Control of Bacteriophage-induced Enzyme Synthesis in Cells Infected with a Temperature-sensitive Mutant

The timing of "early" and "late" protein synthesis in Escherichia coli infected with T-even bacteriophage was studied with a temperature-sensitive phage mutant, T4 tsL13. This strain was completely unable to direct the synthesis of phage deoxyribonucleic acid (DNA) at 44 C because it makes a deoxycytidylate hydroxymethylase which cannot act at that temperature. However, the mutant did multiply normally at 30 C. No detectable formation of the late protein, lysozyme, occurred at 44 C, in agreement with the idea, proposed by several workers, that DNA replication is necessary for activation of late genetic functions. However, the formation of an early enzyme, thymidylate synthetase, was shut off at about 10 min, as in normal infection. This implied that separate mechanisms were responsible for cessation of early functions and activation of late ones. That the infected cell at 44 C retained the capacity for synthesis of early enzymes was shown by the fact that DNA synthesis occurred after a culture was transferred from 44 to 30 C as late as 30 min after infection. This synthesis was inhibited by chloramphenicol, indicating that de novo synthesis of an early enzyme can take place at a late period in development. It is suggested that cells infected under normal conditions maintained an appreciable rate of early enzyme synthesis throughout the course of infection.     

Nucleic Acid Synthesis in Bacteriophage SPO2c1-Infected Bacillus subtilis

The synthesis of host macromolecules was shut off very slowly and incompletely by bacteriophage SPO2c(1). No change in the rate of incorporation of radioactive precursors into protein and ribonucleic acid (RNA) could be detected after infection, and the rate of incorporation of thymidine was increased only slightly. The relative proportions of phage and host species of nucleic acids at various intervals in the latent period were determined by means of nucleic acid hybridization. Phage-specific RNA populations synthesized early were different from those synthesized late in the latent period. Host deoxyribonucleic acid (DNA) replication continued until 8 to 10 min after SPO2c(1) infection and then decreased markedly as phage-specific DNA synthesis was initiated. Host DNA was not degraded to trichloroacetic acid-soluble fragments, and its nucleotides were not found in either newly synthesized intracellular phage DNA or in progeny phage particles. The average burst size of SPO2c(1) was approximately 200 plaque-forming units per cell.     

Morphology and Physiology of the Intracellular Development of Bacillus subtilis Bacteriophage φ25

The morphology of the intracellular development of bacteriophage phi25 in Bacillus subtilis 168M has been correlated with nucleic acid synthesis in infected cells. Host deoxyribonucleic acid (DNA) synthesis was shut off by a phage-induced enzyme within 5 min after infection, and another phage-mediated function extensively degraded host DNA at the time of cell lysis. Synthesis of phage DNA in infected cells began within 5 min and continued until late in the rise period. After phage DNA synthesis and coinciding with lysis, much of the unpackaged, newly synthesized phage DNA was degraded. Studies of thin sections of phi25 infected cells suggested that unfilled capsids may be precursors to filled capsids in the packaging process. To assess dependence of capsid formation on phage DNA replication, cells were either treated with mitomycin C and infected with normal phage or infected with ultraviolet-irradiated (99% killed) phi25. Only empty capsids were found in these cells, indicating that capsid production may be independent of the presence of newly synthesized viral DNA.     

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."     

Bacteriophage T4-related macromolecular synthesis under restriction of plasmid Rts1.

Rts1 is a plasmid which confers upon the host bacteria the capacity to restrict T4 bacteriophage growth at 32 degrees C but not at 42 degrees C. Pulse-labeling of phage-infected cells showed that Rts1 restricts the synthesis of T1 DNA. Despite efficient restriction of T4 phage growth and DNA synthesis, infected Escherichia coli 20SO harboring Rts1 synthesized both early and late T4 phage RNA. Synthesis of early T4 phage RNA under restrictive conditions (32 degrees C) was almost equal to that found under nonrestrictive conditions, and a lesser, but significant, amount of late T4 phage RNA was made in almost complete absence of T4 DNA synthesis. Moreover, very little, if any, T4 phage-coded lysozyme was detected in the infected E. coli 20SO/Rts1 at 32 degrees C, whereas normal amounts of lysozyme were present at 42 degrees C.     

Replication Process of the Parvovirus H-1 III. Factors Affecting H-1 RF DNA Synthesis

Replication of the single-stranded DNA parvovirus H-1 involves the synthesis of a double-stranded DNA replicative form (RF). In this study, the metabolism of RF DNA was examined in parasynchronous hamster embryo cells. The initiation of RF DNA replication was found to occur late in S phase, as was the synthesis of the DNA upon which subsequent viral hemagglutinin synthesis is dependent. Evidence is presented which indicates that initiation of RF replication requires proteins synthesized in late S phase, but that concomittant protein synthesis is not required for the continuation of RF replication. The data also suggest a requirement for viral protein(s) for progeny strand synthesis. Incorporation of 5-bromo-2'-deoxyuridine (BUdR) into viral DNA resulted in an "all-or-none" inhibition of viral hemagglutinin and viral antigen synthesis. BUdR inactivation of viral protein function was used to explore the time of synthesis of viral DNA serving as template for viral RNA synthesis and the effect of viral protein on RF replication and progeny strand synthesis. Results of this study suggest that parental RF DNA is synthesized shortly after infection, and that viral mRNA is transcribed from only a few copies of the viral genome in each cell. They also support the conclusion that viral protein is inhibitory to RF DNA replication. Density labeling of RF DNA with BUdR, allowing separation of viral strand DNA (V) from viral complementary strand (C), provided additional data in support of the above findings.     

T3 and T7 Bacteriophage Deoxyribonucleic Acid-Directed Enzyme Synthesis In Vitro

The T3 phage enzymes S-adenosyl methionine cleaving enzyme and lysozyme and the T7 lysozyme were synthesized in a deoxyribonucleic acid (DNA)-dependent, cell-free system derived from uninfected Escherichia coli. The data presented suggest that these enzymes are encoded in that portion of the DNA which is transcribed early after infection.     

Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1⁺ dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.     

Macromolecular synthesis in mycobacteriophage I3 infected cells

DNA-, RNA- and protein synthesis have been studied inMycobacterium smegmatis cells infected with phage 13. The macromolecular synthesis continued until the end of latent period. Early RNA and protein synthesis were necessary prior to the commencement of DNA replication. The infecting phage DNA sedimented as larger than unit length of genome, after initiation of DNA synthesis. Although the host DNA was not degraded, 90 percent of the RNA synthesized after phage infection hybridized to phage DNA.     

Deoxycytidylate deaminase. Evidence for a new enzyme in cells infected by the virus of herpes simplex

The activities of dCMP deaminase and DNA polymerase I increased twofold and fivefold in BHK-21/C13 cells after infection by the virus of herpes simplex. The increases were greatly diminished, and under certain conditions prevented, by inclusion of actinomycin D or cycloheximide in the cell-virus system during the infective cycle. The dCMP deaminase purified from infected cells harvested 8h after infection differed from the deaminase purified from non-infected cells inasmuch as (a) it was more resistant to heating at 37 degrees C; (b) the substrate (dCMP) concentration at half-maximum velocity was lower; (c) maximum activation was achieved by a lower concentration of dCTP; (d) it was more resistant to inhibition by dTTP; and (e) it behaved differently when assayed in the presence of a herpes-virus-specific antiserum. The DNA polymerase activity in the infected cells was markedly decreased in the presence of the herpes-virus-specific antiserum.     

Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.     

Control of synthesis of mRNA''s for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase.

A 30 degrees C, functional messengers for dCMP hydroxymethylase first appeared 3 to 6 min postinfection and reached their maximum levels at 12 min. Chloramphenicol, added before the phage, reduced the rate of mRNA accumulation. When the antibiotic was added 6 min postinfection, mRNA levels increased at their normal rate but there was no obvious repression of messenger accumulation. Delaying the addition of drug until 8 or 12 min had progressively less effect on the pattern of hydroxymethylase mRNA metabolism. When chloramphenicol was present from preinfection times or from 6 min postinfection, all hydroxymethylase mRNA's synthesized were stable; at later times, however, the ability of the drug to stabilize mRNA decreased with its ability to delay the turnoff of mRNA production. An overaccumulation of hydroxymethylase mRNA was also seen when phage-specific DNA synthesis was inhibited either by mutational lesion in an essential viral gene or by 5-fluorodeoxyuridine. By min 20 of a DNA-negative program, hydroxymethylase mRNA synthesis was repressed to the point where it no longer compensated for decay. However, a finite level of hydroxymethylase mRNA synthesis was maintained at later times of a DNA-negative infection. Such results indicate that replication of the phage chromosome is necessary but not sufficient for a complete turnoff of hydroxymethylase mRNA production. Functions controlled by the maturation-defective proteins (the products of genes 55 and 33) played only a minor role in the regulation of hydroxymethylase mRNA, metabolism. Thus, we favor the hypothesis that a complete turnoff of hydroxymethylase messenger production requires one or more new proteins as well as an interval of DNA replication. The absence of DNA synthesis had no particular effect upon dihydrofolate reductase messenger production. The preinfection addition of chloramphenicol likewise had little effect on dihydrofolate reductase messenger metabolism. These latter data imply that prior synthesis of a phage-coded protein synthesis may not be required for the turnoff of reductase messenger production.