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.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type2.

Fourteen temperature-sensitive mutants of human adenovirus type2, which differed in their plaquing efficiencies at at the permissive and nonpermissive temperatures by 4 to 5 orders of magnitude, were isolated. These mutants, which could be assigned to seven complementation groups, were tested for their capacity to synthesize adenovirus DNA at the nonpermissive temperature. Three mutants in three different complementation groups proved deficient in viral DNA synthesis. The DNA-negative mutant H2ts206 complemented the DNA-negative mutants H5ts36 and H5ts125, whereas mutant H2ts201 complemented H5ts36 only. Among the DNA-negative mutants, H2ts206 synthesized the smallest amount of viral DNA at the nonpermissive temperature (39.5 C). Data obtained in temperature shift experiments indicated that a very early function was involved in temperature sensitivity. In keeping with this observation, early virus-specific mRNA was not detected in cells infected with H2ts206 and maintained at 39.5 C. Prolonged (52 h) incubation of cells infected with H2ts206 at the nonpermissive temperature led to the synthesis of a high-molecular-weight form of viral DNA.     

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.     

A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1

The effect of restrictive temperature on ubiquitin conjugation activity has been studied in cells of ts20, a temperature-sensitive cell cycle mutant of the Chinese hamster cell line E36. Ts20 is arrested in early G2 phase at nonpermissive temperature. Immunoblotting with antibodies to ubiquitin conjugates shows that conjugates disappear rapidly at restrictive temperatures in ts20 mutant but not in wild type E36 cells. The incorporation of 125I-ubiquitin into permeabilized ts20 cells is temperature-sensitive. Addition of extracts of another G2 phase mutant, FM3A ts85, with a temperature-sensitive ubiquitin activation enzyme (E1), to permeabilized ts20 cells at restrictive temperatures fails to complement their ubiquitin ligation activity. This indicates that the lesions in the two mutants are similar. Purified E1 from reticulocytes restores the conjugation activity of heat-inactivated permeabilized ts20 cells. Ubiquitin conjugation activity of cell-free extracts of ts20 cells was temperature-sensitive and could be restored by adding purified reticulocyte E1. Purified reticulocyte E2 or E3, on the other hand, did not restore the ubiquitin conjugation activity of heat-treated ts20 extracts. These results are consistent with the conclusion that ts20 has temperature-sensitive ubiquitin-activating enzyme (E1). The fact that two E1 mutants (ts20 and ts85) derived from different cell lines are arrested at the S/G2 boundary at restrictive temperatures strongly indicates that ubiquitin ligation is necessary for passage through this part of the cell cycle. The temperature thresholds of heat shock protein synthesis of ts20 and wild type E36 cells were identical. The implications of these findings with respect to a suggested role of ubiquitin in coupling between protein denaturation and the heat shock response are discussed.     

Adenovirus transcription. IV. Synthesis of viral-specific RNA in human cells infected with temperature-sensitive mutants of adenovirus 5.

Cytoplasmic RNA sequences produced in HeLa cells infected with the adeno-virus 5 temperature-sensitive mutants ts1, ts2, ts9, ts17, ts18, ts19, ts20, ts22, ts49, ts36, and ts125 were characterized by hybridization to DNA probes generated by strand separation of restriction endonuclease fragments of adenovirus 5 DNA. Two "early' mutants defective in DNA synthesis, ts125 and ts36, fail to make wild-type levels of all previously reported classes of late RNA at the nonpermissive temperature. At 40.5 degrees C, both ts125 and ts36 synthesize a wild-type complement of early cytoplasmic RNA 16 h after infection. Under these conditions, no "late' cytoplasmic RNA sequences were observed. Similarly, nuclear RNA present in these cells resembled early cytoplasmic RNA rather than late nuclear RNA. All the late adenovirus 5 temperature-sensitive mutants synthesized normal wild-type levels of late cytoplasmic RNA at the nonpermissive temperature, except ts2, which appears to overproduce certain cytoplasmic species.     

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.     

The function(s) provided by the adenovirus-specified, DNA-binding protein required for viral late gene expression is independent of the role of the protein in viral DNA replication

The adenovirus type 2 (Ad2) host range mutant Ad2hr400 grows efficiently in cultured monkey cells at 37 degrees C, but is cold sensitive for plaque formation and late gene expression at 32.5 degrees C. After nitrous acid mutagenesis of an Ad2hr400 stock, cold-resistant variants were selected in CV1 monkey cells at 32.5 degrees C. One such variant, Ad2ts400, was also temperature sensitive (ts) for growth in both CV1 and HeLa cells. Marker rescue analysis has been used to show that the two phenotypes, cold resistant and temperature sensitive, are due to two independent mutations, each of which resides in a different segment of the gene encoding the 72-kilodalton DNA binding protein (DBP). The cold-resistant mutation (map coordinates 63.6 to 66) is a host range alteration that enhances the ability of the virus to express late genes and grow productively in monkey cells at 32.5 degrees C. The temperature-sensitive mutation is in the same complementation group and maps to the same segment of the DBP gene (map coordinates 61.3 to 63.6) as the well-characterized DBP mutant Ad5ts125. Like Ad5ts125, Ad2ts400 is unable to replicate viral DNA or to properly shut off early mRNA expression at the nonpermissive temperature. Two sets of experiments with Ad2ts400 suggest that DBP contains separate functional domains. First, when CV1 cells are coinfected at the nonpermissive temperature with Ad2 plus Ad2ts400 (Ad2 allows DNA replication and entry into, but not completion of, the late phase of infection), normal late gene expression and productive growth occur. Second, temperature shift experiments show that, although DNA replication is severely restricted at the nonpermissive temperature in ts400-infected monkey cells, late gene expression occurs normally. These results indicate that the DBP activity required for normal late gene expression in monkey cells is functional even when the DBP's DNA replication activity is disrupted.     

Cytoplasmic regulation of two G1-specific temperature-sensitive functions

tsAF8 and ts13 cells are temperature-sensitive (ts) mutants of BHK cells that specifically arrest, at nonpermissive temperature, in the G1 phase of the cell cycle. These two mutants can complement each other. Both cell lines can be made quiescent by serum deprivation (G0). When subsequently stimulated by serum, they can enter S phase at 34 degrees C but not at 39.5 degrees-40.6 degrees C. We have used these mutants to determine whether the nucleus is needed during the G0 leads to S transition for the expression of the G1 ts functions. For this purpose, we fused cytoplasts of G0-tsAF8 with whole ts13 cells in G0, and cytoplasts of G0-ts13 with whole tsAF8 cells in G0. Serum stimulation at the nonpermissive temperature induced DNA synthesis in both types of such fusion products. No DNA synthesis was induced by serum stimulation at the nonpermissive temperature in fusion products constructed between either G0-tsAF8 cytoplasts and whole G0-tsAF8 cells or G0-ts13 cytoplasts and whole G0-ts13 cells. These results demonstrate that the information for these two ts functions, which are required for entry of serum-stimulated cells into the S phase, are already present in the cytoplasm of G0 cells--that is, before serum stimulation commits them to the transition from the nonproliferating to the proliferating state.     

Human ubiquitin-activating enzyme (E1): compensation for heat-labile mouse E1 and its gene localization on the X chromosome.

We have constructed interspecific somatic cell hybrids between a temperature-sensitive (ts) mutant cell line of mouse FM3A cells, ts85, that has a heat-labile ubiquitin-activating enzyme (E1) and a human diploid fibroblast cell line, IMR-90. A hybrid clone that could grow stably at a nonpermissive temperature (39 degrees C) was obtained. Segregation of the hybrid cells at a permissive temperature (33 degrees C) gave rise to temperature-sensitive clones. The electrophoresis of extracted histones and karyotype analysis of the segregants revealed a close correlation of the ability to grow at 39 degrees C, the presence of uH2A (ubiquitin-H2A semihistone) at 39 degrees C, and the presence of the human X chromosome. One of the hybrid clones that could grow at the nonpermissive temperature contained the X chromosome as the only human chromosome. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of affinity-purified E1 showed that this hybrid clone contained both human and mouse type E1. Thus we conclude that the functional gene for human E1 is located on the X chromosome.     

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.     

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.     

Alterations to controls of cellular DNA synthesis by adenovirus infection.

Human adenovirus type 5 and temperature-sensitive mutants ts36, ts37, and ts125 induced cellular DNA synthesis in quiescent rodent cells at both permissive and nonpermissive temperatures. Cellular DNA synthesis induced by adenovirus type 5 or by serum required protein synthesis for both initiation and continuation, whereas viral DNA synthesis was not dependent upon continued protein synthesis once it was initiated. Both cellular and viral DNA replication was induced in adenovirus type 5-infected cells in the presence of dibutyryl cyclic AMP at concentrations which inhibited induction by serum which suggested that some of the controls of DNA synthesis in serum-treated and virus-infected cells are different. After adenovirus infection of quiescent cells, there was a decrease in the number of cells with G1 DNA content and an increase in cells with G2 diploid and greater DNA contents. Thus, adenovirus type 5 induces a complete round of cellular DNA replication, but in some cells, it induces a second round without completion of a normal mitosis. These results suggest that adenovirus type 5 is able to alter cell growth cycle controls in a way which may be related to its ability to transform cells.     

Characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain A59 with maturation defects in the spike protein.

Two temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59, ts43 and ts379, have been described previously to be ts in infectivity but unaffected in RNA synthesis (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). We present a detailed analysis of the protein synthesis of the mutant viruses at the permissive (31 degrees C) and nonpermissive (39.5 degrees C) temperatures. It was found that synthesis of the nucleocapsid protein N and the membrane protein M of both viruses was insensitive to temperature. However, the surface protein S of both viruses was retained in the endoplasmic reticulum at the nonpermissive temperature. This was shown first by analysis of endoglycosidase H-treated and immunoprecipitated labeled S proteins. The mature Golgi form of S was not present at the nonpermissive temperature for the ts viruses, in contrast to wild-type (wt) virus. Second, gradient purification of immunoprecipitated S after pulse-chase labeling showed that only wt virus S was oligomerized. We conclude that the lack of oligomerization causes the retention of the ts S proteins in the endoplasmic reticulum. As a result, ts virus particles that were devoid of S were produced at the nonpermissive temperature. This result could be confirmed by biochemical analysis of purified virus particles and by electron microscopy.     

Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovrius defective in viral DNA synthesis.

Infection of African green monkey kidney cells with type 5 adenovirus leads to the synthesis of two infected, cell-specific proteins with approximate molecular weights of 72,000 and 48,000, that bind specifically to single-stranded but not double-stranded DNA. The production of these two proteins was studied after infection with two DNA-negative adenovirus mutants belonging to different complementation groups (H5 ts36 and H5 ts 125). Both DNA binding proteins were detected in cells infected with either mutant at the permissive temperature (32 C) AND ALSO IN H5 ts36-infected cells at the nonpermissive temperature (39.5 C). In H5 ts125-infected cells at 39.5 C, however, less than 5% of the normal wild-type level of these DNA binding proteins was detectable. When H5 ts125-infected cells were labeled with radioactive leucine at 32 C and subsequently shifted to 39.5 C in the presence of unlabeled leucine (chase), the level of DNA binding proteins found in these infected cells was markedly reduced compared to cultures not shifted to 39.5 C. These data suggest that the DNA binding proteins themselves were temperature sensitive. This conclusion was confirmed by experiments in which the DNA binding proteins were eluted from DNA cellulose with buffers of increasing temperatures (thermal elution). The H5 ts 125 proteins were shown to elute at lower temperatures than either wild-type or H5 ts36 proteins. These results are taken to indicate that the H5 ts125 mutant codes for a DNA binding protein that is thermolabile for continued binding to single-stranded DNA.     

Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins.

The vaccinia virus B1 gene encodes a 34-kDa protein with homology to protein kinases. In L cells infected nonpermissively with mutants containing lesions in the B1 gene (ts2 and ts25), the infectious cycle arrests prior to DNA replication. In this report, we demonstrate that DNA synthesis ceases when cultures infected with these mutants at 32 degrees C are shifted to the nonpermissive temperature (39.5 degrees C) in the midst of DNA replication. We also show that B1 protein is synthesized transiently during the early phase of infection, even when the progression to later stages of gene expression is prevented. Although wild-type (wt) B1 is stable, the ts B1 proteins are markedly labile in both L and BSC40 cells at both permissive and nonpermissive temperatures. These results suggest that the ts phenotype of the mutants is complex and may in part reflect a temperature-dependent requirement for kinase activity, an induction of temperature sensitivity in B1 substrates under nonpermissive conditions, and/or ts complementation by host factors. To facilitate biochemical analyses, recombinant wt B1, ts2 B1, and ts25 B1 were produced in Escherichia coli. The wt protein was able to phosphorylate serine and threonine residues on several exogenous substrates in vitro. The activity of ts25 B1 was 3% that of the wt enzyme, and no detectable kinase activity was associated with ts2 B1. In light of the inactivity of the ts2 B1 protein in vitro and its extreme lability in vivo, we attempted to isolate a vaccinia virus B1 null mutant by targeted interruption of the B1 gene at 32 degrees C. No null mutants were isolated. These results indicate that the B1 protein kinase provides a vital function which cannot be supplied by the host or circumvented by incubation at 32 degrees C.     

Ubiquitin metabolism in ts85 cells, a mouse carcinoma line that contains a thermolabile ubiquitin activating enzyme

Red blood cell-mediated microinjection was used to introduce radioiodinated ubiquitin into ts85 cells, a mouse cell line that contains a thermolabile ubiquitin-activating enzyme (E1). The proportion of ubiquitin present as histone conjugates, high molecular weight conjugates, and free molecules was then determined by gel electrophoresis and autoradiography. When ts85 cells were incubated at the nonpermissive temperature, 39.5 degrees C, high molecular weight conjugates accumulated. This unexpected result was confirmed by Western blot analyses. To determine whether ubiquitin conjugates formed under nonpermissive conditions or merely persisted after the temperature increase, ts85 cells were incubated at 39.5 degrees C to generate large amounts of conjugates and then shifted to 42 degrees C. The higher temperature resulted in a 25% reduction in conjugates, but upon return to 39.5 degrees C, the ubiquitin conjugates were restored to pre-42 degrees C amounts. Since all changes in ubiquitin conjugate levels occurred above 39.5 degrees C, ts85 cells can couple ubiquitin to cellular proteins even after prolonged culture at nonpermissive temperatures. Western blot analyses showed that less than 10% of the E1 molecules present in ts85 cells at 31 degrees C remained after 2 h at 39.5 degrees C. However, when 125I-ubiquitin was added to extracts from heated ts85 cells an apparent high molecular weight form of E1 and thiol ester adducts between ubiquitin and the E2 carrier proteins were detected by electrophoresis at 4 degrees C. Considering both in vivo and in vitro demonstrations that heated ts85 cells retain the ability to conjugate ubiquitin to endogenous proteins, considerable caution must be exercised in the design and interpretation of proteolysis experiments using this mutant cell line.     

Incubation at the nonpermissive temperature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a temperature-sensitive ubiquitin-activating enzyme (E1).

In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization. Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.     

DNA polymerase-primase complex in wild-type and ts A1S9 mouse L-cells, temperature-sensitive for DNA replication during cell cycle progression

ts A1S9 mutant cells, derived from wild type WT-4 mouse L-cells, are temperature-sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase-alpha and DNA primase as a function of time. Forty-seven hours after temperature upshift DNA polymerase-alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT-4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase-alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]-labelled cells, with a specific monoclonal antibody directed against DNA polymerase-alpha. We have found that, irrespective of temperature of cultivation of WT-4 or ts A1S9 cells, this antibody precipitated polypeptides of 220, 186, 150, 110, 68-70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5 degrees C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha-polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase-alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase-alpha complex and polymerase delta complex synthesize co-ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase-alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.