Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis

Tribby, Ilse I. E. (University of Chicago, Chicago, Ill.), and James W. Moulder. Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis. J. Bacteriol. 91:2362-2367. 1966.-Uninfected L cells and the meningopneumonitis agent propagated in L cells utilized exogenous adenine, guanine, and their ribonucleosides and deoxyribonucleosides for synthesis of both deoxyribonucleic acid (DNA) and ribonucleic acid. Cytosine, cytidine, and uridine were also incorporated into the nucleic acids of both host and parasite. L cells and the meningopneumonitis agent incorporated uracil, thymine, and deoxyuridine very poorly. L cells utilized thymidine and deoxycytidine almost exclusively for DNA synthesis, but the meningopneumonitis agent did not incorporate these nucleosides at all. Since the L cell had previously been shown to convert added thymidine to its nucleotides, mainly the triphosphate, it was concluded that the meningopneumonitis agent can utilize neither the thymidine nor the thymidine nucleotides of the L-cell pool, and that it can probably synthesize the thymidine triphosphate needed for DNA synthesis from the uridine of the L-cell pool.     

Effect of Infection with the Meningopneumonitis Agent on Deoxyribonucleic Acid and Protein Synthesis by Its L-Cell Host

Cycloheximide, which had already been shown to inhibit protein synthesis in Earle's L cells (mouse fibroblasts) without having any effect on the multiplication or protein synthesis in Chlamydia psittaci (strain meningopneumonitis) infecting these host cells, also caused greater than 90% inhibition of deoxyribonucleic acid (DNA) synthesis in L cells after a 3-hr exposure to the drug. L cells infected with the meningopneumonitis agent and treated with cycloheximide were used to follow meningopneumonitis-specific DNA synthesis during intracellular growth of the parasite. The rate at which labeled precursors were incorporated into parasite DNA doubled every 2 hr. The effect of meningopneumonitis infection on L-cell DNA and protein synthesis was investigated in logarithmically growing and in stationary-phase (nondividing) populations of L cells. Host-specific DNA and protein synthesis appeared to be inhibited in infected L cells when compared with logarithmically growing control cells, whereas no inhibition was apparent when the comparison was made with stationary-phase control cells. The maximal amount of protein and DNA synthesis that occurred in meningopneumonitis-infected L cells was equal to the amount of DNA and protein synthesized in logarithmically growing, uninfected L cells. A possible explanation of these results is given.     

Separation of Protein Synthesis in Meningopneumonitis Agent from That in L Cells by Differential Susceptibility to Cycloheximide

Cycloheximide had no effect on multiplication of the meningopneumonitis agent in L cells in concentrations which eliminated over 90% of the protein synthesis in the host cells. Infected L cells treated with cycloheximide, however, incorporated labeled amino acids into the trichloroacetic acid-insoluble fraction. This incorporation was attributed to the biosynthetic activity of the meningopneumonitis agent. Synthesis of meningopneumonitis protein was abolished by chloramphenicol and chlortetracycline, inhibitors of bacterial protein synthesis, at concentrations which did not inhibit protein synthesis in L cells. Protein synthesis in the meningopneumonitis agent was sustained at a high rate when the host cells remained viable and declined as the L cells died. Overall host protein synthesis was not inhibited by multiplication of the meningopneumonitis agent.     

Changes in macromolecular synthesis in Xanthomonas oryzae infected with bacteriophage XP-12.

Phage XP-12, which has complete substitution of the cytosine residues in its DNA with 5-methylcytosine residues, was shown to inhibit incorporation of uracil into host DNA and RNA during the latent period. This apparent inhibition of host macromolecular synthesis was not accompanied by extensive degradation of the host chromosome. Phage DNA synthesis in infected cells occurred at a faster rate than host DNA synthesis in analogous uninfected cells. However, phage DNA synthesis could not be accurately monitored by incorporation of [methyl-3H]thymidine into DNA because, soon after infection, there was a marked inhibition of utilization of exogenous thymidine for DNA synthesis. Phage infection conferred upon a thymine auxotrophic host the ability to synthesize thymine nucleotides for phage DNA synthesis. It is suggested that a phage-induced thymidylate synthetase activity is partially responsible for the inhibition of thymidine incorporation.     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

Autoradiographic study of deoxyribonucleic acid synthesis in L cells infected with the agent of meningopneumonitis

Moore, Dorothy E. (University of Chicago, Chicago, Ill.), and James W. Moulder. Autoradiographic study of deoxyribonucleic acid synthesis in L cells infected with the agent of meningopneumonitis. J. Bacteriol. 92:1128-1132. 1966.-L cells infected with the agent of meningopneumonitis were labeled with H(3)-cytidine at 5-hr intervals after infection, and cell samples were fixed every 5 hr after labeling. These preparations were then digested with ribonuclease, stained by the Feulgen procedure, and examined by autoradiography. Labeled meningopneumonitis inclusions were first seen 15 hr after infection. Deoxyribonucleic acid (DNA) was synthesized in both L-cell nuclei and meningopneumonitis agent for as long as 40 hr after infection. Nuclear DNA synthesis was unaffected until 25 hr after infection, at which time synthesis of agent DNA reached its peak. After 25 hr, both meningopneumonitis and L cell DNA synthesis declined.     

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.     

Ribonucleotide Reductase Activity of Synchronized KB Cells Infected with Herpes Simplex Virus

The replication of herpes simplex virus (HSV) is unimpeded in KB cells which have been blocked in their capacity to synthesize deoxyribonucleic acid (DNA) by high levels of thymidine (TdR). Studies showed that the presence of excess TdR did not prevent host or viral DNA replication in HSV-infected cells. In fact, more cellular DNA was synthesized in infected TdR-blocked cells than in uninfected TdR-blocked cells. This implies that the event which relieved the TdR block was not specific for viral DNA synthesis but allowed some cellular DNA synthesis to occur. These results suggested that HSV has a means to insure a pool of deoxycytidylate derivatives for DNA replication in the presence of excess TdR. We postulated that a viral-induced ribonucleotide reductase was present in the cell after infection which was not inhibited by thymidine triphosphate (TTP). Accordingly, comparable studies of the ribonucleotide reductase found in infected and uninfected KB cells were made. We established conditions that would permit the study of viral-induced enzymes in logarithmically growing KB cells. A twofold stimulation in reductase activity was observed by 3 hr after HSV-infection. Reductase activity in extracts taken from infected cells was less sensitive to inhibition by exogenous (TTP) than the enzyme activity present in uninfected cells. In fact, the enzyme extracted from infected cells functioned at 60% capacity even in the presence of 2 mm TTP. These results support the idea that a viral-induced ribonucleotide reductase is present after HSV infection of KB cells and that this enzyme is relatively insensitive to inhibition by exogenous TTP.     

Regulation of mitosis onset and thymidine kinase activity during the cell cycle of Physarum polycephalum plasmodia: effect of hydroxyurea

The effects of hydroxyurea have been investigated on three events of the cell cycle, S-phase, mitosis, and the cyclic synthesis of thymidine kinase, in the synchronous plasmodium of the myxomycete Physarum. DNA synthesis was slowed down with limited action on other macromolecular syntheses and any increase of thymidine kinase that had already been triggered was indistinguishable from that of the control. When DNA synthesis was inhibited, the onset of the following cyclic increase of thymidine kinase synthesis occurred at the same time as in the control, but mitosis was delayed in a very early prophase stage. The arrest of thymidine kinase synthesis occurred after completion of the delayed mitosis. All these effects were suppressed when the action of hydroxyurea was prevented by the addition, to the medium, of the four deoxyribonucleosides. These observations show that (1). The blockage of S-phase does not prevent the nuclei from entering a very early prophase stage but does prevent them from proceeding through metaphase. (2) The transient blockage of DNA synthesis does not perturb the normal timing of the triggering of thymidine kinase synthesis. (3) The signal which triggers the arrest of thymidine kinase synthesis is postmitotic but does not require extensive DNA synthesis. The effect of hydroxyurea is not limited to an inhibition of S-phase. The blockage of DNA replication also led to the dissociation of the normal coordination between two other events of the cell cycle, mitosis and thymidine kinase synthesis. This observation could have strong implications in cell synchronization with chemical agents.     

Consequences of herpes simplex virus type 2 and human cell interaction at supraoptimal temperatures.

The consequences of herpes simplex virus type 2 (HSV-2) and human embryonic fibroblast cell interaction at different temperatures (37, 40, and 42 degrees C) were investigated. Incubation at 37 or 40 degrees C was permissive for HSV-2 inhibition of host DNA synthesis, induction of virus-specific DNA replication, and infectious virus production. The amount of [methyl-3H]thymidine incorporated into viral DNA and the final yield of new infectious virus were significantly reduced at 40 degrees C compared to 37 degrees C. At 42 degrees C, detectable virus-specific DNA synthesis was totally blocked. Maximum stimulation of host cell DNA synthesis at 42 degrees C was measured after a multiplicity of infection of 0.5 to 1.0 PFU/cell. By autoradiography, data indicated that HSV-2 stimulates host cell chromosomal DNA synthesis. Stimulation of thymidine kinase activity with thermostability properties in common with a virus enzyme was detected during the first 24 h of infection at 42 degrees C, after 24 h the enhanced thymidine kinase activity had properties in common with host cell isozymes. The data obtained during this investigation indicated that stimulation of host cell DNA synthesis does not require viral DNA synthesis.     

Expression of the Viral Thymidine Kinase Gene in Herpes Simplex Virus-Transformed L Cells

In these studies, the expression of thymidine kinase (TK) in normal and herpes simplex virus (HSV)-transformed L cells has been compared. In asynchronously dividing cultures of L cells, the TK activity rose and declined rapidly and coordinately with DNA synthesis. When net cell increase stopped, TK activity was at a minimum. In contrast, TK activity of HSV-transformed cells remained at a minimum during rapid DNA synthesis and gradually increased as the rate of DNA synthesis decreased. When net cell increase stopped, TK activity was at a maximum. In synchronous cultures of L cells, TK activity rose and fell coordinately with the rate of DNA synthesis. In synchronous cultures of HSV-transformed cells, no increase in TK activity was observed during the period of rapid DNA synthesis, i.e., the S phase. These findings indicated that the viral TK gene in HSV-transformed cells was not placed under the control of the cellular mechanisms which normally modulate the host cell TK gene. Lytic infection of HSV-transformed cells with a TK(-) mutant of HSV-1 induced a four-to fivefold increase in viral TK. The TK of HSV-1 was induced in the HSV-1-transformed cells and HSV-2 in the HSV-2-transformed cells by this TK(-) mutant. The same infection of normal L cells decreased the cellular TK activity by 80%. This stimulation, rather than inhibition, suggest that the viral gene in HSV-transformed cells retain some of its original viral characteristics.     

Stability of the Nucleic Acids of L Cells After Infection with the Meningopneumonitis Agent

The stability of host nucleic acids in L cells infected with Chlamydia psittaci (strain meningopneumonitis) was studied. The L cells were prelabeled with either (32)P-orthophosphate, (3)H-uridine, or (3)H-thymidine. After infection, the redistribution of each label among the different fractions of host and parasite was quantitatively determined and compared. There were no signs of accelerated degradation of host nucleic acid as the consequence of meningopneumonitis infection. Comparison of the specific activities of the meningopneumonitis nucleic acids with that of the acid-soluble fraction of host cell cytoplasm suggested that the major source of precursors for parasite nucleic acid synthesis was the common cytoplasmic pool of the infected host cell.     

Acquisition of thymidylate by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

The pathway for the acquisition of thymidylate in the obligate bacterial parasite Rickettsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3H]thymidine added to the culture. In the thymidine kinase-negative host cells, the label available to the rickettsiae in the host cell cytoplasm would have been thymidine, and in the thymidine kinase-positive host cells, it would have been both thymidine and TMP. Further support for the inability to utilize thymidine was the lack of thymidine kinase activity in extracts of R. prowazekii. However, [3H]uridine incorporation into the DNA of R. prowazekii was demonstrable (973 +/- 57 dpm/3 x 10(8) rickettsiae). This labeling of rickettsial DNA suggests the transport of uracil, uridine, uridine phosphates (UXP), or 2'-deoxyuridine phosphates, the conversion of the labeled precursor to thymidylate, and subsequent incorporation into DNA. This is supported by the demonstration of thymidylate synthase activity in extracts of R. prowazekii. The enzyme was determined to have a specific activity of 310 +/- 40 pmol/min/mg of protein and was inhibited greater than or equal to 70% by 5-fluoro-dUMP. The inability of R. prowazekii to utilize uracil was suggested by undetectable uracil phosphoribosyltransferase activity and by its inability to grow (less than 10% of control) in a uridine-starved mutant cell line (Urd-A) supplemented with 50 microM to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 microM uridine (117% of control). However, no measurable uridine kinase activity could be measured in extracts of R. prowazekii. Normal rickettsial growth (92% of control) was observed when the host cell was blocked with thymidine so that the host cell's dUXP pool was depressed to a level inadequate for growth and DNA synthesis in the host cell. Taken together, these data strongly suggest that rickettsiae transport UXP from the host cell's cytoplasm and that they synthesize TTP from UXP.     

The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation

The activities throughout the cell cycle of thymidine kinase (EC 2.7.1.21), dihydrothymine dehydrogenase (EC 1.3.1.2), thymidine phosphorylase (EC 2.4.2.4) and dTMP phosphatase (EC 3.1.3.35) were measured in the Epstein-Barr virally transformed human B lymphocyte line LAZ-007. Cells were synchronised at different stages of the cell cycle using the technique of centrifugal elutriation. The degree of synchrony in each cycle-stage cell population was determined by flow microfluorimetric analysis of DNA content and by measurement of thymidine incorporation into DNA. The activity of the anabolic enzyme thymidine kinase was low in the G₁ phase cells, but increased many-fold during the S and G₂ phases, reaching a maximum after the peak of DNA synthesis, then decreasing in late G₂ + M phase. By contrast, the specific activities of the enzymes involved in thymidine and thymidylate catabolism, dihydrothymine dehydrogenase, thymidine phosphorylase and dTMP phosphatase remained essentially constant throughout the cell cycle, indicating that the fate of thymidine at different stages of the cell cycle is governed primarily by regulation of the level of the anabolic enzyme thymidine kinase and not by regulation of the levels of thymidine catabolising enzymes.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Thymidine kinase from Tetrahymena thermophila. Purification and immunological analysis

Thymidine kinase is an enzyme involved in DNA precursor metabolism and DNA replication. The synthesis of this enzyme is highly regulated during the cell cycle and the activity of the enzyme is also regulated by feedback inhibition. Genes encoding thymidine kinase have been extremely useful as selectable markers for introducing DNA into a number of cells. In order to study cell cycle regulation of thymidine kinase, the gene which encodes this enzyme, as well as aspects of DNA replication in the ciliated protozoan Tetrahymena thermophila, we have purified thymidine kinase from Tetrahymena. Two forms of thymidine kinase with native molecular masses of 59 kDa and 80 kDa have been identified and purified 6800- and 4600-fold, respectively. The 59-kDa enzyme, a homodimer of 30-kDa subunits, has been purified to near homogeneity and polyclonal antibodies have been raised against the 30-kDa subunit. Serological studies indicate that the two enzymes are antigenically distinct. The antibody against the Tetrahymena protein cross-reacts with a polypeptide in Chinese hamster ovary (CHO) cell extracts of 26 kDa which corresponds to the reported size of Chinese hamster thymidine kinase protein.     

Reversible G1 arrest of a human lung epithelial cell line by staurosporine.

Staurosporine, a microbial-derived protein kinase inhibitor, reversibly blocked non-synchronized, replicating cultures of the human lung epithelial cell line EKVX in the G1 phase of cell cycle and inhibited DNA synthesis and cell replication. The mechanism of this cell-cycle arrest in EKVX cells by staurosporine was likely due to inhibition of protein kinase C (PKC) because: 1) dose-dependent inhibition of DNA synthesis occurred at levels of staurosporine that inhibit phosphorylation of PKC substrate, 2) inhibition of DNA synthesis was also seen after treatment with another PKC inhibitor H7, but not by the chemically similar HA1004, which has a relative inhibitory specificity for cAMP-dependent protein kinase, and 3) the DNA synthesis was not inhibited by specific tyrosine kinase inhibitors Genistein and Lavendustin A at concentrations that inhibit tyrosine kinase activity. Removal of staurosporine from cell culture media resulted in a rebound in PKC activity and synchronized DNA synthesis in EKVX cultures. The reversibility of the inhibition was noted even after 5 days of treatment with staurosporine, and DNA synthesis remained synchronized for at least two rounds of cell replication after removal of staurosporine. Flow cytometric analysis confirmed that more than 90% of the cell population was blocked in the G1 phase after cells were treated with staurosporine for 24 h. Agents such as staurosporine may be useful for synchronizing cell populations to study cell-cycle specific biochemical events important for the regulation of cell replication in the EKVX cell line.