Frameshift mutagenesis in bacteriophage T7.

We have undertaken an initial characterization of frameshift mutagenesis in bacteriophage T7 and have identified a subset of very low reversion frameshift mutations in the T7 ligase gene (gene 1.3). We used this information to construct bacteriophage T7 strains that contain one extra or one less base pair in gene 1.3 such that a frameshift event restores the reading frame of that gene. These events can be quantified and the frameshift mutation isolated within a localized region of the ligase gene. We have also identified a portion of the T7 ligase protein that will accept tracts of nonsense amino acids yet still give a ligase positive phenotype. This allows flexibility in the design of the target DNA sequence with which to study frameshift mutagenesis. These assays for frameshift mutagenesis performed in E. coli cells infected with the appropriate T7 strain, were used to measure the frequency of both plus and minus frameshifts in vivo.     

Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels.

Agarose gel electrophoresis of the following was performed in 0.05 M sodium phosphate-0.001 M MgCl2 (pH 7.4): (i) bacteriophage T7; (ii) a T7 precursor capsid (capsid I), isolated from T7-infected Escherichia coli, which has a thicker and less angular envelope than bacteriophage T7; (iii) a second capsid (capsid II), isolated from T7-infected E. coli, which has a bacteriophage-like envelope; and (iv) capsids (capsid IV) produced by temperature shock of bacteriophage T7. Bacteriophage T7 and all of the above capsids migrated towards the anode. In a 0.9% agarose gel, capsid I had an electrophoretic mobility of 9.1 +/- 0.4 X 10(-5) cm2/V.s; bacteriophage T7 migrated 0.31 +/- 0.02 times as fast as capsid I. The mobilities of different preparations of capsid II varied in such gels: the fastest-migrating capsid II preparation was 0.51 +/- 0.03 times as fast as capsid I and the slowest was 0.37 +/- 0.02 times as fast as capsid I. Capsid IV with and without the phage tail migrated 0.29 +/- 0.02 and 0.42 +/- 0.02 times as fast as capsid I. The results of the extrapolation of bacteriophage and capsid mobilities to 0% agarose concentration indicated that the above differences in mobility are caused by differences in average surface charge density. To increase the accuracy of mobility comparisons and to increase the number of samples that could be simultaneously analyzed, multisample horizontal slab gels were used. Treatment with the ionic detergent sodium dodecyl sulfate converted capsid I to a capsid that migated in the capsid II region during electrophoresis through agarose gels. In the electron microscope, most of the envelopes of these latter capsids resembled the capsid II envelope, but some envelope regions were thicker than the capsid II envelope.     

Spontaneous temperature-sensitive mutations in bacteriophage T7.

Attempts to recover temperature-sensitive mutations affecting genes 13 and 14 (virion proteins) in bacteriophage T7 by analysis of amber revertants were confounded by the frequent occurrence of spontaneous temperature-sensitive mutations in other genes. These incidental temperature-sensitive mutations are physically distinct from but may be functionally related to genes 13 and 14, as shown by complementation and recombination studies. The possibility that these incidental temperature-sensitive mutations represent secondary-site suppressors of the pseudonormal suppressed amber products is discussed.     

Superinfection exclusion by bacteriophage T7.

Only two of the early genes of bacteriophage T7 were found to play a significant role in exclusion of superinfecting bacteriophage T3 particles; genes 0.3 and 1. Protein synthesis by the preinfecting phage particle was not required for efficient exclusion. These findings are discussed with regard to the known functions of these genes during T7 development.     

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.     

Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested.     

Intracellular organization of bacteriophage T7 DNA: analysis of parenteral bacteriophage T7 DNA-membrane and DNA-protein complexes.

After infection of Escherichia coli with bacteriophage T7, the parenteral DNA forms a stable association with host cell membranes. The DNA-membrane complex isolated in cesium chloride gradients is free of host DNA and the bulk of T7 RNA. The complex purified through two cesium chloride gradients contains a reproducible set of proteins which are enriched in polypeptides having molecular weights of 54,000, 34,000, and 32,000. All proteins present in the complex are derived from host membranes. Treatment of the complex with Bruij-58 removes 95% of the membrane lipid and selectively releases certain protein components. The Brij-treated complex has an S value of about 1,000 and the sedimentation rate of this material is not altered by treatment with Pronase or RNase.     

Deletion mutagenesis independent of recombination in bacteriophage T7.

Deletion between directly repeated DNA sequences in bacteriophage T7-infected Escherichia coli was examined. The phage ligase gene was interrupted by insertion of synthetic DNA designed so that the inserts were bracketed by 10-bp direct repeats. Deletion between the direct repeats eliminated the insert and restored the ability of the phage to make its own ligase. The deletion frequency of inserts of 85 bp or less was of the order of 10(-6) deletions per replication. The deletion frequency dropped sharply in the range between 85 and 94 bp and then decreased at a much lower rate over the range from 94 to 900 bp. To see whether a deletion was predominantly caused by intermolecular recombination between the leftmost direct repeat on one chromosome and the rightmost direct repeat on a distinct chromosome, genetic markers were introduced to the left and right of the insert in the ligase gene. Short deletions of 29 bp and longer deletions of approximately 350 bp were examined in this way. Phage which underwent deletion between the direct repeats had the same frequency of recombination between the left and right flanking markers as was found in controls in which no deletion events took place. These data argue against intermolecular recombination between direct repeats as a major factor in deletion in T7-infected E. coli.     

Involvement of DNA gyrase in bacteriophage T7 growth.

We have found that the burst size of bacteriophage T7 was decreased in two Escherichia coli temperature-sensitive gyrase mutants incubated at the restrictive temperature. This reduction in burst size indicates that gyrase may be required for T7 growth.     

Protein kinase of bacteriophage T7. 1. Purification.

A protein kinase, ATP:protein phosphotransferase (EC 2.7.1.37) was detected in Escherichia coli after infection with bacteriophage T7. The enzyme was purified from the ribosomal wash fraction by conventional methods, affinity chromatography on Cibacron blue and on lysozyme coupled to Sepharose, and by cellogel electrophoresis. An approximately 5000-fold purification was achieved.     

Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA.

The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases. AvaI, XbaI, BglII, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites. The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards. As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases. The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA. The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7.     

Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity.

The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences. In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP. Substitution of this residue (Asn) with the corresponding residue found in T3 RNAP (Asp) results in a switch in promoter specificity, and specifically alters recognition of the base pairs (bp) at positions -11 and, possibly, -10 in the promoter. A complementary mutation in T3 RNAP (T3-D749N) results in a similar switch in promoter preference for that enzyme. The hierarchy of bp preference by the mutant and wild-type enzymes for bp at -10 and -11, and the results of previous experiments, lead to a model for specificity in which it is proposed that N748 in T7 RNAP (and D749 in T3 RNAP) make specific hydrogen bonds with bases at -11 and -10 on the non-template strand in the major groove. The specificity determining region of T7 RNAP does not appear to exhibit homology to any known sequence-dependent DNA binding motif.     

Role of gene 2 in bacteriophage T7 DNA synthesis.

Studies have been carried out to elucidate the in vivo function of gene 2 in T7 DNA synthesis. In gene 2-infected cells the rate of incorporation of (3-H)thymidine into acid-insoluble material is about 60% that of cells infected with T7 wild type. Gene 2 mutants do not however produce viable phage after infection of the nonpermissive host. In T7 wild type-infected cells, a major portion of the newly alkaline sucrose gradients. The concatemers serve as precursors for the formation of mature T7 DNA as demonstrated in pulse-chase experiments. In similar studies carried out with gene 2-infected cells, concatemers are not detected when the intracellular DNA is analyzed at several different times during the infection process. The DNA made during a gene 2 infection is present as duplex structures with a sedimentation rate close to mature T7 DNA.     

Binding of ethidium to bacteriophage T7 and T7 deletion mutants

Equilibrium binding of ethidium, quantitated by fluorescence enhancement, to DNA packaged in bacteriophage T7 and T7 deletion mutants has been compared with the binding of this dye to DNA released from its capsid (free DNA). During achievement of apparent equilibrium binding, no change in bacteriophage T7 structure occurred, by the criterion of agarose gel electrophoresis. However, excessive incubation with ethidium bromide caused detectable changes in bacteriophage structure, a possible explanation of disagreements in similar studies previously performed with T-even bacteriophages. Scatchard plots for packaged DNA had a curvature greater than the previously demonstrated [Bresloff, J. L. & Crothers, D. M. (1981) Biochemistry 20 , 3547–3553] curvature for free DNA. By treating plots for packaged DNA as though they were biphasic, it was found that binding to most sites occurred with an apparent association constant (K_ap) 3.3–4.3 times lower than the K_ap of free DNA. The number of these sites increased significantly as the density of packaged DNA was decreased by use of the deletion mutants. Values of ΔH° for these sites were negative and equal to the ΔH° for free DNA; values of ΔS° were positive and about half the ΔS° for free DNA. A second class of sites, roughly 1.2% of the total, had a significantly higher K_ap and more negative ΔH° than those of the majority of sites.