Polysomes in various phases of the cell cycle in synchronized plasmacytoma cells

The fraction of membrane-bound and free polysomes during different phases of the cell cycle was determined in suspension cultures of mouse plasmacytoma cells, synchronized by growth in isoleucine-deficient medium. The membrane-bound polysomes reached a maximum value (about 28 % of total polysomes) during the G 1 phase. In the S phase and G 2 phase only 18 to 20 % of the total polysomes were found to be membrane-bound. A high percentage of membrane-bound polysomes in the G 1 phase of the cell cycle agrees with the earlier finding that maximum synthesis of immunoglobulin light chain takes place on polysomes bound to the membrane in the G 1 phase of the cell cycle. The presence of a significant fraction of membrane-bound polysomes in the S and G 2 phases of the cell cycle would suggest that membrane-bound polysomes are also involved in the synthesis of proteins other than immunoglobulins.The ultrastructure of the cells during the various phases of the cell cycle was also studied. During the G 1 phase the surface of the majority of cells was distinguished by the presence of ruffles and slender villus-like cytoplasmic projections. In the S phase the surface contour tended to become smooth and even. These differences in the surface morphology may reflect the change in function which occurs during the transition from the G 1 to the S phase.     

Ribosomal RNA metabolism in synchronized plasmacytoma cells

Ribosome synthesis and metabolism has been studied in a plasmacytoma cell line synchronized by isoleucine deprivation. Ribosomal RNA (rRNA) was characterized by gel electrophoresis. The rate of ribosome synthesis (as measured by the appearance of labelled rRNA in the cytoplasm) varied greatly during the cell cycle. It was low during the G l phase, increased rapidly during the S phase, remained high during part of the G 2 phase, and dropped to a minimum during mitosis. A slowdown in the increasing rate of RNA synthesis was observed during the middle of the S phase.No significant decrease in the total nucleotide pool per cell could be observed during the S phase. The accumulation of RNA (as determined by absorbance measurements) was highest during the S and G 2 phases.Pulse labelling of rRNA and pulse chase experiments demonstrated that newly synthesized ribosomal subunits entered into free polysomes to the highest extent during the S phase. The percentage of membrane-bound polysomes of total polysomes increased during the G 1 phase, as did the percentage of labelled rRNA in the membrane-bound fraction.     

Initiation of endogenous messenger RNA translation on hen oviduct polysomes.

The eIF-2A fraction of reticulocyte ribosomal salt wash is capable of maximally stimulating the translation of endogenous messenger RNA by hen oviduct polysomes. The factor increases the initiation of protein synthesis 2--3-fold when measured by the factor-dependent synthesis of NH2-terminal peptides. The addition to these polysomes of elongation factor, EF-1, also increases protein synthesis but at a distinctly different rate and Mg2+ concentration optimum than the eIF-2A fraction. Moreover, there is no stimulation of NH2-terminal peptide synthesis with EF-1 alone. In contrast, all the known initiation factors are required for the translation of exogenous globulin mRNA on oviduct polysomes. Reticulocyte polysomes isolated by an identical procedure to that used for oviduct polysomes or by standard methods also require all the initiation factors for the translation of either endogenous mRNA or exogenous ovalbumin mRNA. Addition of 7-methylguanosine 5'-monophosphate does not inhibit the factor-dependent stimulation of oviduct polysomes except at high concentrations (1.0 mM) indicating that the sites with which 7-methylguanosine 5'-monophosphate normally competes are already occupied. These findings suggest that the messenger RNA remains bound to the oviduct polysomes or initiation factors. Hence the addition of exogenous factors which are involved with mRNA recognition and binding to the ribosome are not required. It has been previously shown that eIF-2A is capable of binding in vitro the initiatior tRNA to an existing Ado-Urd-Gua-40 S complex and initiating protein synthesis when such a complex is present. These present studies indicate that such an initiation complex may exist within the oviduct cell on membrane-associated polysomes. Under these circumstances eIF-2A mediates binding of the initiator tRNA and initiates protein synthesis.     

Variation in the relative synthesis of immunoglobulin G and non-immunoglobulin G proteins in cultured MPC-11 cells with changes in the overall rate of polypeptide chain initiation and elongation

The synthesis of individual proteins in the mouse plasmacytoma cell MPC-11 is differentially inhibited when the rate of polypeptide chain initiation is reduced by exposure of cells to hypertonic medium. The synthesis of immunoglobulin G light and heavy chain polypeptides is 3.5 to 4-fold and 1.5 to 2-fold more resistant, respectively, than the synthesis of non-immunoglobulin G proteins when total protein synthesis is reduced by ~90%. In contrast, when polypeptide chain elongation is inhibited, the synthesis of the light and heavy chains is not more resistant than the synthesis of non-immunoglobulin G proteins.The results with MPC-11 cells suggests that: (1) under standard growth conditions the relative synthesis of individual proteins is determined mainly, but not exclusively, by the relative amounts of the individual messenger RNA species present in the cell; (2) under conditions where the overall rate of polypeptide chain initiation is reduced the relative synthesis of individual proteins becomes more dependent upon the intrinsic ability of their corresponding mRNAs to form functional mRNA-ribosome initiation complexes.     

Characterization of cell-free synthesis of collagen by lung polysomes in a heterologous system.

In normal lung growth, post-pneumonectomy lung growth, and in possibly several lung disorders, there are marked alterations in the density of collagen and changes in the rate of synthesis of collagen relative to the synthesis of other lung proteins. To provide a technology to begin to understand these changes at the molecular level, polysomes were prepared from rabbit lung and translated in a heterologous cell-free system including rabbit reticulocyte 0.5 M KCl ribosomal wash fraction and liver tRNA. Collagen was shown in the cell-free product by collagenase sensitivity, hydroxylation of incorporated proline by peptidyl prolyl hydroxylase, agarose gel chromatography, and sodium dodecyl sulfate acrylamide gel electrophoresis. The cell-free system was optimized with respect to K+, Mg2+, amino acids, and ribosomal wash fraction and used under conditions where total protein synthesis and collagen synthesis are linear with respect to time and amount of polysomes. Under these conditions, collagen synthesis was directed almost entirely by polysomes derived from the endoplasmic reticulum. Polysomes isolated from late fetal lung directed collagen synthesis at twice the rate (per polysome) as those polysomes isolated from adult lung. Similar changes were seen if lung tRNA replaced liver tRNA and if lung ribosomal wash fraction replaced reticulocyte wash fraction. Although these changes in cell-free lung collagen synthesis with tissue explants, further studies will have to be carried out to determine whether, in fact, age-related alterations in control of lung collagen synthesis are truly explained by these findings.     

Regulation of fibrinogen assembly. Transfection of Hep G2 cells with B beta cDNA specifically enhances synthesis of the three component chains of fibrinogen

Previous studies indicated that synthesis of B beta chain may be a rate-limiting factor in the production of human fibrinogen since Hep G2 cells contain surplus pools of A alpha and gamma but not of B beta chains, and fibrinogen assembly commences by the addition of preformed A alpha and gamma chains to nascent B beta chains attached to polysomes. To test whether B beta chain synthesis is rate limiting Hep G2 cells were transfected with B beta cDNA, and its effect on fibrinogen synthesis and secretion was measured. Two sets of stable B beta cDNA-transfected Hep G2 cells were prepared, and both cell lines synthesized 3-fold more B beta chains than control cells. The B beta-transfected cells also synthesized and secreted increased amounts of fibrinogen. Transfection with B beta cDNA not only increased the synthesis of B beta chain but also increased the rate of synthesis of the other two component chains of fibrinogen and maintained surplus intracellular pools of A alpha and gamma chains. Transfection with B beta cDNA did not affect the synthesis of albumin, transferrin, or anti-chymotrypsin and had a small inhibitory effect on the synthesis of C-reactive protein. Taken together these studies demonstrate that increased B beta chain synthesis specifically causes increased production of the other two component chains of fibrinogen and that unequal and surplus amounts of A alpha and gamma chains are maintained intracellularly.     

Evidence for the presence of an inhibitor on ribosomes in mouse L cells infected with mengovirus.

After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes. These 80S ribosomes could not be chased back into polysomes under an elongation block. The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells. Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes. The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection. However, the transit times in mock-infected and infected cells remained the same. Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells. The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition. Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected. Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity. In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells. The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant. A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation. The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation.     

A new method to discriminate G1, S, G2, M, and G1 postmitotic cells

A new flow cytometric method combining light scattering measurements, detection of bromodeoxyuridine (BrdU) incorporation via fluorescent antibody, and quantitation of cellular DNA content by propidium iodide (PI) allows identification of additional compartments in the cell cycle. Thus, while cell staining with BrdU-antibodies and PI reveals the G1, S, and G2 + M phases of the cell cycle, differences in light scattering allow separation of G2 phase cells from M phase cells and subdivision of G1 phase into two compartments, i.e., G1A representing postmitotic cells which mature to G1B cells ready to initiate DNA synthesis. The method involves fixation of cells in 70% ethanol, extraction of histones with HC1, and thermal denaturation of DNA. This treatment appears to enhance the differences in chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated on the basis of the 90 degrees scatter. Mitotic cells show much lower scatter than G2 phase cells, and G1 postmitotic cells (G1A) show lower scatter than G1 cells about to enter the S phase (G1B). Light scattering is correlated with chromatin condensation, as judged by microscopic evaluation of cells sorted on the basis of light scatter. The method has the advantage over the parental BrdU/DNA bivariate analysis in allowing the G2 and M phases of the cell cycle to be separated and the G1 phase to be analyzed in more detail. The method may also allow separation of unlabeled S phase cells from mitotic cells and distinguish between labeled and unlabeled mitotic cells.     

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis in Amaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light → phytochrome → G protein → guanylyl cyclase → cGMP.     

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis inAmaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light → phytochrome → G protein → guanylyl cyclase → cGMP.     

Polyamines are necessary for maximum in vitro synthesis of globin peptides and play a role in chain initiation.

The salt wash fraction removed from rabbit reticulocyte ribosomes with 0.5 m KCl contains dialyzable components required for maximum in vitro synthesis of globin peptides. The active substances were identified as spermidine and spermine. Rabbit reticulocyte ribosomes contain spermine and spermidine in a 1:3 ratio of which about 75% is removed in the 0.5 m KCl wash fraction. Dialyzed salt wash can be reactivated for in vitro protein synthesis by addition of either spermine, spermidine, or Mg²⁺ ion. A twofold higher leucine incorporation into protein was obtained with the optimum concentration of either polyamine than with Mg²⁺. Spermidine is effective in lowering the Mg²⁺ requirement for initiation of phenylalanine peptides in the poly(U)-directed system, apparently by formation of an initiation complex. Also, spermidine competitively interferes with edeine inhibition of globin chain initiation. These results indicate that spermidine may play a special role in peptide initiation.     

Initiation of DNA synthesis in the prematurely condensed chromosomes of G1 cells

The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.     

Prespore cell fate bias in G1 phase of the cell cycle in Dictyostelium discoideum

By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Phospholipids are synthesized in the G2/M phase of the cell cycle

Very little is known about the metabolism of phospholipids in the G2 and M phases of the cell cycle, but limited studies have led to the postulation that phospholipid synthesis ceases during this period. To investigate whether phospholipids are synthesized in the G2/M phase of the cell cycle, protocols were developed to produce synchronized MCF-7 cell populations with greater than 80% of the cells in G1/S or G2/M phases that moved in synchrony following removal of the blocking agent. Analysis of the activities of key phosphatidylcholine and phosphatidylethanolamine biosynthetic enzymes in subcellular fractions obtained from MCF-7 cells at different cell cycle phases revealed that there was robust activity of key enzymes in the fractions prepared from MCF-7 cells in G2/M phase. Radiolabeled choline and ethanolamine were rapidly incorporated into cells maintained at G2/M phase with nocodazole, and the rates of incorporation were similar to those obtained in cells allowed to progress into the G1 phase. Furthermore, radiolabeled glycerol was incorporated into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid in MCF-7 cells maintained at G2/M phase with nocodazole. Similar results were obtained in CHO cells. These results demonstrate that glycerophospholipid synthesis is very active in the G2/M phase of these cells. Therefore, the postulated cessation of phospholipid synthesis in G2/M phases is not applicable to all cell types.     

Butyrate inhibits mouse fibroblasts at a control point in the G1 phase

Butyrate block 3T6 cells in the G1 phase of the cell cycle approximately 5--6 h prior to the start of the S phase. Serum factors are required before as well as after the butyrate-sensitive steps in G1 in order to allow cells to start DNA synthesis. 3T6 cells infected with SV40 or with polyoma virus are also blocked at the same stage in G1 in the presence of the fatty acid. However, events before as well as after the butyrate-sensitive step do not require serum in virus-infected cells. The sensitivity of the initiation of cellular DNA synthesis to increasing concentrations of butyrate is the same for serum-stimulated or for virus-infected cells. A similar and parallel effect on DNA synthesis is observed if cells are incubated in the presence of very small amounts of cycloheximide. After release of the cycloheximide-induced G1 arrest about 4--6 h have to pass before cells enter the S phase. Cells stably transformed by SV40 are considerably more resistant to low cycloheximide concentrations and to butyrate. These data are discussed in the light of the hypothesis that both low concentrations of cycloheximide and sodium butyrate block cells at a control point in G1 by interference with the synthesis of one or more rapidly turning over, cell cycle-specific proteins.     

Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma

Fractionation of MOPC 41 DL-1 tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane- bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a heterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same mol wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section.     

Gene-specific and strand-specific DNA repair in the G1 and G2 phases of the cell cycle.

We have analyzed the fine structure of DNA repair in Chinese hamster ovary (CHO) cells within the G1 and G2 phases of the cell cycle. Repair of inactive regions of the genome has been suggested to increase in the G2 phase of the cell cycle compared with other phases. However, detailed studies of DNA repair in the G2 phase of the cell cycle have been hampered by technical limitations. We have used a novel synchronization protocol (D. K. Orren, L. N. Petersen, and V. A. Bohr, Mol. Cell. Biol. 15:3722-3730, 1995) which permitted detailed studies of the fine structure of DNA repair in G2. CHO cells were synchronized and UV irradiated in G1 or early G2. The rate and extent of removal of cyclobutane pyrimidine dimers from an inactive region of the genome and from both strands of the actively transcribed dihydrofolate reductase (DHFR) gene were examined within each phase. The repair of the transcribed strand of the DHFR gene was efficient in both G1 and G2, with no major differences between the two cell cycle phases. Neither the nontranscribed strand of the DHFR gene nor an inactive region of the genome was repaired in G1 or G2. CHO cells irradiated early in G2 were more resistant to UV irradiation than cells irradiated in late G1. Since we found no major difference in repair rates in G1 and G2, we suggest that G2 resistance can be attributed to the increased time (G2 and G1) available for repair before cells commit to DNA synthesis.     

Serum-dependent regulation of proliferation of cultured rat fibroblasts in G1 and G2 phases

We reported that: (i) 3Y1tsF121 cells, a temperature-sensitive (ts) mutant of rat 3Y1 fibroblasts, are reversibly arrested either in the G1 or in the G2 phase, at the nonpermissive temperature. (ii) Cells retain the ability to resume proliferation at the permissive temperature after prolonged arrest in the G1 phase (for 5 days), whereas they lose it after prolonged arrest in the G2 phase (over 24 h). (iii) The G1 arrest is overcome at the nonpermissive temperature by the addition of fresh serum (H. Zaitsu and G. Kimura (1984) J. Cell. Physiol. 119, 82; (1985) J. Cell. Physiol. 124, 177). In the present study, the G2 arrest was overcome by exposing the cells to fresh serum, at the nonpermissive temperature. The G2 arrest occurred only at a higher cell density than that of the G1 arrest. The efficiency of the overcome was higher in the case of the G2 arrest than in case of the G1 arrest. When cells synchronized at the G1/S border by aphidicolin at the permissive temperature were released from the block, they divided in the absence of serum, at the permissive temperature. Even if they had passed through the previous G2 phase in a very high concentration of fresh serum at the permissive temperature, mitotic cells did not enter the S phase in the absence of serum, even at the permissive temperature. When the cells arrested in the G1 phase (not in G0) due to the ts defect were incubated in the absence of serum at the permissive temperature, only 34% entered the S phase and only 15% divided. These results suggest that (i) the ts defect in 3Y1tsF121 limiting cellular proliferation in both the G1 and the G2 phases is probably due to a single mutational event, and is a serum-requiring event. (ii) Preparation of the serum-requiring event which is required for the G2 traverse is completed in the G1 phase, under ordinary conditions. (iii) However, cells are able to fulfill the serum-requiring event in the G2 phase as well as in the G1 phase when the preparation is below the required level. (iv) The commitment to DNA synthesis is not necessarily a commitment to cell division. (v) Cells are arrested in the G1 phase more safely and more effectively than in the G2 phase, by the serum-related mechanism.     

Regulation of protein synthesis in vesicular stomatitis virus-infected mouse L-929 cells by decreased protein synthesis initiation factor 2 activity.

Infection of mouse L-cell spinner cultures by vesicular stomatitis virus (VSV) effected the selective translation of viral mRNA by 4h after viral adsorption. Cell-free systems prepared from mock- and VSV-infected cells reflected this phenomenon; protein synthesis was reduced in the virus-infected cell lysate by approximately 75% compared with the mock-infected (control) lysate. This effect appeared to be specific to protein synthesis initiation since (i) methionine incorporation into protein from an exogenous preparation of initiator methionyl-tRNA gave completely analogous results and (ii) the addition of a ribosomal salt wash (containing protein synthesis initiation factors) stimulated protein synthesis by the infected cell lysate but had no effect on protein synthesis by the control. Micrococcal nuclease-treated (initiation-dependent) VSV-infected cell lysates were not able to translate L-cell mRNA unless they were supplemented with a ribosomal salt wash; a salt wash from ribosomes from uninfected cells effected a quicker recovery than a salt wash from ribosomes from infected cells. When salt wash preparations from ribosomes from uninfected and infected cells were tested for initiation factor 2 (eIF-2)-dependent ternary complex capacity with added GTP and initiator methionyl-tRNA, we found that the two preparations contained equivalent levels of eIF-2. However, initiation complex formation by the factor from virus-infected cells proceeded at a reduced initial rate compared with the control. When the lysates were supplemented with a partially purified eIF-2 preparation, recovery of activity by the infected cell lysate was observed. Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.