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.     

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.     

Cell cycle re-entry of quiescent mammalian nuclei following heterokaryon formation

When 3T3 mouse fibroblasts are made quiescent by serum deprivation and are then fused with tsAF8 hamster fibroblasts synchronized by a combination of high temperature block and hydroxyurea, the nuclei of binucleated heterokaryons which are formed enter S phase asynchronously in media containing low levels of serum. The tsAF8 nuclei of these biphasic heterokaryons enter S phase shortly after fusion, as do the tsAFS nuclei of homokaryons in the same culture. In contrast, the nuclei of the biphasic heterokaryons which have been contributed by quiescent 3T3 enter S phase only after a lag following fusion. This suggests that the quiescent nucleus within the heterokaryon is stimulated by factor(s) from the more advanced cell to re-enter the cell cycle in the absence of serum. In contrast to factors which induce the immediate synthesis of DNA, these factors may be those responsible for the transition of a cell from a non-proliferating to a proliferating state.     

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.     

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.     

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.     

Dominant versus recessive behavior of a cold- and a heat-sensitive mammalian cell cycle variant in heterokaryons

A heat-sensitive (hs, arrested at 39.5 degrees C, termed 21-Ta) and a cold-sensitive (cs, arrested at 33 degrees C, termed 21-Fb) clonal cell cycle variant were isolated from the same clone of the P-815 murine mastocytoma line. At the respective nonpermissive temperatures, both the hs and the cs variant were reversibly arrested in G1 phase, and numbers of cells forming colonies upon reincubation at the permissive temperature remained nearly constant for at least 6 days. Cells arrested in G1 by incubation at the respective nonpermissive temperatures were fused to cells of another P-815 clone (31-S) that had been arrested by serum deprivation. Upon reincubation in medium containing 10% serum for 48 h at 39.5 degrees C, 21-Ta x 31-S heterokaryons, similar to 31-S x 31-S homokaryons, entered the S phase, whereas at 33 degrees C, 21-Fb x 31-S heterokaryons, similar to 21-Fb x 21-Fb homokaryons, remained arrested in G1, indicating a recessive expression of the hs and a dominant expression of the cs phenotype.     

Murine temperature-sensitive DNA polymerase α mutant displays a diminished capacity to stimulate DNA synthesis in senescent human fibroblast nuclei in heterokaryons at the nonpermissive condition

We have investigated the capacity of a murine cell line with a temperature-sensitive (ts) mutation in the DNA polymerase α (Pola) locus and a series of ts non-Pola mutant cell lines from separate complementation groups to stimulate DNA synthesis, in senescent fibroblast nuclei in heterokaryons. In the Pola mutant × senescent heterodikaryons, both human and murine nuclei display significantly diminished levels of DNA synthesis at the restrictive temperature (39.5°C) as determined by [³H]thymidine labeling in autoradiographs. In contrast, all of the non-Pola mutants, as well as the parental (wild-type) murine cells, induced similar levels of DNA synthesis in both parental nuclei at the nonpermissive and permissive temperatures. Similarly, young human fibroblasts are also able to initiate DNA synthesis in heterokaryons with the ts Pola mutant at the two temperatures. In order to determine if complementation of the non-Pola mutants requires induction of serum responsive factors in the senescent cells, fusion studies of similar design were conducted with young and old human fibroblasts incubated in low serum (0.2%) for 48 hr prior to and after cell fusion. Again, a diminished level of DNA synthesis was observed at 39.5°C in the Pola mutant x senescent cell heterokaryons. In these low-serum studies, both parental nuclei in the Pola x young cell heterokaryons and the human nuclei in heterokaryons with one of the non-Pola mutants (FT107) also displayed diminished levels of DNA synthetic activity. All of the other mutants are able to support similar levels of synthetic activity at both temperatures in the presence of reduced serum. The nature of the mutation in three of the non-Pola lines has not been determined but, like the Pola mutant cells, are inhibited in the G₁ phase of the cell cycle when incubated at the nonpermissive temperature (39.5°C). The fourth non-Pola mutant line is known to have at least one ts mutation in the cdc2 gene and is inhibited in the G₂ phase when exposed to 39.5°C. These results suggest that there may be a functional deficiency of pol α in senescent human fibroblasts, and this replication factor may be one of the rate-limiting factors involved in loss of the capacity to initiate DNA synthesis in senescent cells. © 1994 Wiley-Liss, Inc.     

Microinjection of RNA polymerase II corrects the temperature-sensitive defect of tsAF8 cells

Summary tsAF8 cells area temperature-sensitive (ts) mutant of BHK cells that arrest in the G₁ phase of the cell cycle at the non-permissive temperature of 40.6 °C. Previous reports had suggesed that the temperature-sensitivity of these cells was based on a defect in either the synthesis, assembly or turnover of RNA polymerase II. We now show that the direct microinjection of purified RNA polymerase 11 into nuclei of tsAF8 cells corrects the ts defect and allows these cells to enter the S phase of the cell cycle.     

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.     

Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

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.     

Globin synthesis in fibroblasts fused with erythroblasts. II. Human fibroblasts.

Fusion of human (diploid) fibroblast monolayers with erythroblasts from 3-day chick embryos resulted in cultures containing on the average 14% heterokaryons and 8% fibroblast homokaryons. When these heterokaryon-containing cultures were labeled with radioactive amino acids during the first 24 h after fusion, the proportion of labeled proteins found in the globin region of analytical polyacrylamide gels showed a 40-fold increase compared with fibroblast homokaryons (0.08% vs. 4% of protein synthesized). Incorporation of radioactivity into globin decreased sharply during the second 24 h. Purified 35S-methionine-labeled globin from heterokaryon cultures gave rise to a tryptic fingerprint containing peptides characteristic of chick embryonic globins as late as 4 days after fusion. While fibroblasts in the fusion culture continue to go through the cell cycle normally, heterokaryons stop cycling almost completely soon after fusion.     

Reactivation of chick erythrocyte nuclei in heterokaryons with temperature-sensitive Chinese hamster cells.

Chinese hamster cell line K12 is temperature-sensitive for the initiation of DNA synthesis. K12 cells synchronized by serum deprivation were collected in early G1(G0). Heterokaryons were formed by fusing chick erythrocytes with serum-starved K12 cells through the use of UV-irradiated Sendai virus. At the permissive temperature (36.5 degrees C), erythrocyte nuclei in heterokaryons enlarged, the chromatin dispersed, and erythrocyte nuclei synthesized DNA at about the same time as the K12 nuclei. At the restrictive temperature (41 degrees C), erythrocyte nuclei enlarged, but neither erythrocyte nor K12 nuclei initiated DNA synthesis. When erythrocyte nuclei were fused with Wg-1A cells, the wild-type parent for ts K12 cells, both kinds of nuclei synthesized DNA at 36.5 degrees C and 41 degrees C. Activation of erythrocyte nuclei was inefficient in heterokaryons incubated in low-serum medium. The results indicate that serum factors and a cellular function defined by the K12 mutation are required for activation of chick erythrocyte nuclear DNA synthesis.     

Reactivation of chick erythrocyte nuclei in young and senescent WI38 cells

The chromatin of the dormant chick nucleus is dispersed in the heterokaryons made by Sendai virus fusion of phase II WI38 cells with chick erythrocyte nuclei. The erythrocyte nucleus resumes RNA synthesis and enters into DNA synthesis with the host nucleus. In the heterokaryons of phase III WI38 cells and chick erythrocytes, the nuclear chromatin is not dispersed and RNA synthesis occurs at a reduced rate. The differences in the physiological state of the young and senescent cells measured by [³H]uridine incorporation into nuclear RNA is reflected in the extent of reactivation of the chick erythrocyte nuclei in the cytoplasm of these cells. The reactivation of the chick nucleus in enucleated fibroblasts parallels the nucleated cells. The results of these studies are interpreted as evidence that there is a specific loss of nuclear function in the senescent cells.     

Interaction of human and chick DNA repair functions in UV-irradiated xeroderma pigmentosum-chick erythrocyte heterokaryons

Fusion of chick erythrocytes with human primary fibroblasts results in the formation of heterokaryons in which the inactive chick nuclei become reactivated. The expression of chick DNA repair functions was investigated by the analysis of the DNA repair capacity after exposure to ultraviolet (UV) irradiation of such heterokaryons obtained after fusion of chick erythrocytes with normal human or xeroderma pigmentosum (XP) cells of complementation groups A, B, C and D. Unscheduled DNA synthesis (UDS) in normal human nuclei in these heterokaryons is suppressed during the first 2–4 days after fusion. The extent and duration of this suppression is positively correlated with the number of chick nuclei in the heterokaryons. Suppression is absent in heterokaryons obtained after fusion of chicken embryonic fibroblasts with XP cells (complementation group A and C).Restoration of DNA repair synthesis is found after fusion in XP nuclei of all complementation groups studied. It occurs rapidly in XP group A nuclei, starting one day after fusion and reaching near normal human levels after 5–8 days. In nuclei of the B, C and D group increased levels of UDS are found 5 days after fusion. At 8 days after fusion the UDS level is about 50% of that found in normal human nuclei. The pattern of UDS observed in the chick nuclei parallels that of the human counterpart in the fusion. A fast complementation pattern is also observed in chick fibroblast-XP group A heterokaryons resulting within 24 h in a UDS level comparable with that in chick fibroblast-normal human heterokaryons. In heterokaryons obtained after fusion of chick fibroblasts with XP group C cells UDS remains at the level of chick cells. These data suggest that reactivation of chick erythrocyte nuclei results in expression of repair functions which are able to complement the defects in the XP complementation groups A, B, C and D.     

Phenotypic expression in chick erythrocyte X rat myoblast hybrids and in chick myoblast X rat myoblast hybrids

Attempts were made to reprogram chick erythrocyte nuclei to specify the synthesis of chick myosin. Chick erythrocytes were fused with rat myogenic cells with the aid of UV-inactivated Sendai virus. In the heterokaryons and hybrid myotubes which resulted from this fusion, the erythrocyte nuclei resumed RNA synthesis and formed nucleoli. Although some new chick antigens developed in those myotubes which contained fully reactivated chick erythrocyte nuclei, accumulation of chick myosin could not be detected by immunological methods. Neither small heterokaryons nor large hybrid myotubes which were actively synthesizing rat myosin reacted with antibodies directed against chick myosin. A small number of mononucleated cells, believed to be synkaryons formed by mitotic division of heterokaryons, did, however, react strongly with antibodies directed against chick myosin and showed a cross striation typical of skeletal muscle. The frequency of such cells was too low, however, to permit karyological analysis or further characterization of the antigen. Hybrids between chick myoblasts and rat myoblasts produced both chick and rat myosin thus indicating that simultaneous translation of chick and rat mRNA for myosin in a common cytoplasm was possible. In summary the evidence obtained suggested that reprogramming of chick erythrocyte nuclei, if it did occur in the present system, was a rare phenomenon.The possibility that hybrids between chick erythrocytes and rat myoblasts expressed markers typical of an erythroid phenotype was examined by immune staining with antibodies directed against chick haemoglobin. The results suggested that haemoglobin was introduced into hybrid cells by erythrocytes which failed to lyse before fusion. The intensity of this immune fluorescence decreased with increasing time after fusion. The rate at which this decrease occurred was not affected by inhibition of RNA synthesis. Thus, there was no evidence for the accumulation of haemoglobin in the hybrid cells.     

Synthesis of rat myosin light chains in heterokaryons formed between undifferentiated rat myoblasts and chick skeletal myocytes

The control of gene expression during terminal myogenesis was explored in heterokaryons between differentiated and undifferentiated myogenic cells by analyzing the formation of species specific myosin light chains of chick and rat skeletal muscle. Dividing L6 rat myoblasts served as the biochemically undifferentiated parent. The differentiated parental cells were mononucleated muscle cells (myocytes) that were obtained from primary cultures of embryonic chick thigh muscle by blocking myotube formation with EGTA and later incubating the postimitotic cells in cytochalasin B. Heterokaryons were isolated by the selective rescue of fusion products between cells previously treated with lethal doses of different cell poisons. 95-99% pure populations of heterokaryons formed between undifferentiated rat myoblasts and differentiated chick myocytes were obtained. The cells were labeled with [35S]methionine, and whole cell extracts were analyzed on two-dimensional polyacrylamide gels. These heterokaryons synthesize the light chain of chick myosin and both embryonic and adult light chains of rat skeletal myosin. Control homokaryons formed by fusing undifferentiated cells to themselves did not synthesize skeletal myosin light chains. Control heterokaryons formed between undifferentiated rat myoblasts and chick fibroblasts also failed to synthesize myosin light chains. These results indicate that differentiated chick muscle cells provide some factor that induces L6 myoblasts to synthesize rat myosin light chains. This system provides a model for investigating the processes by which differentiated cell functions are induced.     

ACQUISITION OF CHICK CYTOSOL THYMIDINE KINASE ACTIVITY BY THYMIDINE KINASE-DEFICIENT MOUSE FIBROBLAST CELLS AFTER FUSION WITH CHICK ERYTHROCYTES

Chick-mouse heterokaryons were obtained by UV-Sendai virus-induced fusion of chick erythrocytes with thymidine (dT) kinase-deficient mouse fibroblast [LM(TK^-)] cells. Autoradiographic studies demonstrated that 1 day after fusion, [³H]dT was incorporated into both red blood cell and LM(TK^-) nuclei of 23% of the heterokaryons. Self-fused LM(TK^-) cells failed to incorporate [³H]dT into nuclear DNA. 15 clonal lines of chick-mouse somatic cell hybrids [LM(TK^-)/CRB] were isolated from the heterokaryons by cultivating them in selective hypoxanthine-aminopterin-thymidine-glycine medium. LM(TK^-) and chick erythrocytes exhibited little, if any, cytosol dT kinase activity. In contrast, all 15 LM(TK^-)/CRB lines contained levels of cytosol dT kinase activity comparable to that found in chick embryo cells. Disk polyacrylamide gel electrophoresis and isoelectric focusing analyses demonstrated that the LM(TK^-)/CRB cells contained chick cytosol, but not mouse cytosol dT kinase. The LM(TK^-)/CRB cells also contained mouse mitochondrial, but not chick mitochondrial dT kinase. Hence, the clonal lines were somatic cell hybrids and not LM(TK^-) cell revertants. The experiments demonstrate that chick erythrocyte cytosol dT kinase can be activated in heterokaryons and in hybrid cells, most likely as a result of functions supplied by mouse fibroblast cells.