The cytoplasmic appearance of three functions expressed during the G0→G1→S transition is nucleus-dependent

tsAF8, ts13, tsHJ-4, and TK^?ts13 cells are G₁-specific temperature-sensitive (ts) mutants of BHK cells that do not enter S phase when serumstimulated from quiescence at nonpermissive temperature (39.6°-40.6°). TK^?ts13 are, in addition, defective in thymidine kinase. Different G₁ functions must be involved in these cells, since the first three cell lines complement each other when forming heterokaryons. We have used these cells to study the role of the nucleus in the cytoplasmic expression of these G₁ functions during the transition of cells from the non-proliferating to the proliferating state. We fused cytoplasts from either serumstarved (G₀) or serum-stimulated (S) tsAF8 cells with G₀-ts13, G₀-tsHJ-4, and G₀-TK^?ts13 recipient cells and determined, after serum stimulation of the fusion products, which type of cytoplasts could complement the defective G₁ functions. Cytoplasts from S-tsAF8 cells complemented all three functions, i.e., cybridoids between S phase cytoplasts and ts13 or tsHJ-4 recipient cells entered S at the nonpermissive temperature, and TK^?ts13 recipient cells incorporated exogenous thymidine. Cytoplasts isolated from G₀-tsAF8 cells (3 days of serum starvation) complemented ts13 cells but not tsHJ-4 and TK^?ts13 cells. Cytoplasts from 6-day starved tsAF8 cells lost the complementing capacity for ts13 cells. However, when the 6-day starved tsAF8 cells were fused with G₀-ts13 cells, the heterokaryons entered S phase at the nonpermissive temperature. Also, cytoplasts isolated from the 6-day starved cells that were serum stimulated for 40 hr before enucleation regained the capacity to complement ts13 cells. These results demonstrate that three functions required in G₁ cannot be detected in the cytoplasm of serum-starved cells, although they are present in the cytoplasm of S-phase cells. These results suggest that a functional nucleus is required for the cytoplasmic appearance of certain G₁ functions in serumstimulated cells.     

Identification of adenovirus 2 early genes required for induction of cellular DNA synthesis in resting hamster cells.

tsAF8 cells are temperature-sensitive (ts) mutants of BHK-21 cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. When made quiescent by serum restriction, they can be stimulated to enter the S phase by 10% serum at 34 degrees C, but not at 40.6 degrees C. Infection by adenovirus type 2 or type 5 stimulates cellular DNA synthesis in tsAF8 cells at both 34 and 40.6 degrees C. Infection of these cells with deletion Ad5dl312, Ad5dl313, Ad2 delta p305, and Ad2+D1) and temperature-sensitive (H5ts125, H5ts36) mutants of adenovirus indicates that the expression of both early regions 1A and 2 is needed to induce quiescent tsAF8 cells to enter the S phase at the permissive temperature. This finding has been confirmed by microinjection of selected adenovirus DNA fragments into the nucleus of tsAF8 cells. In addition, we have shown that additional viral functions encoded by early regions 1B and 5 are required for the induction of cellular DNA synthesis at the nonpermissive temperature.     

Expression of histone genes in a G1-specific temperature-sensitive mutant of the cell cycle

The expression of genes coding for the four core histones (H2A, H2B, H3, and H4) was studied in tsAF8 cells. These baby hamster kidney-derived cells are a temperature-sensitive (ts) mutant of the cell cycle that arrest in G1 at the restrictive temperature. When serum-deprived tsAF8 cells are stimulated with serum, they enter the S phase at the permissive temperature of 34 degrees C, but are blocked in G1 at the nonpermissive temperature of 39.6 degrees C. Northern blot analysis using cloned human histone DNA probes detected only very low levels of histone RNA either in quiescent tsAF8 cells or in cells serum stimulated at the nonpermissive temperature for 24 h. Cellular levels of histone RNA were markedly increased in cells serum stimulated at 34 degrees C for 24 h. Temperature shift-up experiments after serum stimulation of quiescent populations showed that the amount of histone RNA was related to the number of cells that entered the S phase. Those cells that synthesized histone RNA and entered the S phase were capable of dividing. This is the first demonstration in a mammalian G1-specific ts mutant that the expression of H2A, H2B, H3, and H4 histone genes depends on the entry of cells into the S phase of the cell cycle.     

Expression of thymidine kinase and dihydrofolate reductase genes in mammalian ts mutants of the cell cycle

Thymidine kinase and dihydrofolate reductase mRNA levels and enzyme activities were determined in two temperature-sensitive cell lines, tsAF8 and ts13, that growth arrest in the G1 phase of the cell cycle at the restrictive temperature. The levels of thymidine kinase mRNA and enzyme activity increased markedly in both cell lines serum stimulated from quiescence at the permissive temperature. At the nonpermissive temperature, the levels of thymidine kinase mRNA and enzyme activity remain at the low levels of quiescent G0 cells. The levels of dihydrofolate reductase mRNA as well as the enzyme activity also increase when both cell lines are serum stimulated at the permissive temperature. When ts13 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity declines rapidly and dihydrofolate reductase mRNA is below detectable levels. On the contrary, when tsAF8 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity increases and mRNA levels are detectable slightly above G0 levels, even though the cells are blocked in the G1 phase. Studies with 2 other cDNA clones (one with an insert whose expression is cell cycle dependent and the other with an insert whose expression is not cell cycle dependent) indicate that the results are not due to aspecific toxicity or the effect of temperature. We conclude that the expression of different genes is affected differently by the ts block in G1, even when these genes are all growth-related.     

Time of replication of genes responsible for a temperature-sensitive function in a cell cycle-specific ts mutant from a hamster cell line

A method involving short pulses of 5-bromodeoxyuridine (brUdRib) followed by irraidation with 313 nm light was used to locate the time of replication of certain genes during the cell cycle of two cell lines, AF8 and AL106. AF8, a temperature-sensitive mutant of BHK21/13 cells, grows at 33°C but not at 39.5°C. AL106, a hybrid clone of tsAF8 and SV-40 transformed Lesch-Nyhan fibroblasts (LNSV), which retains all hamster chromosomes and one human chromosome (No. 3), has the ability to grow at 39.5°C. AF8 and AL106 cells synchronized at the G₁-S boundary were released from their block and pulsed with brUdRib for 2-hour periods during the S phase. The cells were subsequently irradiated with 313 nm light. Colony-forming efficiency and revertants frequency were studied. Incorporation of brUdRib during the early S phase (0–4 hours from the begining of S), decreased the colony-forming efficiency of AL106 cells both at 33°C and 39.5°C, and also of AF8 cells at 33°C. No AF8 colonies grew at the nonpermissive temperature regardless of the treatment. Thus the time of replication of genes responsible for colony-forming ability was the same in tsAF8 at the permissive temperature and in AL106 at both temperatures. The time of replication of the genes responsible for the ts function in AF8 cells was located by determining the revertants frequency in synchronized AF8 cells pulsed with brUdRib and irradiated during 1- to 2-hour periods of the S phase. Back-mutants were scored by counting the number of clones capable of growing at 39.5°C (nonpermissive for AF8 cells). The highest frequency of induced back-mutations occurred in synchronized AF8 cells pulsed with brUdRib (and irradiated) between two to four hours from the begining of the S phase. Exposure to brUdRib during other periods of the S phase or during G₁ had no effect on the reversion rate. This method can be used to locate the time of replication (in S) of ts genes in other temperature-sensitive mutants or of other specific genes in other conditional mutants.     

A temperature-sensitive cell cycle mutant of the BHK cell line

A temperature-sensitive growth mutant derived from the BHK 21 cell Line, ts AF8, was found to have greatly reduced DNA synthesis at the nonpermissive temperature. This reduction is mainly due to a decrease in the frequency of cells synthesizing DNA. Upon shift up, ts AF8 becomes blocked in the G1 phase of the cell cycle. The cells acquire elevated cAMP levels and a unimodal distribution of DNA content, equivalent to that of G1 cells at the permissive temperature, Ts AF8 cells blocked at the G1/S boundary with hydroxyurea will enter S when shifted to the nonpermissive temperature. On the other hand, ts AF8 cells arrested m G1 by serum deprivation and shifted to the nonpermissive temperature at the moment of serum addition do not enter S, while those synchronized by isoleucine deprivation and shifted at the time of isoleucine addition will enter S. These data suggest that the cycle arrest point of the ts AF8 mutation is located in G1 between the blocks induced by serum starvation and isoleucine deprivation. The reduction in DNA synthesis caused by the ts AF8 mutation is not reversed by infection or transformation with Polyoma virus. Mitochondrial DNA continues to be synthesized at wild-type levels at the nonpermissive temperature.     

Failure of reactivation of chick erythrocytes after fusion with temperature-sensitive mutants of mammalian cells arrested in G1

Two temperature-sensitive (ts) mutants of mammalian cell lines (AF8 and cs4D3) that arrest in G1 at the nonpermissive temperature were fused with chick erythrocytes and the induction of DNA synthesis was studied in the resulting heterokaryons. While both AF8 and cs4D3 could induce DNA synthesis in chick nuclei at the permissive temperature, they both failed to do so when arrested in G1 at the nonpermissive temperature. When S phase AF8 cells were fused with chick erythrocytes, chick nuclei were reactivated even if the heterokaryons were incubated at the temperature nonpermissive for AF8. A third ts mutant, ts111, that is blocked in cytokinesis but continues to synthesize DNA, reactivated chick nuclei at both permissive and nonpermissive temperature. It is concluded that chick erythrocyte reactivation depends on the presence of S phase-specific factors.     

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.     

Failure of accumulation of cellular RNA in hamster cells stimulated to synthesize DNA by infection with adenovirus 2

AF8 cells are temperature-sensitive mutants of the cell cycle derived from baby hamster kidney (BHK) cells which arrest in the G1 phase when incubated at the nonpermissive temperature. RNA accumulation was studied in these cells by flow cytofluorimetry following serum stimulation or adenovirus 2 infection. Serum stimulation caused an increase in the amount of RNA per cell, which reached a maximum in S and G2 cells, as repeatedly reported in the literature. However, adenovirus 2 infection caused a fraction of cells to enter S phase without any concomitant increase in the amount of RNA per cell.     

Differential expression of Rous Sarcoma virus-specific transformation parameters in enucleated cells.

Chicken embryo fibroblasts transformed with the Ta and ts68 mutants of Rous Sarcoma virus (RSV) were enucleated and studied for their capacity to express reversibly the transformed phenotype in response to temperature changes. After shift to the permissive temperature (35 degrees C), the cytoplasts acquired a transformed morphology and displayed characteristic ruffles and microvilli at their surface. As detected by immunofluorescence, they also lost their actin filament cables and exhibited characteristic changes in the pattern of cell surface structures containing LETS protein. Expression of all these transformation parameters was reversible after shiftback to the nonpermissive temperature (41 degrees C). These results indicate that a whole set of changes characteristic for the transformed phenotype can be expressed independently of the cell nucleus. In contrast, ts mutant-infected cytoplasts were no longer able to respond to temperature shifts with changes in their hexose transport rate. Cytoplasts prepared from cells grown at 41 degrees C retained their low rate of hexose uptake after shift to 35 degrees C, whereas cytoplasts from cells grown at 35 degrees C exhibited a high rate of hexose transport even after 10 hr of shift to 41 degrees C. These results are in accordance with the hypothesis that the product of the src gene of RSV represents a multifunctional protein which acts independently on nuclear and extranuclear sites.     

A nuclear labile protein, p70, is expressed exclusively during G0-S transition but not in G1 phase of a cell cycle ts mutant, tsJT16

tsJT16 is a cell cycle temperature-sensitive (ts) mutant from a Fischer rat cell line. When it is growth-stimulated from G0 phase it enters S phase at the permissive temperature (34 degrees C) but not at the nonpermissive temperature (40 degrees C). It induces a nuclear labile protein, p70, when it is stimulated from G0 phase at 34 degrees C, but not at 40 degrees C. In growing cell cycle it progresses through the S, G2 and M phases at both temperatures but fails to pass through G1 phase at 40 degrees C. Here we described that p70 was synthesized neither in the randomly growing cycle nor in the G1 phase synchronously progressing from M phase. The cells synchronized at early G1 phase by culturing in serum-free medium for 7.5 h from G1/S boundary induced c-fos and c-myc following serum addition, but under the same condition p70 was not synthesized. These results indicate that the synthesis of p70 is not required for progression of the G1 phase of the growing cycle and can be used as an exclusive marker of G0-S transition.     

Changes in RNA polymerase II in a cell cycle-specific temperature-sensitive mutant of hamster cells

tsAF8 cells are a temperature-sensitive mutant of BHK cells that arrest at the nonpermissive temperature in the G₁ phase of the cell cycle. The activity of solubilized RNA polymerase II and its ability to bind [³H]-γ-amanitin decrease in tsAF8 cells at 40.6°, with a half-life of ～ 10 hr. No appreciable changes occur in these two parameters in tsAF8 cells at 34° or in BHK cells at either 34° or 40.6°. Protein synthesis is not appreciably affected for at least 24 hr after tsAF8 cells are shifted to 40.6°. These results indicate that in tsAF8 cells at the nonpermissive temperature, there is a defect in either the synthesis, the assembly, or the stability of RNA polymerase II, and that the loss of RNA polymerase II molecules is not due to widespread cellular damage.     

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content

Cultures of ts BN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [3H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5 degrees. However, when higher (41 degrees - 42 degrees) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of ts BN75 shifted to 42 degrees at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When ts BN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5 degrees C), the restart of DNA synthesis preceded the appearance of mitotic cells. These data suggest that the ts defect of ts BN75 cells might affect primarily the S phase of the cycle rather than the G2 phase.     

Serum-mediated regulation of amino acid transport in cultured chick embryo fibroblasts

Three different temperature sensitive mutants derived from the Syrian hamster cell line BHK 21 were found to have greatly reduced DNA synthesis at the non-permissive temperature. These mutants are distinct by complementation analysis and behave at the non-permissive temperature as cell cycle traverse defective mutants. Microfluorometric analysis of mutant populations arrested at the non-permissive temperature shows an accumulation of cells with G1 DNA content. Mutants ts 13 and ts HJ4 synchronized in G1 by serum or isoleucine deprivation and shifted to the non-permissive temperature at the time of release do not enter the S phase, while in the case of mutant ts 11 preincubation at the non-permissive temperature before release is required to completely prevent its entry into S. Ts 13 and ts 11 are able to traverse the S phase at the non-permissive temperature when synchronized at the boundary G1/S; in this case, preincubation of ts 11 at the non-permissive temperature before release does not affect the ability of these cells to perform DNA synthesis. On the other hand, ts HJ4 appears to traverse S only partially when tested under similar conditions. Temperature shift experiments of mutant populations at different times after isoleucine synchronization suggest that ts 13 and ts 11 are blocked at the non-permissive temperature in early G1, whereas ts HJ4 is probably affected near the initiation of DNA synthesis, or in some early S function.     

Epidermal growth factor has a unique effect in combination with fetal bovine serum to bypass the ts-block of G0-specific ts mutant tsJT60

tsJT60 cells are G0-specific temperature-sensitive mutants of the cell cycle from Fischer rats i.e., they grow exponentially at both 34 degrees and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS) from the resting state (G0) they enter S phase at 34 degrees C but not at 39.5 degrees C. Epidermal growth factor (EGF) also induced DNA synthesis, although weakly, in G0-arrested tsJT60 cells at 34 degrees C but failed at 39.5 degrees C. When G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus EGF, they entered S phase and divided. Somatomedin C, insulin, or transferrin had a weak effect in inducing DNA synthesis in G0-arrested cells when applied at 34 degrees C or with FBS at 39.5 degrees C. Fibroblast growth factor, platelet-derived growth factor, or 12-O-tetradecanoylphorbol 13-acetate had no such stimulatory effect at 39.5 degrees C. Binding of 125I-somatomedin C was not temperature-sensitive. Several other ts mutant cells that were blocked at 39.5 degrees C from entering S phase from the resting state following FBS addition were stimulated by FBS plus EGF at 34 degrees C but not at 39.5 degrees C.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

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.     

The ts41 mutation in Chinese hamster cells leads to successive S phases in the absence of intervening G2, M, and G1.

The ts41 mutation of Chinese hamster cells was first isolated and characterized by Hirschberg and Marcus (1982) who showed that at nonpermissive temperature, cells accumulate up to 16C equivalents of DNA. Here we show that the mutation is recessive and at nonpermissive temperature, cells replicate their genome normally, but instead of going on into G2, M, and G1, they pass directly into a second S phase. Entry into a second S phase does not require serum nor is it inhibited by G2 checkpoints or mitotic inhibitors. Temperature-shift experiments suggest that the ts41 gene product participates in two functions in the cell cycle: entry into mitosis and inhibition of entry into S phase. The ts41 mutation seems to define a class of cell cycle mutant that couples the sequential events of DNA replication and mitosis.     

Temporary complementation of temperature-sensitive mutants of the cell cycle by transfection with a wild-type or a mutant cDNA of ADP/ATP translocase

A number of cell-cycle-specific temperature-sensitive (ts) mutants have been isolated from animal cells, especially Syrian hamster cells. These ts mutants, like cell cycle ts mutants of yeast, can be complemented by specific genes, some of which have been molecularly cloned. We have isolated a cDNA clone that complements TK-ts13 cells, but only temporarily. This clone, called B1, differs from a previously isolated clone (Sekiguchi et al.: EMBO Journal 7:1683-1687, 1988) that specifically complements ts13 cells. In addition, B1 also complemented temporarily three other ts mutants of the cell cycle, tsAF8, ts694, and ts550C cells. These mutants have different mutations since, in cell fusion experiments, they complement each other. Sequencing of the B1 cDNA clone revealed that it was a mutant of human ADP/ATP translocase in which some human sequences at the 5' end have been replaced by SV40 sequences. The wild-type translocase was less effective but could still increase the survival time of cell cycle ts mutants at the restrictive temperature. Using the polymerase chain reaction, it was possible to demonstrate that the B1 plasmid is expressed in TK-ts13 cells undergoing temporary complementation.     

Characterization of ts13 cells a temperature-sensitive mutant of the G1 phase of the cell cycle

ts 13 cells are a temperature-sensitive (ts) mutant of BHK cells that are known to arrest in G1 when shifted to the nonpermissive temperature. We have determined the entry into S of ts13 cells in five different growth conditions, namely: 1) quiescent, sparse cultures stimulated to proliferate by serum. 2) Quiescent, dense cultures stimulated by serum. 3) Quiescent, sparse cultures stimulated by trypsinization and replating. 4) Quiescent, dense cultures stimulated by trypsinization and replating. 5) Mitotic cells collected by mitotic detachment. For each different growth condition we have also determined the execution point of the mutant function, i.e. the time at which a shift-up to the nonpermissive temperature no longer prevents the entry of cells into S. The median time of entry into S and the execution point varied in different growth conditions, but the distance between the median execution point and the median time of entry into S was remarkably constant, i.e. 3.2 hr. In addition we have fused ts 13 cells cells with chick erythrocytes and studied the ability of ts13 cells in heterokaryon formation to induce DNA synthesis in chick nuclei. Although ts13 cells can induce DNA synthesis in chick nuclei at the permissive temperature, they fail to do so when fused and stimulated at the nonpermissive temperature of 39.5 degrees C.