Multiple-Tailed T4 Bacteriophage.

Smith, Kendall O. (Baylor University College of Medicine, Houston, Tex.), and Melvin Trousdale. Multiple-tailed T4 bacteriophage. J. Bacteriol. 90:796-802. 1965.-T4 phage particles which appeared to have multiple-tails were observed. Experiments were designed to minimize the possibility that superimposed particles might account for this appearance. Double-tailed particles occurred at a frequency as high as 10%. Triple- and quadruple-tailed particles were extremely rare. All attempts to isolate pure lines of multiple-tailed phage have failed. Multiple-tailed phage particles were produced in highest frequency by Escherichia coli cells in the logarithmic growth phase which had been inoculated at a multiplicity of about 2.     

Ligase-Defective Bacteriophage T4 II. Physiological Studies

The timing of the suppression of gene 30 (deoxyribonucleic acid ligase) mutations by rII mutations was studied by temperature shift-down experiments with a temperature-sensitive rII mutation. The rII function must remain inactivated for about 5 to 8 min at 37 C for suppression to occur, thus making suppression an early function. This result is in agreement with the timing of expression of other rII functions. A gene 30 defect can also be overcome by replacing the Na(+) cation in the growth medium with the Mg(2+) cation, a result similar to the relief of the lethality of rII mutations in lambda lysogens. Prior infection with bacteriophages T3 or T7, which produce their own deoxyribonucleic acid ligases, can also partially overcome the lethality of gene 30 mutations.     

Consensus methods for finding and ranking DNA binding sites. Application to Escherichia coli promoters

There have been many different approaches employed to define the "consensus" sequence of various DNA binding sites and to use the definition obtained to locate and rank members of a given sequence family. The analysis presented here enlists two of these approaches, each in modified form, to develop a highly efficient search protocol for Escherichia coli promoters and to provide a relative ranking of these sites showing good agreement with in vitro measurements of promoter strength. Schneider et al. have applied Shannon's index of information content to evaluate the significance of each position within the consensus of a family of aligned sequences. In a formal sense, this index is only applicable to a group of sequences, providing at each position a negative entropy value between zero (random) and two bits (total conservation of a single base) for sequences in which all bases are equally represented. A method for evaluating how well an individual sequence conforms to the information content pattern of the consensus is described. A function is derived, by analogy to the information content of the sequence family, for application to individual sequences. Since this function is a measure of conformity, it can be used in a search protocol to identify new members of the family represented by the consensus. A protocol for locating E. coli promoters is presented. The Berg-von Hippel statistical-mechanical function is also tested in a similar application. While the information content function provides a superior search protocol, the Berg-von Hippel function, when scaled at each position by the information content, does well at ranking promoters according to their strength as measured in vitro.     

Bacteriophage T4-induced shut-off of host-specific translation.

To study the mechanism by which bacteriophage T4 inhibits the synthesis of inducible host enzymes we measured the formation of beta-galactosidase from preformed lac mRNA. Beta-Galactosidase was induced with isopropyl-beta-D-thiogalactopyranoside in the presence of 7-azatryptophan, a tryptophan analogue that is incorporated into proteins and renders the beta-galactosidase formed inactive. The accumulated las mRNA was measured by capacity to form active beta-galactosidase after a chase of the analogue with excess tryptophan. After T4 infection the ability to form beta-galactosidase from the preformed lac mRNA was rapidly lost even when T4 infection took place in the presence of rifampin. This restriction was dependent on the multiplicity of infection. At a multiplicity of infection of 8.6, 90% of the ability to express preformed lac mRNA was lost within 30 s. The kinetics of cessation of beta-galactosidase synthesis after T4 infection indicate that infection blocks initiation of lac mRNA translation.     

Bacteriophage Tail Components: II. Dihydrofolate Reductase in T4D Bacteriophage

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.     

Repeating sequences in promoter regions of eukaryotic genes]

Promoter regions of eukaryotic genes were analyzed for the presence of repeated fragments. It was found that the promotor sequences are abundant in direct, symmetrical, and inverted repeats. A computer system for searching and visualizing the repeats was developed.     

Spackle and Immunity Functions of Bacteriophage T4

Cells of Escherichia coli B infected with the immunity-negative (imm2) mutant of bacteriophage T4 are able to develop a substantial level of immunity to superinfecting phage ghosts if the ghost challenge is made late in infection. This background immunity is not seen in infections with phage carrying the spackle (s) mutation in addition to the imm2 lesion. The level of immunity in s⁻ infections is intermediate between that of imm⁻ and wild-type infections under standard assay conditions. With respect to genetic exclusion of superinfecting phage, cells infected with imm⁻ phage are completely deficient, whereas infections with the s⁻ phage are only partially deficient compared to wild-type infections. Whereas s⁻-infected cells are unable to resist lysis from without by a high multiplicity of infection (MOI) of superinfecting phage, cells infected with imm⁻ phage show less than wild-type levels of resistance and the majority of cells remaining intact are unable to incorporate leucine or form infective centers. Under conditions of superinfection by low MOI of homologous phage, imm⁻-infected cells are lysis inhibited, whereas s⁻-infected cells do not show this property. Superinfecting phage inject their DNA into imm⁻-infected cells with the same efficiency as seen in wild-type infections, but this efficiency is reduced when the cells are first infected with s⁻ phage. The s function of T4 appears not only to affect the host cell wall as previously postulated by Emrich, but may also affect the junctures of cell wall and membrane with consequences similar to those of the imm function.