Deoxyribonucleic Acid Synthesis in Synchronized Mammalian KB Cells Infected with Herpes Simplex Virus

We examined the patterns of host cell and virus deoxyribonucleic acid (DNA) synthesis in synchronized cultures of KB cells infected at different stages of the cell cycle with herpes simplex virus (HSV). We found that the initiation of HSV DNA synthesis, we well as the production of new infectious virus, is independent of the S, G1, and G2 phases of the mitotic cycle of the host cell. This is in contrast to data previously found with equine abortion virus. Because HSV replicates independently of the cell cycle, we were able to establish conditions that would permit the study of rates of HSV DNA synthesized in logarithmically growing cells in the virtual absence of cellular DNA synthesis. This eliminates the need for separation of viral and cellular DNA by isopycnic centrifugation in CsCl. We found that HSV DNA synthesis was initiated between 2 to 3 hr after infection. The rate of DNA synthesis increased rapidly, reaching a maximum 4 hr after infection, and decreased to 50% of maximum by 8 hr. Evidence is also presented which suggests that HSV infection can inhibit both the ongoing synthesis of host DNA as well as the initiation of the S phase.     

Deoxyribonucleic Acid Synthesis in Ultraviolet-Light-Irradiated Chinese Hamster Cells

Two mammalian cell lines, Chinese hamster ovary (CHO) which can recover colony-forming ability between fractionated doses of ultraviolet light (UV), and Chinese hamster B-14FAF28 which cannot recover, were tested for the ability to bypass UV-induced photoproducts in DNA during postirradiation DNA synthesis. The molecular weight distributions of newly synthesized DNA in UV-irradiated populations of both cell lines showed evidence for photoproduct bypass. Hence, the bypass mechanism does not correlate with recovery after UV.     

Effect of Nalidixic Acid on Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-infected Bacillus subtilis

The effect of nalidixic acid on deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis cells infected with bacteriophage SPO1 was studied. Nalidixic acid had little inhibitory effect on SPO1 DNA synthesis at concentrations that drastically inhibited B. subtilis DNA synthesis. Inhibition of DNA synthesis, appropriate to the concentration used, was imposed within 1 min after addition of nalidixic acid, suggesting that it acts directly on DNA synthesis in both infected and uninfected cells. The SPO1 DNA synthesized in the presence of high concentrations of nalidixic acid had a density characteristic of normal SPO1 DNA and was packaged into viable progeny phage particles, but its rate of synthesis was reduced and bacterial lysis was delayed.     

Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae Cells Permeabilized with Ether

Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With rho(+) strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-1Brho(+)) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Density-labeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In rho(0) strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperature-sensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment.     

Tests for Deoxyribonucleic Acid Synthesis in Toluenized Bacillus subtilis Cells

Several tests were devised to further characterize deoxyribonucleic acid (DNA) synthesis in toluenized Bacillus subtilis cells. Vigorous agitation of toluenized cells (localization test) demonstrated that the DNA replication is exclusively a cell-associated process. A DNA "repair" condition was also applied to toluenized cells and shown to be distinct from DNA replication in its DNA polymerase I dependency and its ability to synthesize DNA on template which is either cell associated or free, outside the cell. This repair condition was used in conjunction with the localization test to demonstrate the penetration of deoxyribonuclease I and possibly DNA polymerase I into toluenized cells. Therefore, we suggest that the localization test can be used to test the penetration of proteins into toluenized cells for both the DNA repair and replication processes.     

Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-Infected Bacillus subtilis I. Bacteriophage Deoxyribonucleic Acid Synthesis and Fate of Host Deoxyribonucleic Acid in Normal and Polymerase-Deficient Strains

The effect of bacteriophage SPO1 infection of Bacillus subtilis and a deoxyribonucleic acid (DNA) polymerase-deficient (pol⁻) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg²⁺ ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol⁻ mutation had no effect on the synthesis of phage DNA or production of mature phage particles.     

Changes in Deoxyribonucleic Acid Synthesis Regulation in Chinese Hamster Cells Infected with Simian Virus 40

Infection of primary or secondary cultures of Chinese hamster embryo cells with simian virus 40 at a multiplicity of 20 to 50 induced synthesis of the virus-specific intranuclear T antigen in 80 to 90% of the cells within 48 to 72 hr. In the infected cultures, 30 to 50% more cells were recruited into deoxyribonucleic acid (DNA) synthesis than in the controls, whether or not the cultures were confluent. The newly synthesized DNA was mostly cellular, since little virus was produced (as shown by various techniques: immunofluorescence for viral antigen, virus growth curves, and isolation of viral DNA from infected cultures). Transformed cells could be detected a few weeks after infection and produced tumors when inoculated into irradiated animals. Chromosomal changes were observed soon after infection (24 hr). Initially, there was a marked increase in the proportion of polyploid cells (8 to 14%), most of which were chromosomally normal. In a few weeks, a large majority of the infected population was polyploid (30 to 50%). Thus, the polyploid cells have the ability to proliferate. Evidence is presented to suggest that polyploid cells arise by stimulation of cells in the G(1), G(2), or S phases to undergo two or more successive periods of DNA synthesis without an intervening mitosis. With a subsequent loss or redistribution of chromosomal material, this may lead eventually to a biologically transformed cell; thus, it is suggested that the initial event(s) relevant to transformation occurs at the level of control of cellular DNA synthesis.     

Role of Ribonucleic Acid Synthesis in Replication of Deoxyribonucleic Acid

An experiment previously interpreted to show a ribonucleic acid requirement for propagation of deoxyribonucleic replication is reexamined and the earlier interpretation is shown to be incorrect.     

Single-Strand Breaks in Deoxyribonucleic Acid and Viability Loss During Deoxyribonucleic Acid Synthesis Inhibition in Escherichia coli

The effects of deoxyribonucleic acid (DNA) synthesis inhibition brought about in four different ways-thymidine starvation, nalidixic acid, hydroxyurea, and dnaB mutation-were examined in isogenic strains of Escherichia coli K-12. Three parameters were examined to determine whether there are strict correlations among them: (i) the extent of DNA synthesis inhibition; (ii) cell survival; and (iii) the rate of breakage of DNA molecules. There was no significant correlation between the extent of DNA synthesis inhibition and the rate of viability loss caused by the four DNA synthesis inhibitors, nor was there a strict correlation between the rate of occurrence of single-strand breaks in DNA and loss of viability. During treatment with hydroxyurea (0.1 M), no viability loss was observed and little, if any, single-strand breakage of DNA occurred. Both thymidine starvation and nalidixic-acid (20 mug/ml) treatment resulted in viability loss and breakage of DNA. For these latter two inhibitors, the two events appeared to be associated because greater rates of both viability loss and DNA breakage were observed for nalidixic acid compared with thymidine starvation. However, viability loss need not be associated with extensive breakage of DNA as demonstrated with a temperature-sensitive DNA synthesis mutant; at 39 C, viability loss occurred at a high rate without significant DNA breakage. With the other agents, the amount of DNA breakage accumulated when a cell population has sustained an average of one lethal hit was estimated to be about 30 single-strand breaks per genome. Differences in chromosomal and episomal breakage rates were observed.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature.     

Specific Inhibition of Mammalian Ribonucleic Acid C-Type Virus Deoxyribonucleic Acid Polymerases by Rat Antisera

Inhibition of the ribonucleic acid (RNA)- and deoxyribonucleic acid (DNA)-dependent DNA polymerase activities of mammalian C-type viruses was obtained with sera from rats bearing murine leukemia virus-induced transplant tumors. Polymerase activities of nonmammalian (viper) C-type virus and murine mammary tumor virus were not inhibited by such sera nor by serum from a rat immunized with the DNA polymerase of feline leukemia virus purified by isoelectric focusing. The latter serum appeared to inhibit preferentially the DNA-dependent DNA polymerase activity of mammalian C-type viruses showing no inhibition of RNA-dependent DNA synthesis.     

Deoxyribonucleic Acid Synthesis in Synchronously Growing Mycoplasma gallisepticum

Early log-phase cells of Mycoplasma gallisepticum A5969 were synchronized by holding in Eagle minimal essential medium (MEM) for 2 h. When transferred out of MEM into tryptose medium, the cells exhibited synchronous growth. Deoxyribonucleic acid (DNA) synthesis proceeded continuously during this growth but stopped during the period of cell division. One round of DNA replication was observed per cell doubling, and a unique region of DNA was found to be permanently bound to the membrane.     

Mutant of Vesicular Stomatitis Virus Which Allows Deoxyribonucleic Acid Synthesis and Division in Cells Synthesizing Viral Ribonucleic Acid

Some temperature-sensitive mutants of vesicular stomatitis virus were tested for their ability to block the initiation of deoxyribonucleic acid (DNA) synthesis and division in serum-stimulated hamster embryo fibroblasts at the nonpermissive temperature. Although the parental strain blocked these processes, one particular mutant allowed essentially normal DNA synthesis and division. By autoradiography, it was shown that individual cells infected with this mutant could synthesize viral ribonucleic acid and at the same time initiate DNA synthesis and divide. Cells infected with such conditional defective mutants appear to be suitable for studies on the effects of persistent viral infections on molecular and cellular functions in proliferating cell populations.     

Polyamines in the Synthesis of Bacteriophage Deoxyribonucleic Acid. I. Lack of Dependence of Polyamine Synthesis on Bacteriophage Deoxyribonucleic Acid Synthesis

To determine whether polyamine synthesis is dependent on deoxyribonucleic acid (DNA) synthesis, polyamine levels were estimated after infection of bacterial cells with ultraviolet-irradiated T4 or T4 am N 122, a DNA-negative mutant. Although phage DNA accumulation was restricted to various degrees in comparison to cells infected with T4D, nearly commensurate levels of putrescine and spermidine synthesis were observed after infection, regardless of the rate of phage DNA synthesis. We conclude from these data that polyamine synthesis after infection is independent of phage DNA synthesis.     

Integration of Simian Virus 40 Deoxyribonucleic Acid into the Deoxyribonucleic Acid of Permissive Monkey Kidney Cells

Integration of simian virus 40 (SV40) deoxyribonucleic acid (DNA) into cellular DNA occurred when permissive African green monkey kidney (CV-1) cells were infected at a low multiplicity of SV40 in the presence of cytosine arabinoside.     

Relationship Between Protein Synthesis and Viral Deoxyribonucleic Acid Synthesis

Evidence is presented that poxvirus deoxyribonucleic acid (DNA) synthesis required concurrent protein synthesis. The protein requirement in question can be distinguished from viral-induced thymidine kinase and DNA polymerase by virture of the instability of its messenger ribonucleic acid and its stoichiometric rather than catalytic relation to DNA synthesis. The protein(s) required did accumulate in the presence of fluorodeoxyuridine, an inhibitor of DNA synthesis, and, thus, appeared to be an "early" poxvirus function. The protein(s) was stable since it did function several hours after its synthesis had been terminated by puromycin. Two possible roles for such a protein requirement are discussed.     

Integration of Simian Virus 40 Deoxyribonucleic Acid into the Deoxyribonucleic Acid of Primary Infected Chinese Hamster Cells

Simian virus 40 deoxyribonucleic acid (DNA) became associated in an alkaline-stable form with the DNA of Chinese hamster embryo cells at 15 to 20 hr post-infection, at the time when cell DNA synthesis and T antigen were induced. The integration process was not inhibited by d-arabinosyl cytosine and was only partially inhibited by cycloheximide.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.