Recombination of bacteriophage T7 in vivo

Summary Most recombination following infection with T7 was found to coincide with the time of most rapid DNA synthesis, at about 20 min after infection at 30° in minimal medium. Recombining DNA was investigated electron microscopically. Multiply branched DNA structures were observed after infection with T7 wild type, gene 3 ^–, gene 6 ^– and genes 3 ^–, 6 ^– phage, but not after infection with T7 gene 5 ^– phage. Evidence is presented indicating that these structures are T7 DNA molecules in the process of recombining. The detailed structures of these recombinational intermediates suggest mechanisms by which T7 DNA initiates recombination.     

Genetic and physical mapping in the early region of bacteriophage T7 DNA.

A detailed physical map of the early region of bacteriophage T7 DNA has been constructed. This map contains: locations for all the cuts made by the restriction endonucleases HindII, HpaII, HaeIII and HaeII, and many of the cuts by HhaI; the approximate end points for each of 61 different deletions; initiation sites and the termination site for RNAs made by Escherichia coli RNA polymerase; an initiation site for RNA made by T7 RNA polymerase; the five primary RNase III cleavage sites of the early region; and the coding sequences for perhaps nine different early proteins. Virtually all of the non-overlapping coding capacity of the five early messenger RNAs is used, except for untranslated stretches of perhaps 30 or so nucleotides at the ends. It seems likely that each of the nine early proteins is made from its own ribosome-binding and initiation site. The mapped restriction cuts provide fixed reference points, and allow DNA fragments containing specific genetic signals to be identified and isolated.The nucleotide sequences around the ends of three different T7 deletions have been determined. Each deletion eliminated a segment of DNA between repeated sequences of seven, eight or ten base-pairs, located 578 to 2100 base-pairs apart in the wild-type sequence. In each case, one copy of the repeated sequence was retained in the deletion mutant. This is consistent with the deletions having arisen by a genetic crossover between the repeated sequences. The approximate frequency of genetic recombination per base-pair has been estimated within two early genes; in both cases, the value was close to 0.01% recombination per base-pair, consistent with the value expected from the total length of the T7 genetic map. Genetic recombination between non-overlapping deletions appears to be severely depressed when the distance between the deletions is closer than about 40 to 50 base-pairs, but recombination between a point mutation and a deletion does not appear to be similarly depressed. This suggests that efficient genetic recombination in T7 may require a base-paired “synapse” of some minimum size between the recombining DNA molecules.     

Early to late switch in bacteriophage T7 development: functional decay of T7 early messenger RNA

Infection of ultraviolet light-irradiated Escherichia coli with T7 phage in the presence of chloramphenicol results in synthesis of T7 early messenger RNA but not late mRNA. T7 early mRNA accumulates in terms of acid-insoluble, T7 DNA-hybridizable RNA. However, messenger activity of the same RNA decays rapidly with a half-life of about 6.5 minutes at 30 °C when tested for the ability to direct in vitro protein synthesis. This functional decay of T7 early mRNA is attributable to a loss of structural integrity of the RNA. Polyacrylamide-agarose gel electrophoresis shows that T7 early mRNAs are cleaved, generating smaller-size RNAs. Kinetics of the appearance of T7-specific RNA polymerase, one of the early gene products, during normal T7 infection show that the capacity of the cells to produce the enzyme decays very rapidly when early mRNA synthesis is terminated either by rifampicin or by a natural mechanism programmed by T7. Preferential synthesis of late proteins in the presence of chemically stable early mRNA late in T7 infection may be explained by the observed functional decay of early mRNA.     

Purification and characterization of the gene 1.2 protein of bacteriophage T7

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.