T4 gene encoding pyrimidine dimer-DNA glycosylase (PD-glycosylase), was introduced and expressed via duplication insertion in D. radiodurans wild-type, and single and double UV endonuclease mutants. The strain deficient in UV endonuclease-alpha has wild-type UV resistance, and the expression of PD-glycosylase exerted no survival effect on this strain or wild-type. Expression of denV increased survival of both the markedly UV-sensitive double mutant and the moderately UV-sensitive strain deficient only in UV endonuclease-beta. In endonuclease-beta-deficient cells phenotypic complementation by denV was almost complete in restoring UV resistance to wild-type levels. These results suggest that UV endonuclease-alpha (which is present in the endonuclease-beta-deficient cells) does not recognize one or more types of cyclobutane dimer incised by the PD-glycosylase or UV endonuclease-beta.

Extracellular nonreplicating bacteriophage T4 particles accumulate mutations as functions of temperature, time, pH, and ionic environment via two mechanisms: 5-hydroxymethylcytidine deamination produces G.C----A.T transitions while a guanosine modification produces transversions. Neither frameshift mutations nor mutations at A.T base pairs are appreciably induced. We now show that heat induces G.C----T.A transversions which we suggest may arise via a G*.A mispair, in which G* is a modified guanosine that has experienced a glycosylic bond migration. The rate of this reaction at 37 degrees C is sufficient to present a genetic hazard, particularly to large genomes; thus, the lesion is probably efficiently repaired in cellular genomes.

Mismatched and modified base pairs are central to questions of DNA mutation and repair. NMR and X-ray crystallography of mispairs indicate little to no local helical distortion, but these techniques are not sensitive to more global distortions of the DNA molecule. We used polyacrylamide gel electrophoresis and thermal denaturation to examine A.C, G.T, and O6-methylG.T and O6-methylG.C mismatches synthesized in place of either of two adjacent G.C base pairs in synthetic DNA duplexes. Substitution for G.C at either position decreased the stability of the duplex; O6-methylguanine was more destabilizing in place of the 5'G than in place of the 3'G. Comparisons between polymers synthesized so that lesions occurred regularly spaced on the same side of the helix and polymers synthesized so that the lesions alternated from side to side on the helix showed that these lesions introduced helical distortion composed of (i) a symmetric frictional component, probably caused by localized bubble formation, and (ii) an asymmetric component indicative of a more global effect on the DNA molecule. Comparisons between these effects at the two adjacent positions show that the extent of structural perturbation depends on sequence context.

The mutator phenotype of Escherichia coli dam mutants was found to be reversed by introduction of the bacteriophage T4 gene for DNA adenine methyltransferase. This precludes a direct role for the E. coli DNA adenine methyltransferase in mismatch repair, in addition to its role in strand discrimination, as suggested by earlier studies (S. L. Schlagman, S. Hattman, and M. G. Marinus, J. Bacteriol. 165:896-900, 1986).

The tsx-206 allele encodes an altered Tsx protein, Tsx-206, that can no longer function as the T6 receptor. We show here that this allele also confers resistance to the Tsx-specific phages H1, H3, H8, K9, K18 and Ox1 but not to colicin K. The Tsx-206 protein still mediates the efficient permeation of deoxyadenosine across the outer membrane at low substrate concentration. A host-range mutant of phage T6, T6h3.1, was isolated which can use both the Tsx-206 and the Tsx wild-type protein as its receptor. Cloning and DNA sequence analysis of the tsx-206 allele showed that the phage resistant phenotype was associated with an Asn to Tyr substitution at position 254 of the 272-residue Tsx protein.

Seven point mutations were introduced into region I of the herpes simplex virus type 1 DNA polymerase, which is most highly conserved among DNA polymerases and has no drug sensitivity markers mapped to it. The functional consequences of these mutations were studied in an in vitro transcription-translation system in which T7 transcripts of cloned polymerase genes were used to generate enzymatically active polypeptides in reticulocyte lysate. Analysis of labeled polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to show any alterations of stability caused by these mutations. The mutations G885R, D886N, T887K, D888A, and G896V lacked polymerase activity and failed to be stimulated by cotranslation of the herpes simplex virus 65-kilodalton DNA-binding protein, whereas R881G and S889A retained both polymerase activity and the capacity to be stimulated by the 65-kilodalton DNA-binding protein.

The active site sequence of T4 thioredoxin, Cys-Val-Tyr-Cys, has been modified in two positions to Cys-Gly-Pro-Cys to mimic that of Escherichia coli thioredoxin. The two point mutants Cys-Gly-Tyr-Cys and Cys-Val-Pro-Cys have also been constructed. The mutant proteins have similar reaction rates with T4 ribonucleotide reductase as has the wild-type T4 thioredoxin. Mutant T4 thioredoxins with Pro instead of Tyr at position 16 in the active site sequence have three to four times lower apparent KM with E. coli ribonucleotide reductase than wild-type T4 thioredoxin. The KM values for these mutant proteins which do not have Tyr in position 16 are thus closer to E. coli thioredoxin than to the wild-type T4 thioredoxin. The bulky tyrosine side chain probably prevents proper interactions to E. coli ribonucleotide reductase. Also the redox potentials of these two mutant thioredoxins are lower than that of the wild-type T4 thioredoxin and are thereby more similar to the redox potential of E. coli thioredoxin. Mutations in position 15 behave more or less like the wild-type protein. The kinetic parameters with E. coli thioredoxin reductase are similar for wild-type and mutant T4 thioredoxins except that the apparent kcat is lower for the mutant protein with Pro instead of Tyr in position 16. The active site sequence of T4 thioredoxin has also been changed to Cys-Pro-Tyr-Cys to mimic that of glutaredoxins. This change does not markedly alter the reaction rate of the mutant protein with T4 ribonucleotide reductase or E. coli thioredoxin reductase, but the redox potential is lower for this mutant protein than for wild-type T4 thioredoxin.

Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.

The effect of growth of Escherichia coli BE, prior to infection, on multiplication of double amber mutant amN54-amNG71 in gene 31, mutant amN131-amNG114 in gene 26 and T4D wild-type at different temperatures has been studied. In the case of gene 31 mutant the increase in phage burst size, along with increase in growth temperature, was only observed. And this dependence seems to have the same character as the known dependence of growth temperature on cellular levels of heat shock proteins. Possibly, the product of gene 31 might be substituted to some extent by some heat shock protein. An antiserum against gene 31 protein immunoprecipitates heat shock protein, the molecular weight of which is close to the molecular weight of gene 31 protein. So, it seems likely that, in addition to supposed ability of this heat shock protein for functional substitution of gene 31 protein, these proteins might have some structural homology as well.

Bacteriophage T7 and many closely related phages abortively infect plasmid F-containing (F+) strains of Escherichia coli. However phage T3, which is also closely related to T7, grows normally in F+ hosts. Mutants of phage T3 that, like T7, are subject to F-mediated restriction have been isolated. These T3 mutants lack or are defective in one or both of two genes that are nonessential for phage growth in F-, wild-type strains. Our results show that the products of phage T3 gene 1.1 or 1.2, or both, are essential for growth and suggest that the comparable phage T7 genes are naturally defective in their ability to counteract the inhibitory effects of F-encoded proteins.

The bacterial RNA polymerase sigma factor is necessary throughout T4 development. T4 can develop in the E. coli RpoD800 mutant cells only at permissive temperature. RNA synthesis in T4-infected mutant cells remains temperature-sensitive throughout infection as in uninfected mutant bacteria. This shows that bacterial sigma factor is necessary for all types of RNA synthesis in infected E. coli. The data obtained suggest also that active sigma factor is necessary for early, but not for late T4 DNA replication.

An electrophoretic mutation of bacteriophage T4, eph1, appears to code for a missense hoc (highly antigenic outer capsid) protein. This is based on the observation that particles lacking hoc protein (hoc- particles), after incubation in a crude extract of Escherichia coli infected with phage carrying the eph1 mutation acquired the electrophoretic mobility of the eph1 strain (the electrophoretic mobility of the eph1 strain itself is slower than that of hoc- particles). Thus, it is likely that during infection of E. coli with the eph1 strain, a hoc protein is made that has a lower negative charge than normal hoc protein but can nevertheless bind to particles lacking hoc protein. These results confirm that eph1 is a hoc mutation.

The product of gene 31 is normally required for assembly of the T4 capsid. Two mutations that each bypass that requirement are shown to be located at separate sites in gene 23, which encodes the major structural protein of the capsid. A second phenotypic effect that characterizes both bypass31 mutant strains is the ability to multiply in host-defective strains, such as hdB3-1 and groEL mutants, on which wild-type T4 is unable to assemble capsids. The genetic data indicate that both phenotypic effects are due to the bypass31 mutation. Elimination of the requirement for both the phage protein, gp31, and the host protein, GroEL, by either of two single mutations in gene 23 indicates that GroEL and gp31 are normally needed to interact with gp23 in capsid assembly of wild-type T4.

This article discusses ideas about the ways in which the high fidelity of DNA replication is achieved: base selection, exonucleolytic editing, and postreplicative proofreading. I also review possible mechanisms for the enhanced mutation rate associated with SOS induction. The concept of environmental control of mutation rate and other modes of genetic variation is also considered from the point of view that SOS induction is an example of "genetic revolution".

Monoadducts and interstrand cross-links are formed in DNA after psoralen plus light treatment of bacteriophage lambda . Survival and clear plaque mutation frequency of lambda after photosensitization with 8-methoxypsoralen (8-MOP) are increased when the wild type host is slightly UV-irradiated (W-reactivation and W-mutagenesis). The recA13, lexA1 and uvrA6 mutations block W-reactivation and W-mutagenesis of lambda treated with 8-MOP plus light. Using the technique of "repeated irradiation" we showed that the mutagenic effect of 8-MOP plus light treatment on phage is due mainly to formation of cross-links in DNA. The mutagenic activity of monoadducts had been studied by using angular furocoumarin, angelicin which forms mainly monoadducts in DNA. Upon W-mutagenesis of phage lambda treated with angelicin plus light a high mutagenic effect is observed. The results indicate that the mutagenic activity of monoadducts is 15-20 fold slower as compared to that of cross-links. W-reactivation and W-mutagenesis of UV-irradiated (254 nm) bacteriophage lambda are also observed after 8-MOP plus light treatment of Escherichia coli uvrA and wild type hosts. It is possible that the difference in mutagenic activity of psoralen adducts could depend on the repair mechanism of adducts: cross-links repair in bacterial and lambda DNA is controlled by lexA gene (error-prone SOS-repair mechanism), while monoadducts can be efficiently repaired by error-free excision and recombination.

Secondary mutations which increase the efficiency of suppression of nonsense mutations in the rIIB cistron of bacteriophage T4 have been isolated. These secondary mutations, called context mutations, map at sites very close to the nonsense codon, possibly on the promotor distal side. In context-nonsense double mutants, the amount of suppressed gene product is increased approximately 10-fold. The context mutations examined can act on the UAA (ochre) nonsense allele as well as on the UAG (amber) nonsense allele at a given site. These context mutations affect all suppression mechanisms analyzed (genetic suppressors, 5-fluorouracil suppression and spontaneous suppression). We suggest that context mutations affect information which is significant to the termination of polypeptide chains. According to our view, context mutations change the immediate neighborhood of nonsense mutations and so reduce the degree of resemblance to the sequences normally used for the termination of translation.

Bacteriophage T4 infection is known to induce the formation of a complex of enzymes effecting the de novo synthesis of deoxyribonucleoside triphosphates, which in turn are channeled into T4 DNA replication. The first step in this pathway is catalyzed by a ribonucleoside diphosphate reductase, comprised of subunits coded by T4 genes nrdA and nrdB. Maximum rates of synthesis of the pyrimidine deoxyribonucleotides and of DNA replication in vivo also require a type II DNA topoisomerase encoded by T4 genes 39, 52, and 60. We report the identification of a unique mutant, nrdB93, and the suppression of its defective deoxyribonucleotide synthesis by a gene 39 mutation, 39-01. After infection by 39-01, DNA synthesis and plaque formation were temperature-sensitive, but nearly wild type rates of deoxyribonucleotide synthesis were retained at all temperatures. The nrdB93 mutation had a profound effect on deoxyribonucleotide synthesis at 41 degrees C; even at the permissive temperature of 30 degrees C, synthesis was reduced to 30% of that of wild type or 39-01. However, on infection at 30 degrees C by the double mutant, 39-01 nrdB93, the level of deoxyribonucleotide synthesis again reached that of wild type phage infections; involvement of the comparable host enzyme in the suppression process has been excluded. Suppression of the effect of nrdB93 by 39-01 implicates the gene 39 product in the regulation of nrdB expression. The accompanying paper (Cook, K. S., Wirak, D. O., Seasholtz, A. F., and Greenberg, G. R. (1988) J. Biol. Chem. 263, 6202-6208) examines the nature of the suppression process at the molecular level.

Size distributions of mutant clones can reveal important aspects of the mutation process. Previously published data on mutant clones induced by ethyl methanesulfonate (EMS) in bacteriophage T4 generated a distribution that was essentially flat, implying a mutagenic mechanism involving only rare mispairing by reacted bases. Here, methods for estimating the spontaneous component of such a distribution are used to generate a corrected distribution. The corrected distribution is strongly peaked, implying frequent (but not obligatory) mispairing. Frequent mispairing is in accord with current views of the fates of DNA lesions believed to mediate EMS-induced mutagenesis.

Formation of both the tail fiber and the baseplate of bacteriophage T4 depends on the product of T4 gene 57. A single amber mutation in that gene causes loss of two T4-specific proteins. Their molecular weights are 18,000 and about 6,000, respectively, based on their electrophoretic mobilities in SDS-polyacrylamide gels. E. coli carrying a cloned T4 DNA fragment of about 700 basepairs, which directs the synthesis of the smaller protein only, specifically supports the growth of gene 57 amber mutants. We conclude that the small protein is a functional product of gene 57.

Modified polyribonucleotides carrying a number of alkylating groups were used for alkylation of complementary sites of DNA to induce gene-directed mutations. Polyfunctional alkylating mustard, N,N,N'-tri-(beta-chloroethyl)-N'-(p-formylphenyl)-propylene diamine-1,3, moieties were attached to T7 phage early transcripts. The transcripts modified to 4-5% were hybridized with T7 DNA to form R-loops, and RNA molecules were covalently bound to the complementary DNA sites as a result of the activation of RNA carried alkylating groups. T7 DNA molecules alkylated by modified RNAs transcribed from 0.3 and 1.1 T7 phage genes were used for transfection of Escherichia coli. In the progeny of phages generated by the transfection of 24 negative colonies, 3 contained double amber mutants in amounts of 2-10%. Complementation tests permitted to assign all mutations to the same complementation group. Recombination analysis established that one of the mutations concerns gene 0.3. The results of complementation studies indicate that the second mutation, with high probability, concerns gene 1.1.

The allelic forms of the phage genes T4 chi and fdsA, as well as T4y and fdsB are compared in terms of their thymidine incorporation in high or low concentrations of thymidine, sensitivity of DNA synthetic capacity to mitomycin C, and sedimentation rates of DNA replicative intermediates. The results show differences among these mutants for the incorporation of thymidine; however all exhibit mitomycin C-sensitive DNA synthesis and have identical aberrant sedimentation rates for their DNA replicative intermediates.

During viral development bacteriophage T4 modifies the valyl-transfer RNA synthetase of its host Escherichia coli, but the function of the modification has remained elusive. A strain of Escherichia coli has now been identified which is nonpermissive for wild-type bacteriophage T4, but permissive for bacteriophage mutants impaired in the modification reaction. A comparison with other bacteria suggests that nonpermissiveness is due to synthesis of a thermolabile valyl-transfer RNA synthetase and relaxed control of RNA accumulation.

A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesized and cloned by ligation of 26 deoxyribooligonucleotide fragments in two steps with a linearized plasmid followed by transformation. On selection by colony hybridization and DNA sequence analysis, clone pTLY.10 was identified to contain a complete T4 lysozyme synthetic DNA. On expression under lac-promoter, unfused T4 lysozyme was obtained in approximately 4-6% yield. The design and synthesis of two putative folding mutants, flexible (Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75, were based on hierarchical principles. Both mutants lost enzymatic activity of the wildtype. These results are readily understandable if the hierarchical organization of the structure is taken into account. A possible explanation is that the catalytic sites are blocked in both mutants.

We describe a simplified method for the in vitro synthesis of mutated RNA molecules. The method makes use of an oligodeoxyribonucleotide (T7-oligo) which contains the T7RNA polymerase promoter sequence. In combination with a second oligonucleotide, a series of transcripts initiating and terminating at any chosen position on a cloned ss DNA (e.g. M13 phage DNA) can be generated. The phage DNA represents the non-coding DNA strand for the desired transcript; the T7-oligo determines the transcription start site, whereas the second oligonucleotide permits the choice of the transcription termination site. The synthesis of the required template DNA is achieved by hybridizing the two oligonucleotides to the phage DNA and subsequently synthesizing the coding DNA strand by a fill-in reaction with Klenow enzyme. The reaction product is used directly as a template for T7RNA polymerase; cloning of mutants is not required.

The effects of neighbouring base pairs and of temperature on mutation frequencies were measured at nonsense sites in the T4rII region. 2AP-induced AT leads to GC transition frequencies are insensitive to nearest-neighbour effects, while 5BU-induced ones are promoted by GC neighbours on the 5' side. The effect of temperature on 2AP- and 5BU-induced mutation frequencies shows no simple dependence on nearest neighbours. These results are incompatible with a unitary mechanism as explanation for the effects of nearest neighbours and temperature on base-analogue-induced mutagenesis.

The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector. The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities. The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity. The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector. These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity. The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant. On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.(ABSTRACT TRUNCATED AT 250 WORDS)

By use of rate-zonal centrifugation, followed by either one- or two-dimensional agarose gel electrophoresis, the forms of intracellular bacteriophage T7 DNA produced by replication, recombination, and packaging have been analyzed. Previous studies had shown that at least some intracellular DNA with sedimentation coefficients between 32S (the S value of mature T7 DNA) and 100S is concatemeric, i.e., linear and longer than mature T7 DNA. The analysis presented here confirmed that most of this DNA is linear, but also revealed a significant amount of circular DNA. The data suggest that these circles are produced during DNA packaging. It is proposed that circles are produced after a capsid has bound two sequential genomes in a concatemer. The size distribution of the linear, concatemeric DNA had peaks at the positions of dimeric and trimeric concatemers. Restriction endonuclease analysis revealed that most of the mature T7 DNA subunits of concatemers were joined left end to right end. However, these data also suggest that a comparatively small amount of left-end to left-end joining occurs, possibly by blunt-end ligation. A replicating form of T7 DNA that had an S value greater than 100 (100S+ DNA) was also found to contain concatemers. However, some of the 100S+ DNA, probably the most branched component, remained associated with the origin after agarose gel electrophoresis. It has been found that T7 protein 19, known to be required for DNA packaging, was also required to prevent loss, probably by nucleolytic degradation, of the right end of all forms of intracellular T7 DNA. T7 gene 3 endonuclease, whose activity is required for both recombination of T7 DNA and degradation of host DNA, was required for the formation of the 32S to 100S molecules that behaved as concatemers during gel electrophoresis. In the absence of gene 3 endonuclease, the primary accumulation product was origin-associated 100S+ DNA with properties that suggest the accumulation of branches, primarily at the left end of mature DNA subunits within the 100S+ DNA.

Double exbB tolQ mutants of Escherichia coli were completely resistant to bacteriophages T1 and phi 80, in contrast to strains with exbB or tolQ mutations, which were sensitive. Cells carrying mutations in exbB were partially tolerant to colicins B, D, and M and became fully tolerant by the introduction of tolQ mutations. This suggested involvement of both exbB and tolQ in tonB-dependent uptake.

The Escherichia coli rho 026 mutation that alters the transcription termination protein Rho prevents growth of wild-type bacteriophage T4. Among the consequences of this mutation are delayed and reduced T4 DNA replication. We show that these defects can be explained by defective synthesis of certain T4 replication-recombination proteins. Expression of T4 gene 41 (DNA helicase/primase) is drastically reduced, and expression of T4 genes 43 (DNA polymerase), 30 (DNA ligase), 46 (recombination nuclease), and probably 44 (DNA polymerase-associated ATPase) is reduced to a lesser extent. The compensating T4 mutation goF1 partially restores the synthesis of these proteins and, concomitantly, the synthesis of T4 DNA in the E. coli rho mutant. From analyzing DNA synthesis in wild-type and various multiply mutant T4 strains, we infer that defective or reduced synthesis of these proteins in rho 026-infected cells has several major effects on DNA replication. It impairs lagging-strand synthesis during the primary mode of DNA replication; it delays and depresses recombination-dependent (secondary mode) initiation; and it inhibits the use of tertiary origins. All three T4 genes whose expression is reduced in rho 026 cells and whose upstream sequences are known have a palindrome containing a CUUCGG sequence between the promoter(s) and ribosome-binding site. We speculate that these palindromes might be important for factor-dependent transcription termination-antitermination during normal T4 development. Our results are consistent with previous proposals that the altered Rho factor of rho 026 may cause excessive termination because the transcription complex does not interact normally with a T4 antiterminator encoded by the wild-type goF gene and that the T4 goF1 mutation restores this interaction.

Dependence of multiplication of 42 single and double amber mutants in 16 phage head genes on the incubation temperature was studied in the cells of non-permissive host. For amber mutants in 6 head genes the birst size decreases by several orders, with the increase of the incubation temperature. Among amber mutants of the above mentioned genes, mutants in genes 4 and 65 can be distinguished as those with considerably large burst size at low temperature. Phage head genes form the groups, according to temperature sensitivity of multiplication of amber mutants. These groups, together with corresponding groups of phage tail genes, constitute common temperature-sensitive and non-sensitive gene groups on the phage genomic map.

Saturation of a specific region of a chromosome with conditional lethal mutations becomes increasingly difficult as the genome size of the organism increases. We show that mutagenesis of cloned genes followed by their re-integration into a non-mutagenized organism is a practical way to circumvent this difficulty.

Endonuclease V of bacteriophage T4 has been purified to physical homogeneity from T4D-infected Escherichia coli 1100. The enzyme, whose molecular weight is 16,000, possessed two distinct catalytic activities, a pyrimidine dimer-DNA glycosylase and an apurinic/apyrimidinic endonuclease. They acted on UV-irradiated poly(dA) . poly(dT) in a sequential manner; the glycosylase cleaved the N-glycosyl bond between the 5'-pyrimidine of a dimer and the corresponding sugar and then the endonuclease hydrolyzed a phosphodiester bond on the 3'-side of the apyrimidinic site. The 5'-termini thus generated were phosphorylated by T4 polynucleotide kinase only after they had been subjected to direct photoreversal and then treated with alkaline phosphatase. By using two phage mutants, uvs-5 and uvs-13, it was shown that occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities. Suppression of the mutation of uvs-5 rendered both activities partially active. When the mutation of uvs-13 was suppressed, a mutant form of enzyme that possessed only a glycosylase activity was produced. This suggests that there are two distinct domains in a single enzyme, each of which corresponds to one of the activities.

Bisulfite-induced deamination of cytosine produces uracil, a thymine analog reported to be mutagenic both in vitro and in vivo. Although deamination of cytosine in DNA should produce G:C----A:T transitions, treating bacteriophage T4 particles with 0.9 M bisulfite at pH 5 at 37 degrees C produced no more mutations than did the equivalent buffer without bisulfite. Lack of bisulfite mutagenicity is fully consistent with the reported resistance of 5-substituted cytosines to bisulfite-induced deamination, since T4 DNA contains glucosylated 5-hydroxymethylcytosine. However, bisulfite also failed to induce mutations in T4 particles whose DNA contained unmodified cytosine. The lack of mutagenesis persisted in E. coli hosts deficient in uracil glycosylase, an enzyme expected to participate in the repair of the putative bisulfite-generated uracil. Cytosine in T4 DNA may be largely protected from bisulfite attack within phage particles.

Mutation frequency responses produced by ultraviolet light are compared in 4 closely related strains of E. coli B/r having the same tyr(Oc) allele and different excision-repair capabilities: uvr+ (excision repair initiated by wild-type UvrABC activity), uvrA (excision repair defective), uvrA/pdenV-7 (excision repair initiated by endonuclease V of bacteriophage T4, DenV activity), and uvr+/pdenV-7 (excision repair initiated by UvrABC and DenV activities). The production of Tyr+ prototrophic mutants is classified into back-mutations and de novo or converted glutamine tRNA suppressor mutations to indicate different mutation events. Cells transformed with the plasmid pdenV-7 require larger exposures than the parent strains to produce comparable mutation frequency responses, indicating that DenV activity can repair mutagenic photoproducts. When damage reduction by UvrABC or DenV is compared for each of the specific categories of mutation, the results are consistent with the idea that pyrimidine dimers infrequently or never target back-mutations of this allele, frequently target the de novo suppressor mutations, and extensively or exclusively target the converted suppressor mutations. This analysis is based on the distinction that UvrABC-initiated excision repair recognizes dimer and non-dimer (pyrimidine (6-4) pyrimidone) photoproducts but that DenV-initiated repair recognizes only pyrimidine dimers.

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity. The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo. The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo. The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.

We have previously demonstrated that the sequence 5'TGGCAA 3' located at codons 32-33 of the rIIB gene of bacteriophage T4 is a hotspot for transition mutations (Nelson et al. 1981). Here I report the properties of the same TGGCAA sequence introduced into the gene at codons 11-12. The sequence is highly mutable in both locations, suggesting that its high mutability is due to features of the TGGCAA itself and is not dependent on the immediate juxtaposition of additional external sequences. Within this sequence, at either location, only the transition at the central G:C pair frequently arises spontaneously or by 2-aminopurine or ethylmethane sulfonate mutagenesis. However, the 3' G:C pair, in addition, is highly mutable after nitrous acid or hydroxylamine treatment. This suggests that, within the TGGCAA sequence, there are two hotspots which are targeted by different mutagens.

The thermodynamics of melting of bacteriophage T4 lysozyme and four of its mutants have been measured by van't Hoff methods. The effect of pH has been explored and utilized to obtain the dependence of the enthalpy on temperature as suggested by Privalov and co-workers. The enthalpy change is a steep linear function of temperature. delta Cp is large and constant within experimental error. Changes in delta Hu are as large as 30% for a single point mutation. Changes in enthalpy are largely compensated by changes in entropy. Changes in stability, as measured by the free energy of unfolding, are smaller than those of delta H, but are very large in a relative sense, since delta G is very much smaller than delta H. Origins of the destabilization caused by mutations are discussed.