Effects of temperature changes on thymidine kinase in heat- and cold-sensitive cell-cycle mutants and ‘wild-type’ murine P-815 cells

Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide.     

A mammalian somatic "cell cycle" mutant defective in G1

Variants or “mutants” temperature-sensitive (ts) for growth have been isolated by selection from a near-diploid mouse cell line. Thus far. 10 ts mutants which grow normally at 33° C, but not at 39° C, have been isolated. These ts mutants were then studied to determine if any manifested their defect at a unique point or stage in the cell cycle. This type of ts mutant is termed a “cell cycle” mutant. The first screen involves observing individual cells of an asynchronous culture for residual division after a shift from 33° C (permissive temperature) to 39° (nonpermissive temperature). A cell cycle mutant should show some fraction of the cells dividing only once at a normal rate after the shift. The ts variant B54 met this first criterion for a cell cycle mutant (i.e., 50% residual division) and was further analyzed. The second screening technique monitors (1) the rate of entry into S, (2) the length of G₂, and (3) the rate and duration of cells entering mitosis after a shift of an asynchronous culture to 39°. This experiment with B54 revealed that cells in G₁ at the time of the shift to 39° failed to enter S while cells already into S completed the cycle at 39°. These results suggest that B54 is defective in a G₁ function which is required for entry into S, but which is no longer needed once cells have entered S. Other results are presented which also support this hypothesis. In addition the ts function of B54 is apparently required for recovery from a “high density” G₁ arrest.     

Flavonoids in the Flowers of Gnaphalium affine D. Don

Thermonsenstivie division mutants were derived from Bacillus subtilis Marburg 168 thy trp₂ by means of membrane filtration after nitrosoguanidine mutagenesis. Among them, ts42 requiring uracil for normal growth at 48°C was investigated.

In the absence of uracil, the mutant cells grew normally at 37°C and stopped dividing after temperature shift to 48°C resulting in filaments of two to four times length of normal rods. The total cell number after temperature shift from 37 to 48°C, increased two to three fold in 90 min and remained constant thereafter. The viable count after the temperature shift to 48°C, increased 1.5 to 2 fold in initial 60 min and then decreased exponentially. A rapid restoration of colony forming ability was shown when the mutant cells were shifted back to the permissive temperature after 120 to 180 min of incubation at 48°C or when uracil was introduced to the culture at 48°C. This recovery of viability was partly observed even in the presence of chloramphenicol. The synthesis of RNA of this mutant was shown to decline 20 min after the temperature shift to 48°C whereas the syntheses of DNA and protein proceeded for more than 80 min at that temperature.

No newly isolated uracil requiring mutants formed filaments in the medium lacking uracil or showed growth pattern like ts42.     

A yeast lysis mutant: potential biotechnological applications

The conditional yeast lysis mutant cly8 was studied for potential biotechnological applications. The strain stops to grow immediately after a shift to elevated temperatures ( > 30°C). Cell viability (colony forming capacity) decreases at 37°C at a rate depending on the composition of the medium. However, at the elevated temperature cells still consume glucose and incorporate [¹⁴C]leucine into cell material. With decreasing viability the mutant cells become leaky for small, predominantly cytoplasmic components such as leucine or uridine but not for vacuolar storage products like arginine. No trichloroacetic acid-precipitable material could be detected in the medium after the shift to the elevated temperature indicating that leakiness was restricted to low molecular weight compounds. On acetate medium mutant cells became permeable only after prolonged incubation at 37°C but could be used for the oxidation of exogenous NADH. In comparison to the wild type the mutant also produced more glycerol. When the mutant cells were immobilized, glycerol production was in the same range at room temperature and at 28°C and could be maintained for several days.     

Properties of ts Cl mouse L cells which exhibit temperature-sensitive DNA synthesis.

ts Cl mouse L cells are temperature-sensitive (ts) in DNA synthesis. The protein involved undergoes inactivation at 38.5 °C, with an apparent half-life of 3–4 h. A variety of experimental approaches yield data indicating that the ts Cl gene product acts directly during the DNA-synthesis period, probably late during the duplication of chromosomal DNA. The specificity of the ts lesion is reflected in the fact that replication of mitochondrial DNA is unaffected for many hours after nuclear DNA synthesis is almost totally inhibited. Temperature inactivation is not due to degradation or to loss of template capacity of preformed DNA. ts Cl cells are able to enter a DNA-synthesis phase at the higher temperature, as indicated by radioautographic experiments and by studies in which cells, blocked at the permissive temperature (34 °C) in a pre-DNA synthesis phase by isoleucine deprivation, are subsequently incubated at 38.5 °C. Cells arrested early in DNA synthesis by hydroxyurea treatment at 34 °C continue such synthesis for a short interval after up-shift to 38.5 °C. However, they are then unable to complete the S phase in progress nor can they proceed into cell division. The kinetics of DNA synthesis in cells incubated at 38.5 °C and back-shifted to 34 °C are compatible with the model that the ts Cl locus encodes an S phase function.     

Characterization of a Chinese hamster cell with a temperature-sensitive mutation in folate metabolism

The Chinese hamster cell line, tsAUXB1, is auxotrophic for glycine, adenosine, and thymidine when grown at 38.5°C, but is prototrophic at 34°C. The evidence suggests that the temperature-sensitive lesion exists in the enzyme responsible for the addition of glutamyl residues onto intracellular folate derivatives. This enzyme appears to be synthesized constitutively in wild type cells.     

Biosynthesis of plasma membrane components by SV40-virus-transformed 3T3 mouse cells temperature sensitive for expression of some transformed cell properties

We have studied the plasma membranes of an SV40-transformed 3T3 cell line temperature sensitive for the transformed growth phenotype (ts H6-15 cells), and have found that they vary little as a function of temperature of cultivation. Analysis by polyacrylamide gel electrophoresis was performed on plasma membranes prepared from ts H6-15 cell cultured at the permissive (32 °C) and non-permissive (39 °C) temperatures and radioactively-labelled in several ways. No significant differences were seen when the electrophoretic patterns of polypeptides of the plasma membranes of ts H6-15 cells, grown through 3–4 generations in medium containing radioactive leucine (32 °C and 39 °C temperatures) were compared. Plasma membranes derived from cells similarly grown in medium with radioactive glucosamine indicated that extensive alterations in the intrinsic glycopeptides occurred in association with alteration in growth phenotype. A shift towards decreased synthesis of large molecular weight (? 100 000–160 000) glycopeptides occurred in cells grown at the temperature of non-transformed growtn (39 °C). A decrease in amount of a 1200 000 molecular weight glycopeptide at 39 °C was the most prominent of these alterations.We have studied the surface exposure of polypeptides and glycopeptides of intact cells grown at 32 and 39 °C, using lactoperoxidase-catalyzed iodination, NaBH₄ reduction of galactose oxidase-treated cells, and metabolic-labelling with glucosamine of trypsin-sensitive molecules. We found no major qualitative differences between whole cell extracts or between plasma membrane preparations of cells cultivated at the permissive and non-permissive temperatures. Of special interest was the observation that the formation and surface exposure of a trypsin-sensitive, 240 000 molecular weight polypeptide appeared not to be ts in ts H6-15 cells. The significance of these observations will be discussed.     

A novel temperature-sensitive mammalian cell line exhibiting defective prophase progression

A temperature-sensitive mammalian cell line has been isolated which grows and divides normally at the permissive temperature of 33°C. When incubated at 39°C, the nonpermissive temperature, interphase cells continue to enter a prophase-like state. Chromatin-like material condenses and coalesces into dark-staining clumps rather than into discernible chromosomes. Disappearance of the nuclear boundary is observed, but re-formation of the boundary around the clumps fails to occur. Incorporation of labeled precursors reveals a decrease in protein synthesis which is accompanied by a slower decrease in DNA synthesis. Approximately 0.2% of the mutant cells revert in their capability of growth and cell division at 39°C. These “revertants” are found to contain a higher number of chromosomes. The isolation of this mutant is based on the initial observation that the cells become rounded at the nonpermissive temperature. The cell-rounding process characteristic of mitotic cells should serve as a useful marker in the isolation of mitotic mutants.     

Production of non-defective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus

Cultures of three lines of mouse 3T3 cells transformed independently by the thermosensitive ts-a mutant of polyoma virus yield virus upon lowering their incubation temperature to 31°C. At 31°C, the internal pools of DNA of all three lines contain not only superhelical viral monomers, but also a small proportion of viral oligomers.From one of these three cell lines, several sublines of different clonal morphology were isolated at 38.5°C. The viral DNA synthesized at 31°C by each different subline displayed a unique oligomer pattern which has been stable through many cell passages and further reclonings. In contrast to the parental line, the monomer in most of these sublines is a minor component of the viral DNA pool. In one subline, more than 80% of the viral DNA consists of superhelical molecules about 1.6-times the size of a monomer. The specific infectivity of these molecules is only about one-tenth that of monomers, whereas the efficiency in transforming hamster (BHK21) cells is about twice that of monomers.     

A mammalian DNA- mutant decreasing nuclear DNA polymerase alpha activity at nonpermissive temperature.

A temperature-sensitive Dna^? mutant (ts T244) of the mouse FM3A cell line was characterized. ts T244 showed no increase in cell number and rapid decrease in the rate of DNA synthesis after temperature upshift (33°C→39°C). The activity of nuclear DNA polymerase α decreased as rapidly as the rate of DNA synthesis, whereas the activities of thymidine kinase and thymidylate kinase decreased more slowly. The results suggest that the level of nuclear DNA polymerase α has close relation to the ts defect of this Dna^? mutant.     

Unlinking of Cell Division from Deoxyribonucleic Acid Replication in a Temperature-sensitive Deoxyribonucleic Acid Synthesis Mutant of Escherichia coli

A new type of temperature-sensitive deoxyribonucleic acid (DNA) synthesis mutant, which can divide without a completion of DNA replication, was isolated from a thymidine-requiring Escherichia coli strain by means of photo-bromouracil selection after nitrosoguanidine mutagenesis. In this mutant, in spite of the fact that DNA synthesis stopped immediately after the temperature shift from 30 to 41 C, cells could continue to divide, though at a reduced rate. This cell division without DNA synthesis at 41 C is further supported by the following results. (i) Cell division took place at high temperature without addition of thymidine but not at all at 30 C. The parent strain of the mutant did not divide at 41 C without thymidine. (ii) Smaller cells isolated from the culture grown at 41 C did not contain DNA. This was shown by chemical analysis of the smaller cells and on electron micrographs. Ability of cells to divide was examined according to sizes of cells. By using the culture at 30 C, cells of various sizes were separated by means of sucrose-density gradient centrifugation. It was found that all cell fractions, including the smallest one, could divide at high temperature. These results suggest that in this mutant the completion of DNA replication is not required for triggering cell division at high temperature. Heat sensitivity of a factor which links cell division with DNA replication appears to be responsible. Some possible mechanisms of the coordination between cell division and DNA replication are discussed.     

A temperature sensitive mutation that reduces mitotic rate inDrosophila melanogaster

Summary A temperature-sensitive cell autonomous mutation ofDrosophila, l(1)ts-1126 (1–16±2), that affects the rate of cell division is described. When mutant animals are exposed to the restrictive temperature of 29°C during the first and second larval stages, the growth rate of the larvae is retarded. A delay in pupariation occurs during which larvae reach their full size, and the resulting flies are normal. When mutant animals are exposed to restrictive temperature during the third larval stage, growth is also retarded but no delay in pupariation occurs, and the resulting flies are reduced in size. Their small size is due in part to a decreased number of cells and in part to a smaller size of the cells.X-ray induced, marked, homozygousl(1)ts-1126 clones in an otherwise normal animal, are smaller in animals exposed to pulses of restrictive temperature when compared to clones in animals kept at permissive temperature of 22°C. Clone size decreases as pulse length increases. Clones on the wing blade induced 24 h after oviposition are smaller than clones induced at 48 h in animals grown at restrictive temperature. This result is interpreted as an inability of the slower dividingl(1)ts-1126 cells to survive when in competition with wildtype cells. The distribution of survivingl(1)ts-1126 clones in gynandromorphs grown at restrictive temperature supports this conclusion.     

Temperature-dependent internalization of virus glycoproteins in cells infected with a mutant of Semliki Forest virus.

When the ts-1 mutant of Semliki Forest virus (SFV) was grown in chick embryo or BHK 21 cells at the restrictive temperature (39 degrees C), its membrane glycoproteins were arrested in the endoplasmic reticulum, but started to migrate to the cell surface once the cultures were shifted to the permissive temperature (28 degrees C). If the temperature of infected cells was raised back to 39 degrees C, ts-1 glycoproteins disappeared from the cell surface as evidenced by loss of surface immunofluorescence and by radioimmunoassay based on the binding of 125I-labeled protein A. This phenomenon was specific for ts-1 at 39 degrees C as it was observed neither in cells infected with wild-type SFV at 39 degrees C nor with ts-1 at 28 degrees C. The disappearance of the ts-1 glycoproteins was due to internalization. The internalized proteins were digested, as shown by specific decrease of virus glycoproteins labelled with [35S]methionine at 39 degrees C before shift to 28 degrees C, and by concomitant release of acid soluble 35S-activity into the culture medium. Ts-1 infected cells were treated before shift back to 39 degrees C with Fab' fragments, prepared from IgG against the viral membrane glycoproteins. After shift back to 39 degrees C, the Fab' fragments disappeared from the cell surface. In the presence of chloroquine, they could be visualized in vesicular structures, using an anti-IgG-fluorescein isothiocyanate conjugate. The internalization of ts-1 glycoproteins was not inhibited by carbonylcyanide p-trifluoromethoxy phenylhydrazone, chloroquine, cytochalasin B, vinblastine, colcemid, or monensin.     

Lysine tRNA and cell division: a G1 cell cycle mutant is temperature sensitive for the modification of tRNA5Lys to tRNA4Lys.

Ts-694 is a temperature sensitive mutant of hamster cells which is blocked in the G1 phase of the cell cycle at the restrictive temperature of 39 degrees. A comparison of the Lys-tRNA isoacceptors by RPC-5 chromatography showed a decrease in tRNA5Lys and an increase in tRNA4Lys at 39 degrees. This was identical to the changes seen in confluent cultures at the permissive temperature of 33 degrees. These Lys-tRNA changes were not seen in ts-694 cells blocked in G1 by isoleucine deficiency, nor in two other G1 ts mutants at the restrictive temperature. Cells trapped in S phase by a thymidine block also contained decreased levels of tRNA4Lys when raised to 39 degrees. Both tRNA4Lys levels and cell division increased when the cells were returned to the permissive temperature. An in vitro assay was established for the modification of tRNA5Lys to tRNA4Lys with tRNA6Lys and tRNA2Lys as intermediates. The first reaction is the synthesis of tRNA6Lys which involves the introduction of a modified uridine at the third position of the anticodon. Extracts of 694 cells grown at 33 degrees were able to modify rat liver [3H] tRNA5Lys to tRNA6Lys and tRNA4Lys in vitro when assayed at 25 degrees but not at 39 degrees. Extracts of Balb/c 3T3 cells, however, were more active at 39 degrees than at 25 degrees showing that the normal enzyme is not temperature sensitive. Ts-694 cell tRNA, isolated from cells grown at 33 degrees was aminoacylated at both 25 degrees and 39 degrees with rat liver synthetases. tRNA4Lys was present at both temperatures indicating that ts-694 cells do not contain a temperature sensitive tRNA4Lys.     

The influence of fission line expression on the number and positioning of oral primordia in the cdaA1 mutant of Tetrahymena thermophila

During cytokinesis, furrowing creates new boundaries for daughter cells. Following a shift to a restrictive temperature, cells of the temperature-sensitive cell-division-arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis. When maintained under weak restrictive conditions (35°C), some of the chains were arrested prior to the start of fission line formation (D-shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B-shaped chains). In their second cell cycle following the temperature shift, the D-shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain. The B-shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle. These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes. They also indicate that, during cell division cycles, pre-existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity. © 1992 Wiley-Liss, Inc.     

Cell division cycle in mammalian cells : VIII. Mapping of G1 into six segments using temperature-sensitive cell cycle mutants

The G1 blocks in three temperature-sensitive (ts) Syrian hamster cell-cycle mutants have been mapped in relation to other G1 landmarks. Two mutants reported here, ts-559 and ts-694, show defective progression only in G1. When shifted from the permissive temperature of 33 degrees C to the non-permissive temperature of 39 degrees C, G1 cells of these two mutants show no further cell cycle progression, while cells in S, G2 and mitosis progress through the cell cycle but become blocked after entering G1. The two mutants complement each other, and also complement the previously reported mutant ts-550C with blocks in both G1 and G2 of the cell cycle. The locations of the G1 blocks in both ts-559 and ts-694 are before the hydroxyurea arrest point. The G1 ts point in ts-694 is prior to the isoleucine deprivation and serum starvation points, while the G1 block in ts-559 is after the serum starvation point but before the isoleucine block. Other G1 block points which have been reported are in mutants of different species and isolated in different laboratories, causing difficulties for relative positioning of the blocks in G1. The mutants for mapping in this study have been isolated from the same cell line. The G1 ts arrest points of ts-559 and ts-694, and that found in ts-550C, together with nutritional deprivations and metabolic inhibitors, provide seven reference points which divide G1 into six segments, each of which is bracketed by two adjacent points: mitosis, ts-694 block, serum starvation arrest point, ts-559 block, isoleucine deprivation arrest point, ts-550C block, hydroxyurea or excess-thymidine arrest segment.     

Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway

Cells of a Saccharomyces cerevisiae mutant that is temperature-sensitive for secretion and cell surface growth become dense during incubation at the non-permissive temperature (37°C). This property allows the selection of additional secretory mutants by sedimentation of mutagenized cells on a Ludox density gradient. Colonies derived from dense cells are screened for conditional growth and secretion of invertase and acid phosphatase. The sec mutant strains that accumulate an abnormally large intracellular pool of invertase at 37°C (188 mutant clones) fall into 23 complementation groups, and the distribution of mutant alleles suggests that more complementation groups could be found. Bud emergence and incorporation of a plasma membrane sulfate permease activity stop quickly after a shift to 37°C. Many of the mutants are thermoreversible; upon return to the permissive temperature (25°C) the accumulated invertase is secreted. Electron microscopy of sec mutant cells reveals, with one exception, the temperature-dependent accumulation of membrane-enclosed secretory organelles. We suggest that these structures represent intermediates in a pathway in which secretion and plasma membrane assembly are colinear.     

Partition kinetics and thermosensitive replication of pT169, a naturally occurring multicopy tetracycline resistance plasmid of Staphylococcus aureus

The temperature-sensitivity of a naturally occurring plasmid encoding inducible tetracycline resistance in Staphylococcus aureus has been examined in terms of incorporation of thymidine into plasmid DNA. The plasmid, pT169, has been found to have a multiplicity of about 15 copies per cell and to reduce sharply its replication rate immediately upon a shift to 42.5 °C. Plasmid replication at 42.5 °C continues at a rate equivalent to about 5% of the rate at 32 °C—sufficient to maintain a multiplicity of two to three copies per cell. Since under these conditions the plasmid is hereditarily stable, we conclude that there is a specific segregation mechanism, independent of replication, that ensures equal distribution of plasmid molecules to daughter cells during cell division.     

Temperature-dependent transmembrane potential changes in cells infected with a temperature-sensitive moloney sarcoma virus

Normal rat kidney cells (NRK) infected with the temperature-sensitive (ts) transformation mutant of Moloney murine sarcoma virus yielded a clone of cells, 6m2, that exhibited a transformed morphology at 33°C and a normal morphology at 39°C. Transmembrane potential (Em) was measured fluorometrically using a cyanine dye diS-C₃-(5). Fluorescence was inversely correlated with Em. Cells at 33°C had lower Em. Em changes were recorded within 15 minutes of temperature shift from 33°C to 39°C in both directions, increasing in the 33°C to 39°C direction and decreasing in the 39°C to 33°C direction. Uninfected NRK cells when shifted under the same condition exhibited small fluorescence changes in the 33°C to 39°C direction. Shifting from 39°C to 33°C resulted in Em changes similar to those in 6m2 cells. Also studied was a cell line infected with a spontaneous revertant of the ts mutant, designated 54-5A4; it was transformed at both temperatures. Shifting from 33°C to 39°C in both directions yielded small changes. Transmembrane potential changes in 6m2 cells precede other transformation-specific changes that occur after a temperature shift.     

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.