Reversion of temperature-sensitive mutation by inhibition of proteasome-mediated degradation of mutated D123 protein.

A temperature-sensitive cell-cycle mutant of the 3Y1 rat fibroblast cell line, 3Y1tsD123 has in the D123 gene coding region a point mutation which causes instability of the D123 protein. Temperature-sensitive G1 arrest of the mutant is caused by increased degradation of the D123 protein at restrictive temperature. In this study we found that the selective proteasome inhibitors lactacystin and MG132 inhibited degradation of the mutated D123 protein in cell lines overexpressing the mutated D123 protein, followed by accumulation of a modified form (increased molecular weight other than by ubiquitination) of the D123 protein. Although a temperature-resistant revertant of the mutant had no further mutation in the D123 gene coding region, the modification of the mutated D123 protein was inhibited and the mutated D123 protein was rendered stable. The modification was also inhibited in the hybrid cell lines between the revertant and the cell line overexpressing the mutated D123 protein. These facts imply that the mutated D123 protein receives unidentified modification before degradation in the proteasome, and that the revertant expresses a gene inhibiting this modification.     

Induction of DNA synthesis by fibroblast growth factor in temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts arrested at restrictive temperature.

Four temperature-sensitive cell-cycle mutants of rat 3Y1 clonal fibroblasts representing separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125 and 3Y1tsH203) are arrested at restrictive temperature, primarily with a G1-phase DNA content (temperature arrest). We examined various factors affecting signal transduction for activity which induces DNA synthesis at the restrictive temperature when added to the temperature-arrested cultures of these mutants. The factors examined were theophylline, dibutyryl cyclic AMP, cholera toxin (CT), dibutyryl cyclic GMP, sodium nitroprusside, phorbol 12-myristate 13-acetate, 1-oleoyl 2-acetylglycerol, bombesin, vasopressin, basic fibroblast growth factor (FGF), platelet-derived growth factor, A23187, monensin, epidermal growth factor (EGF), insulin and fetal calf serum (FCS). None of these factors induced DNA synthesis in 3Y1tsH203. In one mutant (3Y1ts121), FGF, EGF and FCS individually induced DNA synthesis. In the other 2 mutants (3Y1tsD123 and 3Y1tsG125), FGF and CT individually induced DNA synthesis. The FGF-induced DNA synthesis was suppressed by islet-activating protein (IAP) in 3Y1tsD123 and 3Y1tsG125, but not in 3Y1tsF121. The CT-induced DNA synthesis was also suppressed by IAP, as previously shown. When temperature-arrested cultures were shifted to a permissive temperature, all 4 mutants initiated DNA synthesis in the presence of IAP. These results suggest that (1) a cell can prepare for the initiation of DNA synthesis by using several independent signal transduction pathways, and (2) in a given situation, the cell uses a particular pathway because of its availability, which depends on the culture conditions.     

Transformation by v-H-ras does not restore proliferation of a set of temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts

Three temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsG125, and 3Y1tsH203, each belonging to distinct complementation groups) were transformed with plasmid DNA carrying Harvey murine sarcoma virus cDNA. The criteria for transformation were increase in saturation cell density, capability to clone in soft agar, and alteration in the cellular morphology. At 39.8 degrees C (restrictive temperature of the parental cell lines), all the transformed sublines of each mutant ceased to proliferate and were arrested reversibly in the G1 phase of the cell cycle like the parental lines. At both 39.8 degrees C and 33.8 degrees C (permissive temperature for the parental lines), all the untransformed parental lines synthesized p21ras at low rate. At 33.8 degrees C, all the transformed sublines synthesized p21ras at much higher rate and expressed the morphological phenotype characteristic to v-H-ras-induced transformation. At 39.8 degrees C, the rate of p21ras synthesis was not changed in the transformed sublines of 3Y1tsD123 and 3Y1tsG125, and the morphology of transformed phenotype also remained intact. In the transformed subline of 3Y1tsH203, the rate of p21ras synthesis was lowered at 39.8 degrees C to that seen in the untransformed parental line, and the transformed phenotype in morphology disappeared. In all of the transformed sublines, the amount of v-H-ras mRNA markedly expressed at both 33.8 degrees C and 39.8 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abortive transformation of temperature-sensitive mutants of rat 3Y1 cells by simian virus 40: effect of cellular arrest states on entry into S phase and cellular proliferation

Four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, representing independent complementation groups, cease to proliferate predominantly with a 2n DNA content, at the restrictive temperature (39.8 degrees C) (temperature arrest) or at the permissive temperature (33.8 degrees C) at a confluent cell density (density arrest) (Ohno et al., 1984). We studied the temperature- or the density-arrested cells of these mutants infected with simian virus 40 (SV40) or its mutants affecting large T or small t antigen with respect to kinetics at 39.8 degrees C of entry into S phase and cellular proliferation. Three mutants, 3Y1tsD123, 3Y1tsF121 and 3Y1tsG125, expressed T antigen and entered S phase at 39.8 degrees C from both the arrested states after infection with either wild-type, tsA mutants, or a .54/.59 deletion mutant of SV40, whereas in the density-arrested 3Y1tsH203, expression of T antigen and entry into S phase were inefficient and ts. Following the WT-SV40 induced entry into S phase, the temperature-arrested 3Y1tsD123 detached from the substratum with no detectable increase in cell number, whereas the density-arrested ones completed a round of the cell cycle and then detached. 3Y1tsF121 and 3Y1tsG125 in the both arrested states proliferated through more than one generation. 3Y1tsF121 and 3Y1tsG125 in the density-arrested state infected with tsA mutants once proliferated and then ceased to increase in number as the percentage of T-antigen positive population decreased. These results suggest that wild-type and tsA-mutated large T antigens are able to overcome the cellular ts blocks of entry into S phase in the 3 ts mutants of 3Y1 cells in both the arrested states, and that small t antigen is not required to overcome the blocks. It is also suggested that cellular behaviors subsequent to S phase (viability, mitosis, and proliferation in the following generations) depend on cellular arrest states, on traits of cellular ts defects, and on the duration of large T antigen expression.     

Serum-independent regulation of initiation of DNA synthesis relating to temperature-sensitive defect in rat 3Y1tsD123 fibroblasts and its compensation by simian virus 40

Randomly proliferating 3Y1tsD123 cells are arrested in G1 phase within 24 h after a shift up to 39.8 degrees C (temperature arrest), yet the density-arrested cells (prepared at 33.8 degrees C) enter S phase at 39.8 degrees C with serum stimulation, with or without preexposure to 39.8 degrees C for 24 h (Zaitsu and Kimura 1984a). When the density-arrested 3Y1tsD123 cells were preexposed to 39.8 degrees C for 96 h, they lost the ability to enter S phase at 39.8 degrees C by serum stimulation and required a longer lag time to enter S phase at 33.8 degrees C by serum stimulation than did the cells not preexposed to 39.8 degrees C. Simian virus 40 induced cellular DNA synthesis at 39.8 degrees C in the density-arrested 3Y1tsD123 preexposed to 39.8 degrees C for 96 h. In the absence of serum after a shift down to 33.8 degrees C, the temperature-arrested 3Y1tsD123 cells entered S phase and then divided once. We postulate from these results that (1) the ts defect in 3Y1tsD123 is involved in a serum-independent process. Once this process is accomplished, its accomplishment is invalidated slowly with preexposure to 39.8 degrees C. This and the serum-dependent processes occur in parallel but not necessarily simultaneously. The accomplishment of both (all) processes is required for the initiation of S phase. The density-arrested 3Y1tsD123 cells have accomplished the serum-independent process related to the ts defect, but have not accomplished serum-dependent processes. In case of the temperature-arrested 3Y1tsD123 cells, the reverse holds true. The lag time for entry into S phase depends on the preparedness for the initiation of DNA synthesis (on the extent of accomplishment of each of all processes required for entry into S phase). (2) To induce cellular DNA synthesis, simian virus 40 stimulates directly the serum-independent process. However, we do not rule out the possibility that simian virus 40 stimulates serum-dependent processes simultaneously.     

Effect of temperature and cell density on cellular protein content in temperature-sensitive mutants of rat 3Y1 diploid fibroblasts

Summary We examined cellular protein content in four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) under various conditions of culture that affect cell proliferation. When proliferation of the ts mutants was inhibited at a nonpermissive temperature (39.8°C) in the G₁ phase, prominent accumulation of cellular protein occurred in three mutants (3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) but not in 3Y1tsD123. The over-accumulation of protein at 39.8°C in the former three mutants was inhibited at high cell densities. At low cell densities there was an upper limit in the protein accumulation at 39.8°C. When the three mutants, proliferation-arrested at high cell densities at 33.8°C, were replated sparsely in fresh medium and shifted to 39.8°C, proliferation was completely inhibited whereas over-accumulation of protein occurred. These results indicating dissociation of protein accumulation and cell proliferation suggest that the two events are regulated by different mechanisms. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (1984) to K. Y. from the Ministry of Education, Science, and Culture, Japan.     

Temperature-sensitive clones of herpes simplex virus type 1 from laboratory and clinical strains. I. Cloning and basic pathogenetic and immunogenic properties]

Two HSV-1 strains were used in the study: McIntyre laboratory strain and "eye" strain isolated from a patient. Temperature-sensitive clone of HSV-1 was isolated from McIntyre strain as a consequence of virus replication carried out at lowered temperature (28 degrees C). Temperature-resistant clones were obtained from both strains through passages at 39 degrees C and through heating for four times at 45 degrees C. Pathogenic properties of the temperature clones obtained were determined in inbred mice Balb/c and CFw/Pzh. A loss of pathogenicity for mice of temperature-sensitive clone and an increase of pathogenicity of temperature-resistant clones were noted as compared to parental strains. It was found that an introduction of temperature-sensitive clone, with lowered virulence immunizes against highly virulent temperature-resistant clone.     

Arrest states in a set of mutants of rat 3Y1 cells temperature-sensitive for entering S phase

Four temperature-sensitive (ts) mutants of rat 3Y1 cells (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested at 39.8°C mainly with a 2N DNA content (temperature-arrested cells). The states of these cells were compared with findings in case of cells arrested at 33.8°C at saturation density (density-arrested cells), with regard to the ability to enter S phase after release from arrest or after serum stimulation at 39.8°C. With the 3Y1tsD123, the ts defect is an event which seems essential for the initiation of S phase and occurs after mitosis but not after release from the density arrest. The defect in 3Y1tsF121 related to the efficiency of utilization of serum component(s). In case of 3Y1tsG125, the state of temperature arrest appeared to locate between the state of density arrest and the beginning of S phase. There was no significant difference between the density- and the temperature-arrested cells, in case of 3Y1tsH203.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. I. Rescue of a Simian Virus 40 Temperature-Sensitive Mutant by Passage in Permissive Transformed Monkey Lines

Passage of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 at the permissive temperature in each of three permissive lines of SV40-transformed monkey CV1 cells resulted in the emergence of temperature-insensitive virus, which plated like wild-type SV40 at the restrictive temperature on normal CV1 cells. In independent experiments, the amount of temperature-insensitive virus that appeared after passage on transformed cells was from 10(3)- to 10(6)-fold greater than the amount of ts-revertant virus that appeared after an equal number of passages in nontransformed CV1 cells. The virus rescued by passage on transformed cells bred true upon sequential plaque purification, plated on normal CV1 cells with single-hit kinetics at the restrictive temperature, and displayed no selective growth advantage on transformed cells compared to non-transformed cells. Hence, the reversion of the ts phenotype is neither due to complementation effects nor to the selection of preexisting revertants, which grow better on transformed cells. In the accompanying article (T. Vogel et al., J. Virol. 24:541-550, 1977), we present biochemical evidence that the rescue of tsD202 mediated by passage on transformed cells is due to recombination with the resident SV40 genome. Parallel experiments in which tsA, tsB, and tsC SV40 mutants were passaged in each of the three permissive lines of SV40-transformed monkey cells resulted in either only borderline levels of rescue (tsA mutants) or no detectable rescue (tsB and tsC mutants). Evidence is presented that the resident SV40 genome of the transformed monkey lines is itself a late ts mutant, and we suggest that this accounts for the lack of detectable rescue of the tsB and tsC mutants. We furthermore suggest that the borderline level of rescue observed with two tsA mutants is related to a previous finding (Y. Gluzman et al., J. Virol. 22:256-266, 1977) which indicated that the resident SV40 genome of the permissive transformed monkey cells is defective in the function required for initiation of viral DNA synthesis.     

Genetics of resistance to phosphonoacetic acid in strain KOS of herpes simplex virus type 1.

A DNA- temperature-sensitive mutant of herpes simplex virus type 1 exhibiting thermolabile DNA polymerase activity, tsD9, was shown to be resistant to phosphonoacetic acid (PAA) when plated at the permissive temperature. ts+ revertants of tsD9 were PAA sensitive and exhibited DNA polymerase activity intermediate between that of the wild-type virus and tsD9, indicating that both temperature sensitivity and sensitivity to PAA are controlled by the same gene. Since the position of tsD9 on the existing herpes simplex virus type 1 linkage map is known, the locus for PAA resistance--and therefore for the structural gene for viral DNA polymerase--has been identified.     

Induction of endoreduplication in temperature-sensitive mutants of rat 3Y1 fibroblasts

Four temperature-sensitive mutants of rat 3Y1 fibroblasts belonging to separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly with a 2C DNA content, when cells proliferating at 33.8 degrees C are shifted up to 39.8 degrees C (Ohno et al., 1984). Zaitsu and Kimura (submitted for publication) showed that 3Y1tsF121 cells synchronized in the early S phase were arrested with a 4C DNA content at 39.8 degrees C. We studied the traverse through the S and G2 phases at 39.8 degrees C in the four ts mutants synchronized at the early S phase and found that 3Y1tsG125 and 3Y1tsH203 cells were arrested with a 4C DNA content as 3Y1tsF121, while 3Y1tsD123 cells went through S and G2 phases and underwent mitosis. When 3Y1tsF121 and 3Y1tsG125 mutants arrested at 39.8 degrees C were shifted down to 33.8 degrees C, a substantial fraction of the cells with a 4C DNA content started, with a certain lag period, DNA synthesis without intervening mitosis and underwent the first mitosis with a lag period similar to that in the cells arrested with a 2C DNA content. The tetraploid cells thus generated had a proliferating ability lower than that of diploid cells.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. II. Biochemical Evidence for Genetic Exchange

The genome of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 rescued by passage on transformed permissive monkey lines (see accompanying paper [Y. Gluzman et al., J. Virol. 24:534-540, 1977]) was analyzed by restriction endonuclease cleavage mapping to obtain biochemical evidence that the rescue of the ts phenotype results from recombination with the resident SV40 genome of the transformed cell. It was demonstrated that the endonuclease R. HaeIII cleavage site, which is located at 0.9 map unit in the standard viral genome (and which is in the proximity of the known map position of the tsD lesion), is missing in the DNAs of the parental tsD202 virus and of three independent revertants of tsD202. In contrast, this cleavage site was shown to be present in the DNAs of four out of five independently derived rescued D202 populations and in the DNA of the SV40 strain, 777, used to transform the monkey cells. Comparison of the endonuclease R. Hin(II + III) cleavage patterns of SV40 strain 777 DNA and tsD202 DNA revealed differences in the electrophoretic mobilities of Hin fragments A, B, and F. However, the corresponding Hin fragments from all four rescued D202 genomes were identical in their mobilities to those of tsD202 DNA, indicating that these regions of the rescued D202 genome are characteristic of the tsD202 parent. We conclude, therefore, that the genome of the rescued D202 virus is a true recombinant, since it contains restriction endonuclease cleavage sites characteristic of both parents, the endogenous resident SV40 genome of the transformed monkey cells and the exogenous tsD202 mutant.     

Genetic Analysis of Simian Virus 40 IV. Inhibited Transformation of Balb/3T3 Cells by a Temperature-Sensitive Early Mutant

The temperature-sensitive early mutant, ts(*)101, was characterized during productive infection in monkey cells, and the results are presented in an accompanying paper. This paper demonstrates that although 101 mutant virions adsorb normally to confluent Balb/3T3 mouse cells at both permissive (33 C) and restrictive (38.5 C) temperatures, T antigen synthesis and transformation, abortive and stable, are inhibited at both temperatures (host-range inhibition). T antigen synthesis is temperature sensitive, whereas abortive and stable transformation are not. Clones of 101-transformed Balb/3T3 cells were isolated, and virus was rescued from all clones at both permissive and restrictive temperatures. The rescued virus was as temperature sensitive as the original transforming 101 virions.     

Herpes simplex virus DNA polymerase as the site of phosphonoacetate sensitivity: temperature-sensitive mutants.

Temperature-sensitive (ts) mutants in a number of complementation groups of herpes simplex virus type 1 (HSV-1) are deficient in DNA polymerase induction at the restrictive temperature. Twenty-two mutants in 15 complementation groups were tested for sensitivity to phosphonoacetate (PAA), a compound that inhibits HSV replication in vivo and the DNA polymerase in vitro. One mutant, tsD9, was resistant to PAA (Pr), whereas all others were sensitive. Revertants of tsD9 to the ts+ phenotype simultaneously lost PAA resistance. Additional Pr mutants were isolated from ts mutants belonging to several complementation groups of HSV-1. Double mutants (ts Pr phenotype) were used in three-factor recombination analyses to locate the PAA locus on the genetic map at a position indistinguishable from the ts lesion in tsD9. In all cases, resistance or sensitivity to PAA in vivo was correlated with resistance or sensitivity of DNA polymerase in vitro. These data are compatible with the temperature-sensitive lesion of tsD9 and the determinant of PAA sensitivity both residing in the structural gene for DNA polymerase.     

Simian virus 40 functions required for the establishment and maintenance of malignant transformation.

Members of the five classes of temperature-sensitive simian virus 40 mutants were tested for their ability to transform Chinese hamster lung cells. Two criteria for transformation were used: the ability to form clones in medium with low serum concentrations and the ability to overgrow a monolayer. Only A mutants failed to transform at the restrictive temperature when subconfluent Chinese hamster lung monolayers were used. However, both A and D mutants failed to transform at the restrictive temperature when confluent monolayers and depleted medium were used. When transformed clones were selected, purified by recloning, and examined at both temperatures, only cell lines induced by A mutants lost the transformed phenotype at the higher temperature. Thus, A function is required for maintenance of the transformed phenotype in Chinese hamster lung cells. A function is known to be required for the initiation of viral DNA synthesis in permissive cells. Therefore, transformation may be a consequence of the introduction into a cell of the capacity for aberrant initiation of DNA replication.     

Functional analysis of Rpn6p, a lid component of the 26 S proteasome, using temperature-sensitive rpn6 mutants of the yeast Saccharomyces cerevisiae

Rpn6p is a component of the lid of the 26 S proteasome. We isolated and analyzed two temperature-sensitive rpn6 mutants in the yeast, Saccharomyces cerevisiae. Both mutants showed defects in protein degradation in vivo. However, the affinity-purified 26 S proteasome of the rpn6 mutants grown at the permissive temperature degraded polyubiquitinated Sic1p efficiently, even at a higher temperature. Interestingly, their enzyme activity was even higher at a higher temperature, indicating that once made mutant proteasomes are stable and have little defect in the proteolytic function. These results suggest that the deficiency in protein degradation observed in vivo is rather due to a defect in the assembly of a holoenzyme at the restrictive temperature. Indeed, both rpn6 mutants grown at the restrictive temperature were defective in assembling the 26 S proteasome. A striking feature of the rpn6 mutants at the restrictive temperature was that there appeared a protein complex composed of only four of the nine lid components, Rpn5p, Rpn8p, Rpn9p, and Rpn11p. Altogether, we conclude that Rpn6p is essential for the integrity/assembly of the lid in the sense that it is necessary for the incorporation of Rpn3p, Rpn7p, Rpn12p, and Sem1p (Rpn15p) into the lid, thereby playing an essential role in the proper function of the 26 S proteasome.     

Temperature-sensitive multicellular mutants of Wangiella dermatitidis.

Three temperature-sensitive morphological mutants of Wangiella dermatitidis were isolated and characterized. The mutants grew in the yeastlike morphology at the permissive temperature (25 degrees C) but expressed a multicellular (Mc) phenotype at the restrictive temperature (37 degrees C). Cultures of Mc 2 and 3 incubated at the restrictive temperature showed rapid reductions in the percentage of budded cells in the population. In contrast, budding continued for several generations in cultures of Mc 1. Incubation of cultures of Mc 2 and 3 at the restrictive temperature for 48 h resulted in nearly total conversion of yeastlike cells to the multicellular form; about 50% of the cells of Mc 1 had converted to multicellular forms after 48 h at the restrictive temperature. Studies using radiolabeled compounds documented that DNA, RNA, and protein synthesis continued at the restrictive temperature. The results suggest that multicellularity is the result of inhibition of bud emergence and cell separation without inhibition of growth nuclear division, and cytokinesis.     

Isolation of Temperature-Sensitive Mutants of Arabidopsis thaliana That Are Defective in the Redifferentiation of Shoots

Three temperature-sensitive mutants of Arabidopsis thaliana that were defective in the redifferentiation of shoots were isolated as tools for the study of organogenesis. M3 lines were constructed by harvesting M3 seeds separately from each M2 plant. Comparative examination of shoot redifferentiation in root explants of 2700 M3 lines at 22[deg]C (permissive temperature) and at 27[deg]C (restrictive temperature) led to the identification of seven temperature-sensitive mutant lines. Genetic tests of three of the seven mutant lines indicated that temperature-sensitive redifferentiation of shoots in these three lines resulted from single, nuclear, recessive mutations in three different genes, designated SRD1, SRD2, and SRD3. The morphology of root explants of srd mutants cultured at the restrictive temperature suggests that the products of these SRD genes function at different stages of the redifferentiation of shoots.