Temperature-Dependent Transformation of Cells Infected with a Mutant of Bryan Rous Sarcoma Virus

Chick embryo cells infected with a mutant (Ta) of the Bryan high-titer strain of Rous sarcoma virus (RSV-BH) are morphologically transformed at 36 C but appear similar to uninfected cells at 41 C. When cells infected with RSV-BH-Ta are switched from 41 to 36 C, morphological changes characteristic of transformation are observable within 10 min. The transformation is reversible; cells shifted from 36 to 41 C have been observed to lose their transformed morphology within 1 hr. The transformation after a shift in temperature is unaffected by inhibition of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein synthesis, demonstrating that the proteins involved in the morphological change are already present. Transformed cells infected with RSV-BH or RSV-BH-Ta take up hexose and synthesize hyaluronic acid at higher rates than uninfected cells or RSV-BH-Ta-infected cells grown at 41 C. However, inhibition of either protein or RNA synthesis, but not DNA synthesis, prevented the induction of increased hexose uptake and hyaluronic acid synthesis after a shift of RSV-BH-Ta-infected cells from 41 to 36 C. Therefore, these biochemical changes are secondary to a more basic change responsible for morphological transformation.     

Macromolecule synthesis in yeast spheroplasts

Conditions have been established for the preparation of spheroplasts of Saccharomyces cerevisiae which are able to increase their net content of protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), several-fold upon incubation in a medium stabilized with 1 m sorbitol. The rate of RNA and protein synthesis in the spheroplasts is nearly the same as that occurring in whole cells incubated under the same conditions; DNA synthesis occurs at about half the whole cell rate. The spheroplasts synthesize transfer RNA and ribosomal RNA. The newly synthesized ribosomal RNA is incorporated into ribosomes and polysomes. The polysomes are the site of protein synthesis in these spheroplasts. Greater than 90% of the total RNA can be solubilized by treatment of the spheroplasts with sodium dodecyl sulfate or sodium deoxycholate. These spheroplast preparations appear to be a useful subject for the study of RNA metabolism in yeast.     

Effects of large and small T antigens on DNA synthesis and cell division in simian virus 40-transformed BALB/c 3T3 cells.

The roles of the large T and small t antigens of simian virus 40 in cellular DNA synthesis and cell division were analyzed in BALB/c 3T3 mouse cells transformed by wild-type, temperature-sensitive A (tsA), or tsA-deletion (tsA/dl) double mutants. Assessment of DNA replication and cell cycle distribution by radioautography of [3H]thymidine-labeled nuclei and by flow microfluorimetry indicate that tsA transformants do not synthesize DNA or divide at the restrictive temperature to the same extent as they do at the permissive temperature or as wild-type transformants do at the restrictive temperature. This confirms earlier studies suggesting that large T induces DNA synthesis and mitosis in transformed cells. Inhibition of replication in tsA transformants at the restrictive temperature, however, is not complete. Some residual cell division does occur but is in large part offset by cell detachment and death. This failure to revert completely to the parental 3T3 phenotype, as indicated by residual cell cycling at the restrictive temperature, was also observed in cells transformed by tsA/dl double mutants which, in addition to producing a ts large T, make no small t protein. Small t, therefore, does not appear to be responsible for the residual cell cycling and plays no demonstrable role in the induction of DNA synthesis or cell division in stably transformed BALB/c 3T3 cells. Comparison of cell cycling in tsA and tsA/dl transformants, normal 3T3 cells, and a transformation revertant suggests that the failure of tsA transformants to revert completely may be due to leakiness of the tsA mutation as well as to a permanent cellular alteration induced during viral transformation. Finally, analysis of cells transformed by tsA/dl double mutants indicates that small t is not required for full expression of growth properties characteristic of transformed cells.     

Macromolecular syntheses during biosynthesis of prodigiosin by Serratia marcescens.

Amino acids that were utilized as sole sources of carbon and nitrogen for growth of Serratia marcescens Nima resulted in biosynthesis of prodigiosin in non-proliferating bacteria. Addition of alanine, proline, or histidine to non-proliferating cells incubated at 27 C increased the rate of protein synthesis and also caused biosynthesis of prodigiosin. No increase in the rate of protein synthesis was observed upon the addition of amino acids that did not stimulate prodigiosin biosynthesis. Increased rates of synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) (a small amount) also occurred after addition of amino acids that resulted in biosynthesis of prodigiosin. After incubation of 24 h, the total amount of protein in suspensions of bacteria to which alanine or proline was added increased 67 and 98%, respectively. Total amounts of DNA and of RNA also increased before synthesis of prodigiosin. The amounts of these macromolecules did not increase after addition of amino acids that did not induce biosynthesis of progidiosin. However, macromolecular synthesis was not related only to prodigiosin biosynthesis because the rates of DNA, RNA, and protein synthesis also increased in suspensions of bacteria incubated with proline at 39 C, at which temperature no prodigiosin was synthesized. The quantities of DNA, RNA, and protein synthesized were lower in non-proliferating cells than in growing cells. The data indicated that amino acids causing biosynthesis of prodigiosin in non-proliferating cells must be metabolized and serve as sources of carbon and of nitrogen for synthesis of macromolecules and intermediates. Prodigiosin was synthesized secondarily to these primary metabolic events.     

Quantitative Changes in Unscheduled DNA Synthesis in Rat Muscle Cells after Differentiation

THE incorporation of tritiated thymidine (³H-thymidine) into cells not engaged in normal DNA replication has been called unscheduled DNA synthesis¹. The phenomenon has been observed after X-irradiation¹, ultraviolet irradiation² and after exposure to the monofunctional alkylating agent methyl methane sulphonate³ (MMS) and other carcinogens⁴. In all published reports the cells showing unscheduled DNA synthesis had retained their proliferative capacity (and hence at least their potential ability to synthesize DNA). We have investigated whether differentiated cells—that is, cells which presumably will never have to initiate normal DNA synthesis—are still capable of unscheduled DNA synthesis. We used multinucleated rat muscle cells in vitro. Myotubes have been found to form by fusion of separate, mononucleated cells^5,6, the nuclei of which no longer synthesize DNA. YalTe and Gershon⁷ have shown that such cells can reinitiate DNA synthesis after viral infection. They found it necessary, however, for fusion to continue during viral infection; in the absence of further fusion no new DNA synthesis was observed. The trigger for DNA synthesis after viral infection must therefore have come from cells which had been transformed before differentiation and fusion. This left open the question of whether differentiated cells could initiate DNA synthesis in the absence of trigger from transformed cells.     

Enrichment for temperature-sensitive and auxotrophic mutants in Saccharomyces cerevisiae by tritium suicide

Tritium suicide is shown to be an efficient technique for mutant enrichment in Saccharomyces cerevisiae. Decays from incorporated [5-³H]uridine and tritiated amino acids proved equally effective in inducing suicide; in cultures labeled to a specific activity of 50 dpm/cell, the viability fell to 2% after 12 days' storage at 4°. Mutagenized cultures were labeled with either [5-³H]uridine or a mixture of tritiated amino acids under conditions where auxotrophic mutants and temperature-sensitive mutants in RNA or protein synthesis would not incorporate a significant amount of the tritiated percursor. When survival fell to 2%, the percentages of both auxotrophic and temperature-sensitive mutants were 10-fold higher among these survivors than in the original mutagenized culture, regardless of the radioactive precursor used.     

Glioma proliferation modulated in vitro by isothermal radiofrequency radiation exposure

Isothermal (37 +/- 0.2 degrees C) exposure of glioma cells (LN71) for 2 h to 27 or 2450 MHz continuous-wave radiofrequency (RF) radiation in vitro modulated the rates of DNA and RNA synthesis 1, 3, and 5 days after exposure. The alterations indicate effects on cell proliferation and were not caused by RF-induced cell heating. The dose response for either frequency of the radiation was biphasic. Exposure to specific absorption rates (SARs) of 50 W/kg or less stimulated incorporation rates of tritiated thymidine (3H-TdR) and tritiated uridine (3H-UdR), whereas higher SARs suppressed DNA and RNA synthesis. Statistically significant time-dependent alterations were detected for up to 5 days postexposure, suggesting a kinetic cellular response to RF radiation and the possibility of cumulative effects on cell proliferation. General mechanisms of effects are discussed.     

Nucleic Acid and Protein Synthesis in Reconstituted Lyophilized Escherichia coli Exposed to Air

S ummary : Escherichia coli strain BT^-, freeze dried from distilled water, loses viability by exposure to air in the dry state, and decay curves of such bacteria were determined. The amounts of tritiated thymidine incorporated in the DNA of such 'dead', rehydrated bacteria are compatible with an assumption that the synthesis of DNA proceeds till the completion of its replication cycle, but not beyond. Induction of β-galactosidase served as an indicator of RNA and protein synthesis in the dead bacteria. Such induction occurred after a short lag after which it proceeded at a rate similar to that observed in lyophilized, untreated bacteria. The loss of viability of these bacteria cannot be accounted for by defects in the production of DNA or RNA and protein synthesis; it is most probably due to damage to the DNA initiation point.     

Temperature-Sensitive Mutants of a Chinese Hamster Cell Line. I. Selection of Clones with Defective Macromolecular Biosynthesis

Temperature-sensitive clones have been selected from a mutagenized culture of Chinese hamster lung cells by a procedure involving bromodeoxyuridine (BrdU) incorporation and irradiation with black light. The selection procedure used in these studies was adapted from methods developed by others to yield mutants that cease DNA replication within a short time after they are transferred to nonpermissive temperature. After mutagenesis with ethyl methanosulfonate ten clones survived the selection procedure. Three of the clones (mutants) were temperature-sensitive as measured by growth properties. Two mutants ceased DNA synthesis within six hours of being shifted to 39degrees and the third mutant continued to synthesize DNA at nonpermissive temperature at a reduced rate for at least 24 hours. Thus, all three mutants survived the selection procedure for understandable reasons, since each was unable to incorporate sufficient BrdU at 39degrees to lethally protosensitize its DNA during the standard exposure period. The two mutants that cease DNA synthesis at high temperature (clones 115-47 and 115-53) also stop incorporating radioactive amino acids and uridine within six hours at 39degrees. Their complex phenotype, i.e. defective DNA, RNA and protein biosynthesis, is reversible. When these mutants were returned to 33 degrees after 8 hours at 39 degrees, both resumed DNA synthesis immediately (less than 1 hour). Reversal of defective DNA synthesis in both mutants were sensitive to drugs that inhibit protein biosynthesis specifically. Those same drugs, as well as toxic amino acids analogs, also effected a striking mutant phenocopy in wild-type cells. The phenocopy produced by amino acid analogs that are incorporated into mammalian proteins suggested that one or more proteins must be synthesized continuously to support mammalian cells engaged in programmed DNA replication.     

Differential in vitro growth properties of cells transformed by DNA and RNA tumor viruses

Mammalian cells transformed by DNA and RNA tumor viruses are shown to display consistently different growth properties. All SV40, adenovirus type 7 and polyoma virus (DNA viruses) transformed cells propagated to high densities. The same cells transformed instead by RNA viruses: MSV strain Kirsten (MSV-Ki) or MSV strain Maloney (MSV-M) grew to densities which were consistently lower than DNA virus-transformed cells but greater than that of untransformed cells. The capacity to synthesize DNA at increasing densities also differentiated the RNA and DNA virus-transformed cells. As growing cultures of untransformed cells neared saturation density, the fraction of cells synthesizing DNA was minimal. The RNA virus-transformed cells were also contact-inhibited but at a significantly higher density. In contrast the DNA virus-transformed cells propagated to still greater densities and continued DNA synthesis at a high rate even at very high densities. Therefore the DNA virus-transformed cells truly are not contact inhibited. It is suggested that the capacity to continue DNA synthesis at high densities explains the attainment of much greater densities by DNA virus-transformed cells. There were no clear-cut differences in the ability to form colonies in agar, although a few of the RNA virus-transformed lines could not be propagated in semi-solid medium. These results may be explained as a persistence of the capacity of DNA tumor viruses to stimulate host cell DNA synthesis.     

Relation of Macromolecular Synthesis in Streptococci to Efficiency of Transformation by Markers of Homospecific and Heterospecific Origin

In previous studies with Streptococcus sanguis and S. pneumoniae as recipients and donors of transforming deoxyribonucleic acid (DNA), it was found that heating recipients just prior to exposure to DNA caused an increase in the number of transformants induced by heterospecific DNA relative to that induced by homospecific DNA. In the present studies, S. sanguis recipients were found to recover from this effect of heat (48 C, 15 min) when incubated at 37 C before exposure to DNA. Inhibitors of nucleic acid synthesis, such as rifampin, 5-fluorodeoxyuridine, actinomycin, and p-hydroxyphenylazo-uracil, but not inhibitors of protein synthesis, such as chloramphenicol and erythromycin, prevented recovery from the effect of heat. Inhibitors of nucleic acid synthesis caused changes in unheated cells similar to those observed with heat treatment; these changes included increased transformability by genetically hybrid DNA and by low-efficiency markers in homospecific DNA. The effect of a combination of heat and inhibitors on transformation by heterospecific DNA was greater than when single treatments were used. The most effective inhibitor used alone was rifampin: in treated recipient cells, the yield of transformants produced by a given amount of irreversibly bound heterospecific DNA was increased without a significant change in the yield of transformants produced by bound homospecific DNA. A cell being doubly transformed by homospecific and heterospecific DNA was enhanced specifically in its transformability with the latter as a consequence of rifampin treatment. Treatment with rifampin also increased co-transformation by linked heterospecific markers. The period during which recipient cells were sensitive to the effects induced by rifampin and fluorodeoxyuridine lasted from 10 to 20 min after DNA uptake.     

Synthesis of Protein, Ribonucleic Acid, and Ribosomes by Individual Bacterial Cells in Balanced Growth

Relative rates of protein synthesis in individual cells were determined by allowing random populations to incorporate tritiated leucine for very short periods (pulses) and then examining autoradiographs of these cells to assess the amount of incorporation (grains per cell) as a function of cell size. Relative rates of ribonucleic acid (RNA) synthesis were determined in the same way by using tritiated uracil. Unless the uracil pulse was very short (less than 1/20 generation), the RNA labeled during the pulse was predominantly ribosomal. The rate of protein synthesis in individual cells is directly proportional to cell size. The rate of RNA synthesis also increases linearly with size in larger cells, but there appears to be a slight delay in RNA synthesis immediately after cell division. Total cellular content of protein, RNA, and ribosomes is directly proportional to cell size. Thus, we conclude that, in individual cells during the cell cycle (i) the average rate of protein synthesis per ribosome is constant and (ii) the increase in macromolecular mass of the cell is exponential with age.     

Hydroxyurea synchronization of bovine fetal spleen cells

Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.     

Hydroxyurea synchoronization of bovine fetal spleen cells.

Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.     

Construction and analysis of recombinant DNAs containing a structural gene for rat prolactin.

Poly A-containing RNA enriched in prolactin-coding sequences was isolated from female rate pituitaries after induction with diethylstilbesterol. Double stranded cDNA was synthesized from this RNA and inserted into plasmid pBR322 at the Pst I site via the poly(dG):polyy(dC) tailing method. E. coli was transformed with this DNA and the recombinant plasmid in one of the transformants characterized in detail. About half of its 900 base pair cDNA insert was sequenced. The DNA sequence is consistent with most of the reported amino acid sequence of rat preprolactin. In addition, the recombinant plasmids in two of the other transformants appear to contain growth hormone coding sequences.     

Hypoxanthine-xanthine oxidase-related defect in polypeptide chain initiation by endothelium

After exposure to O2 intermediates generated by the hypoxanthine-xanthine oxidase system (HX-XO), the rate of [3H]phenylalanine incorporation into total proteins in cultured endothelial cells was markedly reduced. This reduction, which was prevented by catalase, could not be explained by 1) changes in amino acid pools, 2) increased rate of degradation of newly synthesized proteins, 3) impaired poly(A)+ RNA synthesis and efficiency, 4) decreased rate of amino acylation. On the other hand, the increase in the monoribosome-to-polyribosome ratio suggested that translation was affected at the level of chain initiation. Further analysis indicated that 40S initiation complex formation was normal, whereas the assembly of 80S initiation complex was inhibited. Results from reconstitution experiments showed that both normal and treated ribosomes could support normal protein synthesis in the presence of normal initiation factors (IFs). In contrast, IFs from HX-XO lysates did not support normal protein synthesis with ribosomes from either source. Thus, the effect of XO treatment on protein synthesis appears to be an initiation defect related to decreased IF activity and/or availability.     

Macromolecular Synthesis in Saccharomyces cerevisiae in Different Growth Media

Synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein was determined in Saccharomyces cerevisiae during amino acid and pyrimidine starvation and during shift-up and shift-down conditions. During amino acid starvation, cell mass, cell number, and RNA continued to increase for varying periods. During amino acid and pyrimidine starvation, cell mass and RNA showed little increase, whereas total DNA increased 11 to 17%. After a shift from broth medium to a minimal defined medium, increase in RNA and protein remained at the preshift rate before assuming a lower rate. DNA increase remained at an intermediate rate during shift-down, and then dropped to a low rate. During shift-up from minimal to broth medium, increase in cell number, protein, and DNA showed varying lag periods before increasing to the new rate characteristic of broth medium; each of these quantities exhibited a step sometime in the first 2 hr after transfer to rich medium, suggesting a partial synchronous division. Immediately after shift-up, RNA synthesis assumed a high rate, and then dropped to a rate characteristic of growth in the rich medium after about 1 hr.     

A Comparison of the Killing of Cultured Mammalian Cells Induced by Decay of Incorporated Tritiated Molecules at -196°C

The killing efficiency of tritium disintegrations in frozen mammalian cells labeled with tritiated uridine, histidine, and lysine was compared with the killing efficiency of incorporated tritiated thymidine. In each case, the distribution of tritium in the cells was determined by chemical fractionation as well as by radio-autography. Of all tritium disintegrations, by far the most effective were those occurring in DNA molecules within frozen cells; such incorporated tritium has a killing efficiency of 0.006. When cells were incubated with tritiated uridine for 10 min to label nuclear RNA, the killing efficiency was 0.0015. When the cells were pulse labeled with tritiated uridine and permitted to grow in nonradioactive media for 10 hr before freezing in order to incorporate tritium into cytoplasmic RNA, the killing efficiency was reduced to 0.0010. The results suggest that decay of tritium in nuclear RNA is more effective than that in cytoplasmic RNA. When the cells were labeled with tritiated histidine or lysine for 30 min, tritium atoms were found mainly in the acid soluble rather than in the protein fraction and the killing efficiency in each case was approximately 0.0007. The results of these suicide experiments indicate that the killing efficiency of tritium disintegrations depends on where tritium is located within the cells. Tritium disintegrations in the nucleus are more effective in killing the cell than that in cytoplasm; and tritium disintegrations on DNA in the nucleus is more effective in killing the cell than that of nuclear RNA.     

Mode of Action of Myxin on Escherichia coli

The effect of the new antibiotic, myxin, on the syntheses of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in Escherichia coli (strains B and 15T(-)) was examined. Within 7 min of the addition of myxin at 5 mug/ml, the synthesis of new bacterial DNA was almost completely inhibited. This was followed by an extensive degradation of the pre-existing DNA to an acid-soluble form. All of the evidence indicated that the primary effect of the antibiotic was on cellular DNA. The synthesis of RNA was completely inhibited after 15 min of exposure to myxin (5 mug/ml), and the synthesis of protein was markedly reduced after 30 min. There was no measurable breakdown of either RNA or protein in the myxin-treated cells. A marked stimulation of (14)C-uracil incorporation was found in the presence of myxin in 15T(-) cells only. This did not result from an increased rate of RNA synthesis but was due to an increase in the proportion of exogenous uracil, relative to endogenous uracil, incorporated into cellular RNA. This probably reflected a partial inhibition of the biosynthesis of uridine monophosphate from orotate. At 4.5 mug of myxin per ml and with 0.8 x 10(8) cells per ml, 50% of the antibiotic was reduced in 15 min from the biologically active oxidized form to the biologically inactive state. Under these conditions, a maximum of 0.6% (27 mumug/ml) of the myxin was retained in the cells.     

Macromolecule Synthesis in Temperature-sensitive Mutants of Yeast

Approximately 400 temperature-sensitive mutants of Saccharomyces cerevisiae were isolated. The mutants were unable to form colonies on enriched media at 36 C, but grew normally, or nearly so, at 23 C. The mutants were tested for loss of viability, change in morphology, increase in cell number, and the ability to synthesize protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) after a shift from 23 to 36 C. Mutations were found which resulted in a preferential loss of ability to carry out protein synthesis, RNA synthesis, DNA synthesis, cell division, or cell-wall formation. Diploid cells heterozygous for the temperature-sensitive mutations were constructed and tested for their ability to form colonies at 36 C. Four mutations dominant to their wild-type allele were identified.