Regulation of Expression of Cloned Bacteriophage T4 Late Gene 23

The parameters governing the activity of the cloned T4 gene 23, which codes for the major T4 head protein, were analyzed. Suppressor-negative bacteria carrying wild-type T4 gene 23 cloned into plasmid pCR1 or pBR322 were infected with T4 gene 23 amber phage also carrying mutations in the following genes: (i) denA and denB (to prevent breakdown of plasmid DNA after infection) and (ii) denA, denB, and, in addition, 56 (to generate newly replicated DNA containing dCMP) and alc/unf (because mutations in this last gene allow late genes to be expressed in cytosine-containing T4 DNA). Bacteria infected with these phage were labeled with (14)C-amino acids at various times after infection, and the labeled proteins were separated by one-dimensional gel electrophoresis so that the synthesis of plasmid-coded gp23 could be compared with the synthesis of other, chromosome-coded T4 late proteins. We analyzed the effects of additional mutations that inactivate DNA replication proteins (genes 32 and 43), an RNA polymerase-binding protein (gene 55), type II topoisomerase (gene 52), and an exonuclease function involved in recombination (gene 46) on the synthesis of plasmid-coded gp23 in relation to chromosome-coded T4 late proteins. In the denA:denB:56:alc/unf genetic background, the phage chromosome-borne late genes followed the same regulatory rules (with respect to DNA replication and gp55 action) as in the denA:denB genetic background. The plasmid-carried gene 23 was also under gp55 control, but was less sensitive than the chromosomal late genes to perturbations of DNA replication. Synthesis of plasmid-coded gp23 was greatly inhibited when both the type II T4 topoisomerase and the host's DNA gyrase are inactivated. Synthesis of gp23 was also substantially affected by a mutation in gene 46, but less strongly than in the denA:denB genetic background. These observations are interpreted as follows. The plasmid-borne T4 gene 23 is primarily expressed from a late promoter. Expression of gene 23 from this late promoter responds to an activation event which involves some structural alteration of DNA. In these respects, the requirements for expressing the plasmid-borne gene 23 and chromosomal late genes are very similar (although in the denA:denB:56:alc/unf genetic background, there are significant quantitative differences). For the plasmid-borne gene 23, activation involves the T4 gp46, a protein which is required for DNA recombination. However, for the reasons presented in the accompanying paper (Jacobs et al., J. Virol. 39:31-45, 1981), we conclude that the activation of gene 23 does not require a complete breakage-reunion event which transposes that gene to a later promoter on the phage chromosome. Ways in which gp46 may actually be involved in late promoter activation on the plasmid are discussed.     

Mutation to Overproduction of Bacteriophage T4 Gene Products

R9 was isolated as one of several mutations that enhanced the growth of a leaky amber (am) mutant of bacteriophage T4 gene 62 (product required for phage DNA synthesis) under conditions of partial suppression by ribosomal ambiguity. R9 also enhanced the growth of leaky am mutants of some, but not all, other T4 “early” gene functions. R9 mapped between mutations in genes 43 and 62. By using assays involving polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate, we observed the following. (i) R9 resulted in an overproduction of many T4 “early” proteins in infected cells. The most pronounced effects of R9 were observed when phage DNA synthesis and/or the functions of maturation genes 55 and 33 were not expressed. (ii) In rifampintreated infected cells, the capacity to synthesize T4 “early” proteins decayed more slowly in the presence of the R9 mutation than in the presence of the wild-type counterpart of R9. R9 appeared to have no effect on the rates of RNA synthesis either during early or late times after infection. The results suggest that the R9 mutation leads to increased functional stability of T4 “early” messengers.     

The Riia Gene of Bacteriophage T4. II. Regulation of Its Messenger RNA Synthesis

When the rII genes are first introduced into cells which had been previously infected by T4 phage deleted for these genes, the kinetics of synthesis of rIIA and rIIB RNA are rapid and identical. We show that this rapid synthesis depends on a functional motA gene for rIIB, but not for rIIA, RNA synthesis. By primer-extension mapping of T4 messenger RNA, we find three promoters close to the rIIA gene. One of them is an early promoter just before the rIIA.1 gene; it is used under all conditions tested. Another is in the coding portion of the rIIA.1 gene; it is weak, primarily because of a 19-bp spacing between the -10 and -35 elements, and its use is stimulated by T4 functions. The third is a motA-dependent (middle) promoter which has an unusual CCCGCTT box at -33. We present results which suggest that none of these promoters is likely to be the site at which the motB and motC gene products exercise their major influence on rIIA RNA synthesis.     

Control of Early Gene Expression of Bacteriophage T4: Involvement of the Host rho Factor and the mot Gene of the Bacteriophage

Many early mRNA species of bacteriophage T4 are not synthesized after infection of Escherichia coli in the presence of chloramphenicol. This has been interpreted as a need for T4 protein(s) to be synthesized to allow expression of some early genes, e.g., those for deoxycytidinetriphosphatase, deoxynucleosidemonophosphate kinase and UDP-glucose-DNA beta-glucosyltransferase. In the experiments described here, early mRNA of bacteriophage T4 was allowed to accumulate during chloramphenicol treatment. After the addition of rifampin to inhibit further RNA synthesis, and subsequent removal of chloramphenicol, the accumulated mRNA was permitted to express itself into measured enzyme activities. It was shown that the early mRNA species coding for deoxycytidinetriphosphatase and UDP-glucose-DNA beta-glucosyltransferase could be formed in the presence of chloramphenicol if the E. coli host cell carried a mutation in the structural gene for the RNA chain termination factor rho. This was interpreted to mean that T4 protein(s) with anti-rho activity is normally required for the expression of these two early genes. An altered rho-factor could not, however, relieve the need of phage protein synthesis for the formation of another early mRNA, that coding for deoxynucleosidemonophosphate kinase. In this case the mot gene of T4 seemed to be involved, since the primary infection of E. coli cells with the mot gene mutant tsG1 did not allow subsequent deoxynucleoside monophosphate kinase mRNA synthesis after wild-type phage infection in the presence of chloramphenicol. In control experiments, deoxynucleoside monophosphate kinase mRNA synthesis induced by wild-type phage superinfecting in the presence of chloramphenicol was facilitated by the primary infection with T4 phage containing an unmutated mot gene.     

Involvement of Gene 49 in Recombination of Bacteriophage T4

The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvsX, but recombination between rII markers was not.     

Role of Gene 52 in Bacteriophage T4 DNA Synthesis

In an attempt to elucidate the mechanism of delayed DNA synthesis in phage T4, Escherichia coli B cells were infected with H17 (an amber mutant defective in gene 52 possessing a "DNA-delay" phenotype). The fate of (14)C-labeled H17 parental DNA after infection was followed: we could show that this DNA sediments more slowly in neutral sucrose than wild-type DNA 3 min postinfection. In pulse-chase experiments progeny DNA was found to undergo detachment from the membrane at 12 min postinfection. Reattachment to the membrane was found to be related to an increase in rate of DNA synthesis. A nucleolytic activity that is absent from cells infected by wild-type phage and from uninfected cells could be detected in extracts prepared from mutant-infected cells. In contrast, degradation of host DNA was found to be less extensive in am H17 compared with wild-type infected cells. Addition of chloramphenicol to mutant-infected cells 10 min postinfection inhibited the appearance of a nuclease activity on one hand and suppressed the "DNA-delay" phenotype on the other hand. We conclude that the gene 52 product controls the activity of a nuclease in infected cells whose main function may be specific strand nicking in association with DNA replication. This gene product might directly attack both E. coli and phage T4 DNA, or indirectly determine their sensitivity to degradation by another nuclease.     

Expression and Properties of Bacteriophage T4 Gene Product 11

A plasmid vector for expression of bacteriophage T4 gene product 11 (gp11) in E. coli cells has been constructed. Gp11 is a baseplate protein that connects short tail fibers providing irreversible adsorption of the virus on a cell. A method based on chromatography on hydroxyapatite has been developed for purification of recombinant gp11. The protein is active in an in vitro complementation assay and transforms defective phage particles lacking gp11 into infective ones. Gel filtration data suggest that the biologically active protein is a trimer. According to CD spectroscopy and sequence analysis data, the polypeptide chain of gp11 contains not less than 20% -helical segments, about 30% -structure, and belongs to the class of / structural proteins.     

Intragenic Complementation by Gene 42 Amber Mutations of Bacteriophage T4

Phage T4 amber mutants defective in gene 42 (dCMP hydroxymethylase) were shown by in vivo and in vitro experiments to participate in both positive and negative intragenic complementation. This argues that incomplete polypeptide chains can participate in subunit interaction.     

Properties of Bacteriophage T4 Baseplate Protein Encoded by Gene 8

Gene product 8 (gp8, 344 amino acids per monomer) of bacteriophage T4 is one of the baseplate structural proteins. We constructed an expression vector of gp8 and developed a method for purification of recombinant protein. CD spectroscopy showed that gp8 is an / type structural protein. Its polypeptide chain consists of nearly 40% -structure and 15% -helix. These data agree with results of prediction of secondary structure based on the amino acid sequence of the protein. The sedimentation coefficient under standard conditions (S_20,w) is 4.6S. Analytical ultracentrifugation results demonstrated that gp8 in solution has two types of oligomers—dimer and tetramer. The tetramer of gp8 may be included in the wedge (1/6 of the baseplate), and the dimer may be an intermediate product of association.     

Role of Gene 46 in Bacteriophage T4 Deoxyribonucleic Acid Synthesis

In an attempt to establish whether Escherichia coli B infected with N130 (an amber mutant defective in gene 46) is recombination-deficient, the postinfection fate of (14)C-labeled N130 parental deoxyribonucleic acid (DNA) was followed, its amount in complex with the host cell membrane being determined in sucrose gradients after mild lysis of the infected cells. The parental DNA was found to undergo gradual detachment from the membrane during infection. Pulse-chase experiments similarly showed that newly synthesized DNA is normally attached to the host cell membrane and is detached by endonucleolytic breakage at a late stage of infection. The conclusion is that only attached DNA molecules are replicated by membrane-bound replicase, whereas those detached by endonucleolytic breakage are not. It thus seems that the gene 46 product controls the activity of a nuclease whose main function is recombination of DNA nicked by endonuclease, thereby attaching it to the host cell membrane. The rate of T4 DNA synthesis is apparently governed by the efficiency of recombination. Supporting evidence was found in experiments with the double mutant N130 x N134 (genes 46, 33).     

Degradation of Escherichia coli Chromosome After Infection by Bacteriophage T4: Role of Bacteriophage Gene D2a

Mutations in the D2a gene of bacteriophage T4 have recently been shown to result in the stabilization of cytosine-containing phage deoxyribonucleic acid (DNA) made after infection by phage gene 56 (deoxycytidine triphosphatase) mutants. In the experiments reported here, we investigate the role of the D2a gene in the degradation of the host chromosome. We find that if T4 endonuclease II, a product of the phage gene denA, is active, host chromosome degradation appears normal, regardless of the presence of the D2a gene product. However, if T4 endonuclease II is absent, a small amount of host chromosome degradation occurs, but only if the D2a product is present. These results are interpreted in terms of the hypothesis that D2a controls a nuclease which degrades cytosine-containing DNA. Neither D2a nor denA mutations affect the shut-off of host DNA synthesis.     

Inhibition of Translation of Preformed lac Messenger Ribonucleic Acid by T4 Bacteriophage

Cells infected with bacteriophage T4 are unable to translate beta-galactosidase messenger ribonucleic acid which exists in the cell before infection, unless translation is underway at the time of infection.     

Immediate-Early Expression of the Gene Causing Superinfection Breakdown in Bacteriophage T4B

Superinfection breakdown appears to belong to the "immediate-early" functions induced by T4 phage.     

Revised Location of the rIII Gene on the Genetic Map of Bacteriophage T4

Two- and three-factor crosses showed that the T4 rIII gene is located between genes 31 and 30 rather than between genes 31 and 63.