Cell, Vol. 7, 289-295,

February

1976,

Copyright0

1976 by MIT

Temperature-Sensitive Mutants of Balb/3T3 Description of a Mutant Affected in Cellular and Polyoma Virus DNA Synthesis

Cells:

A temperature-sensitive Dna- mutant (ts-2) of the mouse cell Balb/3T3 is characterized. Studies with synchronized ceils indicate that the defect is in DNA synthesis itself, rather than in progress toward its initiation. ts-2 supports polyoma DNA synthesis after infection at 33°C but not at 38OC. Viral DNA synthesis begun at 33°C is inhibited upon shift to 38OC. A procedure is proposed by which viral DNA synthesis can be used to distinguish different classes of cell Dna- mutants.

upon the DNA replication machinery and some Gl functions of the host cells (Tooze, 1973; Pages et al., 1973). In the case of temperature-sensitive Dnamutants, therefore, studies with polyoma can serve the dual purpose of assessing the nature of reliance of the virus on host functions and also of providing a model for biochemical characterization of the temperature-sensitive defect. This expectation is based on the extensive information already known on the replication of this single replicon viral genome and the availability of cell-free systems which faithfully complete the replication of viral molecules initiated in intact cells (for example, Hunter and Francke, 1974). We describe here the initial characterization of a Balb/3T3 temperature-sensitive mutant, ts-2, affecting cellular and polyoma DNA synthesis.

Introduction

Results

Conditional lethal, temperature-sensitive mutants have proved of great value in the analysis of events in the replication cycle for a variety of microbial systems. For example, studies on the initiation of DNA synthesis and on the components and structure of the “replicase” of bacterial cells were greatly aided by experiments with temperature-sensitive Dnamutants (Hirota, Ryter, and Jacob, 1968; Wechsler and Gross, 1971; Sheckman and Kornberg, 1974). In the case of yeast, temperature-sensitive cell division cycle (cdc-) mutants were instrumental in elucidating the manner in which cells become committed to entry of the mitotic cycle and progress toward the initiation of DNA synthesis (Hartwell, 1974). In the past few years, a number of laboratories have isolated and characterized temperature-sensitive mutants in different mammalian ceils in culture. In a few cases, it has been possible to localize the biochemical defect with precision (Thompson, Harkins, and Stanners, 1973; Toniolo, Meiss, and Basilica, 1973; Haralson and Roufa, 1975). We have previously reported that temperaturesensitive mutants can be isolated in the mouse cell line Balb/3T3 (Wittes and Ozer, 1973). This cell line is of particular interest for temperaturesensitive studies; since its growth is highly serum-dependent, it is subject to “contact inhibition,” and it is susceptible to transformation by a variety of agents. Furthermore, 3T3 cells can be productively infected with the papovavirus polyoma. With the exception of the A function required for the initiation of polyoma DNA synthesis (Francke and Eckhart, 1973), viral DNA replication depends

General Growth Properties A clone, designated ts-2, was isolated at 33°C after mutagenesis of Balb/c 3T3 clone A31 cells with EMS and selection with 3H-thymidine at 38”C, as described in Experimental Procedures. At 33”C, ts-2 and wild-type (3T3) cells exhibit similar growth characteristics with respect to efficiency of colony formation (approximately 20%), cell morphology in sparse and confluent cultures, and sensitivity of DNA synthesis and cell division to contact inhibition and serum concentration. The rate of cell growth is somewhat reduced for ts-2, requiring 35-40 hr for a doubling of cell number; in contrast, 3T3 requires approximately 30 hr at 33°C and 24 hr at 38°C. However, at 38”-39”C, ts-2 exhibits drastically altered growth characteristics. The efficiency of colony formation is less than one colony per 105 cells plated. A typical growth curve is shown in Figure 1. There is less than a 2 fold increase in cell number in 24 hr after shifting cultures in logarithmic growth from 33°C to 38°C. Thereafter, there is a progressive loss of cells from the petri dish. The viability of ts-2 at 38°C is shown in Figure 2. There is no loss of colony-forming ability (tested at 33°C) in the first 16 hr at 38°C; thereafter, however, viability falls. Essentially no change in these growth characteristics has been observed on repeated subcloning of the original isolate of ts-2.

M. L. Slater” and H. L. Ozeri Worcester Foundation for Experimental Shrewsbury, Massachusetts 01545

Biology

Summary

“Present address: Laboratory of Biochemistry and Metabolism, National Institute of Arthritis and Metabolic and Digestive Diseases, Bethesda, Maryland 20014. +To whom requests for reprints should be addressed.

Macromolecular Synthesis The rate of incorporation of radioactive precursors into TCA-precipitable macromolecules was investigated after a culture at 33°C was shifted to 38”C, as shown in Figure 3. There is a gradual decrease in the incorporation of 3H-thymidine (TdR) at 38°C after an initial increase. The latter increase is also seen with wild-type cells. Incorporation of 3H-deoxy-

Cell 290

cytidine is similarly affected (data not shown). Uridine and lysine incorporation per culture increase over the first 16 hr (as seen with growing cells), as shown in the insert of Figure 3. Consequently, there is a preferential decrease in the rate of DNA synthesis. This inhibition of TdR incorporation at 38°C is independent of the cell density (between 104 and 106 cells per 60 mm dish) or the prior duration of incubation at 33°C. The inhibition of thymidine incorporation is reversible even after 25 hr at 38”C, as shown in Figure 4. Incorporation resumes without an apparent lag upon shift to 33”C, indicating that at least some cells are arrested at or within S phase. The data do not, however, indicate a synchronous resumption of DNA synthesis. Cell Cycle Analysis In an effort to determine the basis for the preferential inhibition of DNA synthesis, we assessed the incorporation of 3H-TdR following manipulation of the cell cycle. In the first approach, synchronized cultures were used to search for an “execution time” (Hartwell, 1974) during Gl, that is, a time when the temperature-sensitive function is completed at 33”C, leaving the cells resistant to 38°C for the rest of the cell cycle. Cells were arrested in Go (or early Gl) by incubation in low serum. Serum was added at 33°C and at various times samples were pulse-labeled with 3H-TdR. S phase was reached at 33°C between 15 and 20 hr after the

addition of 30% serum. At various times after addition of 30% serum, parallel cultures were shifted to 38°C and incubated for a total period of 21 hr (time at 38°C plus 33°C). Then all the cultures at 38°C were labeled for 4 hr. Cultures shifted before the execution time should not reach S phase and remain unlabeled. Cultures shifted after the execution time should reach S phase along with the control culture maintained at 33°C for the entire period. Thus if the temperature-sensitive function were

0 0

2 100

Ia

I IO

0 I 0

I 20 HOURS

Figure

2. Viability

I 30 AT

40

5

38“C

of ts-2 at 38’C

Cells from a log phase culture were plated in quadruplicate at 50,250, and 500 cells, incubated at 33°C for 20 hr, and shifted to 38°C. At various times, samples were returned to 33°C and incubated for 2 weeks. Colonies were counted after staining.

125 = 100 k? LL

75

k

50 25

0

+

0

I

I

I

30 HOURS

Figure

1. Growth

Curve

I

I

60 AFTER

of ts-2 at 33°C

I

I,

90 120 INOCULATION

I

I

150

and 38°C

5 x 104 cells were plated at 33OC (o-o). by the arrow), half the cultures were shifted

After 24 hr (indicated to 38°C (O---O).

Figure

4

3. Macromolecular

8 12 HOURS AT Synthesis

16 38%

of ts-2

20

24

at 38°C

105 cells were inoculated at 33°C and shifted to 38°C after 24 hr. At various times, duplicate cultures were incubated for 2 hr with 3H-thymidine (O-O), SH-uridine (O-O, insert), or 3H-lysine (o-0) at 1 @ml, and lysed; TCA-precipitable radioactivity was measured. The sample at 0 hr was at 33°C; the sample for 2 hr at 38°C was designated 100%.

Temperature-Sensitive 291

Mutants

in DNA Synthesis

completed at a specific time in Gl, there should be a discontinuous increase in the ability of cells to reach S phase at 38°C (depending upon the Gl interval spent at 33°C). No such discontinuity was found. Instead, the ability to begin DNA replication increased in a gradual manner (see Figure 5), consistent with the mutant being “leaky” in the sense that the affected function continues for some time at 38”C, although at a declining rate. Furthermore, cells which were shifted to 38°C shortly after the addition of serum failed to enter S phase. It is therefore not necessary to postulate an additional temperature-sensitive defect in the ability of ts-2 to transit through G2 and/or mitosis to explain the temperature-sensitive phenotype of nonsynchronized cells. Distinguishing between partially impaired transit through Gl or a function required in S phase (although synthesized in Gl) would not be possible by the above type of experiment because of incomplete synchrony in the population. Unsynchronized cells were therefore provided the opportunity to reach the Gl/S border at 38”C, and the question was asked whether these cells passed the temperature-sensitive site. Cultures in random growth were shifted to 38°C in the presence or absence of hydroxyurea, an inhibitor of DNA synthesis. After 16

hr at 38”C, the hydroxyurea was removed and all cultures were labeled with 3H-TdR. If the temperature-sensitive function were required in S phase, cells accumulating at the Gl /S border in hydroxyurea would be at a point prior to the temperature-sensitive site. Thus the ability to synthesize DNA would decay as rapidly in the presence of hydroxyurea as in its absence. A partial defect in the ability to transit Gl, on the other hand, would allow cells to “leak” past the temperature-sensitive site and to accumulate at the Gl /S border in the presence of hydroxyurea. These cells would be ex-

200

100 IT ‘0

2

50

!i2

20

cr W a

0

IO

20

30

HOURS AT 33°C PRIOR TO SHIFT TO 38°C Figure l”‘l”‘l

0 HOURS Figure

4. Reversibility

of DNA Synthesis

AT

12 24 33°C

in ts-2

Cultures in log phase were shifted to 38’C and pulsed for 30 min with 10 pc/ml 3H-TdR after 1 hr and 25 hr (O-O). At 25 hr, the cultures were shifted to 33OC and labeled with XH-TdR immediately and at intervals of 3 hr (o----O).

5. Progress

of Synchronized

Cells toward

S at 38°C

The medium in a log phase population of ts-2 was replaced by medium containing 0.5% calf serum. Samples were pulse-labeled with 3H-TdR until the rate of incorporation into TCA-precipitable material was determined to be less than 0.75% of the peak value. At time 0, the medium was replaced by medium containing 30% serum. At various times, beginning immediately (time O), samples were shifted to 38°C and incubated for a total of 21 hr (33°C + 38°C). At this time, all the cultures at 38°C were labeled with 3H-TdR (10 pc/ml) for 4 hr (C--o). A culture maintained at 33°C incorporated 1.2 x 105 cpm.

Cell 292

petted to replicate DNA normally following removal of the hydroxyurea. The data obtained are shown in Table 1. No increase in DNA synthesis is observed in the cultures previously incubated in hydroxyurea at 38”C, as compared to those incubated at 38°C alone, even when the former was shifted to 33°C immediately before labeling. Replicate cultures were labeled with 3H-uridine to control for differential viability or cell number in the presence or absence of hydroxyurea at 38°C. Normalization of DNA synthesis to RNA synthesis by this method did not change the result. Finally, appreciable DNA synthesis was observed in cultures treated with hydroxyurea at 33”C, eliminating the possibility that unusual toxicity of hydroxyurea might affect resumption of DNA synthesis. The results, therefore, indicate that the lesion is a “leaky” S phase function. Other interpretations, although not consistent with the results observed, cannot be rigorously excluded, however; for example, asynchrony in the response to 30% serum could obscure the identification of an “execution time” and alter the interpretation of the experiment with hydroxyurea. We therefore undertook an alternate approach less dependent for interpretation upon synchronization of ts-2, that is, the effect of temperature shift on polyoma viral DNA synthesis. Polyoma Infection of ts-2 A series of preliminary studies were performed with polyoma infection of ts-2 to assess the effect of the temperature-sensitive mutation on the virus-cell interaction. In all these experiments, information was sought only for 3H-TdR incorporation into cellular and/or viral DNA. Polyoma Infection Does Not Correct the Temperature-Sensitive Defect at 38” C The papovaviruses are known to induce multiple cellular enzymes of DNA biosynthesis in 3T3 cells (Kit and Dubbs, 1969). It was therefore conceivable that polyoma infection could correct the temperature-sensitive defect either by increasing the activity of the putative temperature-sensitive function or by Table

1. Effect

of Hydroxyurea

inducing an alternate form. As shown in Table 2, cellular DNA synthesis declined to a similar extent for uninfected and infected log phase cells at 38°C. Polyoma DNA Synthesis at 33°C and 38% Since polyoma did not correct the temperaturesensitive defect at 38”C, it was possible to determine whether polyoma DNA replication occurs at 38°C. Synthesis of viral DNA I was also compared with that in wild-type 3T3 cells at 33°C and 38°C. The data are shown in Table 3. A number of points can be made. First, viral DNA synthesis can be readily detected in infected log phase 3T3 cells by 30 hr at 33°C or 24 hr at 38”C, as expected (Eckhart, 1969). The percentage in viral DNA I of the total 3H-TdR incorporated is relatively low (2-5%) since cellular DNA synthesis continues (Cheevers, Kowalski, and Yu, 1972). The incorporation into viral DNA I observed as a discrete peak at 21s on neutral sucrose gradients (NSG) is nonetheless approximately 1 O-20 fold that obtained with comparable fractions from uninfected cells analyzed in parallel (0.17-0.35% of total cellular TCA-precipitable counts). Second, viral DNA synthesis in ts-2 at 33°C is significantly reduced in comparison to 3T3. No discrete peak was observed at 30 hr on the NSG, although the percentage of total counts in the appropriate gradient fractions was 2 fold that in comparable uninfected ts-2 extracts, suggesting that minimal amounts of DNA I were in fact present. By 47 hr after infection at 33”C, DNA I was readily detectable as a discrete peak, which was 6 fold the uninfected “background.” On a per cell basis, the rate of apparent synthesis was only 20% that of infected 3T3 cells. It should be noted that incorporation of 3HTdR into cellular DNA is only approximately 50% for infected or uninfected ts-2 as compared to 3T3 in a 2 hour pulse, consistent with the slower growth rate of the former. Third, there is little or no detectable DNA I in ts-2 labeled at 38°C. No DNA I was observed at 24 hr in cells shifted immediately after infection to 38°C. Cultures which were infected, incubated at 33°C for 30 hr, shifted to 38°C for 17 hr, and pulse-labeled showed a minimal peak of

on ts-2

Condition Incubation Temperature

Hydroxyuma

Labeling Temperature

3H-Thymidine (x 1O-4 cpm

Incorporation per Culture)

Relative Incorporation (Thymidine/Uridine)

38°C

5.4

38°C

+

38°C

3.4

1.79

38°C

+

33°C

2.9

1.41

33oc

+

33°C

37.9

38°C

1.17

Log phase cultures at 33°C were shifted to 38°C or maintained at 33°C in the presence or absence of 0.5 mM hydroxyurea. After 16 hr, the medium was changed to growth medium with 10 pc/ml of 3H-thymidine or “H-uridine for 4 hr, and TCA-precipitable radioactivity was determined. Cultures shifted to 38OC for 16 hr without hydroxyurea showed a 90% decrease in DNA synthesis.

Temperature-Sensitive 293

Mutants

in DNA Synthesis

DNA I (data not shown), similar to that observed for parallel cultures which were pulse-labeled after 30 hr at 33”C, that is, at the time of temperature shift. Thus, viral DNA synthesis is prevented by the temperature-sensitive defect at 38°C. Effecf of Temperature Shift on Polyoma DNA Synfhesis There are multiple explanations for the failure to synthesize polyoma DNA I, including an interruption of progression of ts-2 through the cell cycle. Pages et al. (1973) studied papovavirus infection in synchronized cells and found that viral DNA synthesis would not start unless the infected cells traversed part of Gl and the first 2 hr of S. On the other hand, they also reported that once viral DNA synthesis began, initiation and completion of new replicative intermediates occurred in all subsequent stages of the cell cycle, including those devoid of cell DNA synthesis. Since our study with synchronized ts-2 led us to conclude that the temperature-sensitive defect affects DNA replication directly, we would predict that polyoma DNA synthesis would be inhibited at 38°C even after it had begun in cells at 33°C. If our interpretation of the synchrony study were incorrect, the rate of viral DNA synthesis should be unaffected or increased after shift to 38°C. Moreover, viral DNA synthesis would be expected to continue whether a defect were “tight” or “leaky” in respect to Gl transit, for example. We therefore infected subconfluent ts-2 with polyoma, incubated the cultures for 48 hr at 33°C to allow accumulation of replicating viral DNA molecules, and then shifted to 38°C. Cultures in triplicate were pulse-labeled with 3H-TdR for 2 hour intervals between 30 min and 12 hr at 38”C, and viral DNA I quantitated on NSG. In all cases, a discrete peak of viral DNA I was observed. The “background” from uninfected cells was negligible, since many of the cells had reached confluence by the time the 3H-TdR was added, minimizing incorporation into cellular DNA. The data normalized to the first time point (052.5 hr at 38°C) are plotted in Figure 6. It can be readily seen that viral DNA synthesis is inhibited and with a time course similar to that of cellular DNA synthesis.

Discussion We have described the properties of a new temperature-sensitive mutant (ts-2) of Balb/3T3 cells. This mutant is genetically stable, and the mutant phenotype is recessive in cell hybrids with non-ts 3T3 (K. K. Jha and H. L. Ozer, unpublished data). The temperature-sensitive phenotype is independent of cell density in contrast to the only other temperature-sensitive mutant in cell growth previously reported in this cell line (Wittes and Ozer, 1973). ts-2 is termed a Dna- mutant because at 38°C the rate of DNA synthesis decreases before that of RNA or protein synthesis, indicating a defect more specifically related to the former. Both cellular and polyo-

1.0

0.5

,,,i

, 0

Figure

(

,

2

4

of Polyoma

Hours after Infection

Hours 38°C

24 47

Virus

at

on ts-2

at 38°C

Cell cpm Cell cpm

38OC 33°C

Uninfected

Infected

24

0.014

0.011

17

0.125

0.109

Log phase ts-2 were infected with polyoma virus at 33”C, incorporation of 3H-TdR (10 Kc/ml) into TCA-precipitable was determined in a 2 hr pulse at the times indicated.

and the material

,

,

,

,

,

,

6 8 IO HOURS AT 38C

6. Viral and Cell DNA Synthesis

3. Polyoma

DNA Synthesis Hours

2. Effect

,

at 38°C

,

I

I2

in ts-2

A log phase population containing 106 ceils was infected with polyoma, incubated at 33°C for 48 hr, shifted to 38OC, and pulse-labeled at various times for 2 hr with SH-TdR (10 +c/ml). Incorporation into viral (O-O) and cell (-0) DNA was measured. At the first point, there were 7.5 X lo4 cpm in viral DNA and 1 x 106 cpm in total cellular DNA.

Table

Table

,

Cells

33°C

3T3 3T3 3T3

30 47

ts-2 ts-2 ts-2

30 47

after

at 33°C

and 38°C

Infection

cpm in Viral DNA I oer 106 Cellsa

38OC

24

63,100 43,100 58,300

24

43:300b 10,360

3T3 cells: description of a mutant affected in cellular and polyoma virus DNA synthesis.

Cell, Vol. 7, 289-295, February 1976, Copyright0 1976 by MIT Temperature-Sensitive Mutants of Balb/3T3 Description of a Mutant Affected in Cellul...
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