Temperature-sensitive Saccharomyces cerevisiae mutant defective in lipid biosynthesis.

A temperature-sensitive mutant of Saccharomyces cerevisiae (DAM303) is described that exhibits an early defect in lipid biosynthesis at the restrictive growth temperature, 37 degrees C. This strain rapidly lost viability after 1 h of incubation at 37 degrees C, and this was accompanied by a significantly reduced incorporation of 32Pi into cellular lipid and an accumulation of [1-14C]acetate into the free fatty acid fraction. The temperature-sensitive DAM303 mutation failed to complement the sec13 mutation described by Novick et al. (Cell 21:205-215, 1980), and from analysis of invertase secretion in the temperature-sensitive DAM303 strain, it is clear that the loss of invertase secretion in the mutant occurs after the loss of phospholipid synthesis. Although the precise nature of the temperature-sensitive lesion in the DAM303 strain has still to be identified, the results from the study of this mutant indicate that a defect in lipid biosynthesis can be correlated with subsequent alterations in extracellular protein secretion and loss of other macromolecular functions including DNA, RNA, and protein syntheses. From studies of this mutant, two procedures of enriching for other temperature-sensitive mutants with defects in lipid biosynthesis have emerged: inositol overproduction and screening for increased buoyant densities.     

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.     

Physiological consequences of mutations in Escherichia coli heat shock dnaK and dnaJ genes.

Mutations in the heat shock genes, dnaK and dnaJ, cause severe defects of several cellular functions. Null dnaJ and dnaKdnaJ mutations can be transduced in a restricted range of temperature. The efficiency of transformation with three unrelated plasmids, viz pACYC184, pBR322 and pSC101, is two times lower in dnaK mutants while the dnaJ mutant is characterized by slightly impaired transformation with pSC101 only. The lack of DnaJ function negatively influences the stability of pSC101 at 42 degrees C, and this plasmid cannot be stably maintained at 30 degrees C in the delta dnaKdnaJ mutant. The double deletion mutant, delta dbaKdnaJ, is characterized by impaired osmoadaptation. The galactokinase content is lower in both mutants tested compared with wild-type strains even at 30 degrees C. The efficient complementation of some of these defects by the wild-type alleles present on low-copy number plasmid was achieved.     

A novel Saccharomyces cerevisiae secretory mutant possesses a thermolabile phosphomannose isomerase.

A temperature-sensitive mutant of Saccharomyces cerevisiae was identified which at the restrictive temperature of 37 degrees C is unable to secrete a number of cell wall-associated proteins and thus resembles previously reported sec mutants. In contrast to other sec mutants, however, both the temperature-sensitive growth and the secretion defects can be repaired by the addition of D-mannose to growth media. We show that the mutant possesses a single, apparently recessive mutation which leads to the production of a thermolabile phosphomannose isomerase.     

epsilon-COP is a structural component of coatomer that functions to stabilize alpha-COP.

We isolated a novel yeast alpha-COP mutant, ret1-3, in which alpha-COP is degraded after cells are shifted to a restrictive temperature. ret1-3 cells cease growth at 28 degrees C and accumulate the ER precursor of carboxypeptidase Y (p1 CPY). In a screen for high copy suppressors of these defects, we isolated the previously unidentified yeast epsilon-COP gene. epsilon-COP (Sec28p) overproduction suppresses the defects of ret1-3 cells up to 34 degrees C, through stabilizing levels of alpha-COP. Surprisingly, cells lacking epsilon-COP (sec28 Delta) grow well up to 34 degrees C and display normal trafficking of carboxypeptidase Y and KKXX-tagged proteins at a permissive temperature. epsilon-COP is thus non-essential for yeast cell growth, but sec28 Delta cells are thermosensitive. In sec28 Delta cells shifted to 37 degrees C, wild-type alpha-COP (Ret1p) levels diminish rapidly and cells accumulate p1 CPY; these defects can be suppressed by alpha-COP overproduction. Mutant coatomer from sec28 Delta cells behaves as an unusually large protein complex in gel filtration experiments. The sec28 Delta mutation displays allele-specific synthetic-lethal interactions with alpha-COP mutations: sec28 Delta ret1-3 double mutants are unviable at all temperatures, whereas sec28 Delta ret1-1 double mutants grow well up to 30 degrees C. Our results suggest that a function of epsilon-COP is to stabilize alpha-COP and the coatomer complex.     

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.     

Ultrastructure of two secretory mutants of Saccharomyces cerevisiae as revealed by freeze-fracture technique

Permissive and restrictive phenotypes of two secretory mutants of Saccharomyces cerevisiae, sec 1 and sec 18, were studied by freeze-fracture technique. The sec 1 mutant, in addition to accumulating secretory vesicles, was characterized by a disappearance of the plasma membrane invaginations and by an aggregation of intra-membrane particles in vacuolar membranes. A prolonged incubation of the cells at 37 degrees C led to pathological fusion of some vesicles with the plasma membrane. After the cells were transferred back to the permissive temperature the invaginations reappeared rapidly while the accumulated vesicles disappeared only after budding had been resumed. The sec 18 mutant, apart from having distended endoplasmic reticulum membranes, also lost the plasma membrane invaginations at 37 degrees C and regained them at 24 degrees C. The described ultrastructural changes are typical for the restrictive phenotypes and represent further manifestations of the pleiotropic effect of the respective sec mutations.     

Characterization of a thermosensitive sporulation mutant of Bacillus subtilis affected in the structural gene of an intracellular protease.

A thermosensitive sporulation mutant (ts-15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42 degrees C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts-15R1 and ts-15R2) and were also transmitted together by transformation into the wild type. Immunological studies have shown that when ts-15 is grown at 42 degrees C it synthesizes a 'cryptic' protein with apparently the same antigenic properties as the wild type or as ts-15 mutant grown at the permissive temperature (30 degrees C). The intracellular proteases from the wild type and from ts-15 grown at 30 degrees C and 42 degrees C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts-15 grown at 30 degrees C was 64000--65000 in the absence of sodium dodecylsulfate and 31000--32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts-15 grown at 42 degrees C showed the same molecular weight of 32000--34000 in the presence or in the absence of sodium dodecylsulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts-15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form.     

A novel mutation in ribosomal protein S4 that affects the function of a mutated RF1

Release factors (RF) 1 and 2 trigger the hydrolysis of the peptide from the peptidyl-tRNA during translation termination. RF1 binds to the ribosome in response to the stop codons UAG and UAA, whereas RF2 recognizes UAA and UGA. RF1 and RF2 have been shown to bind to several ribosomal proteins. To study this interaction in vivo, prfA1, a mutant form of RF1 has been used. A strain with the prfA1 mutation is temperature sensitive (Ts) for growth at 42 degrees C and shows an increased misreading of UAG and UAA. In this work we show that a point mutation in ribosomal protein S4 can, on the one hand, make the RF1 mutant strain Ts(+); on the other hand, this mutation increases the misreading of UAG, but not UAA, caused by prfA1. The S4 mutant allele, rpsD101, is a missense mutation (Tyr51 to Asp), which makes the cell cold sensitive. The behaviour of rpsD101 was compared to the well-studied S4 alleles rpsD12, rpsD14, and rpsD16. These three mutations all confer both a Ts (44 degrees C) phenotype and show a ribosomal ambiguity phenotype, which rpsD101 does not. The three alleles were sequenced and shown to be truncations of the S4 protein. None of the three mutations could compensate for the Ts phenotype caused by the prfA1 mutation. Hence, rpsD101 differs in all studied characteristics from the three above mentioned S4 mutants. Because rpsD101 can compensate for the Ts phenotype caused by prfA1 but enhances the misreading of UAG and not UAA, we suggest that S4 influences the interaction of RF1 with the decoding center of the ribosome and that the Ts phenotype is not a consequence of increased readthrough.     

Simian virus 40 mutants carrying two temperature-sensitive mutations.

Five temperature-sensitive mutants of simian virus 40 containing two temperature-sensitive mutations were isolated. The double mutant of the A and D complementation groups, like the D mutants, failed to complement by conventional complementation analysis and did not induce host DNA synthesis at 40 degrees C. However, under conditions that suppressed the D defect, the A:D double mutant expressed only the A defect. Thus, viral DNA replication dropped rapidly after this mutant was shifted from permissive to restrictive temperatures. The A:D double mutant failed to transfrom at the restrictive temperature when subconfluent Chinese hamster lung monolayers were used. Double mutants of A:B, A:C, and A:BC complementation groups, like their A parent, were defective in viral DNA replication, in the induction of host DNA synthesis and in the transformation of secondary Chinese hamster lung cells at the nonpermissive temperature.     

The Characteristics and Genetic Map Location of a Temperature Sensitive DNA Mutant of E. COLI K12

A temperature sensitive strain of E. coli K12 has been isolated in which residual DNA synthesis occurs at the 40 degrees C restrictive temperature; syntheses of RNA, protein and DNA precursors are not directly affected. The mutation has been designated dna-325 and is located at 89 min on the E. coli map in the same region where the dnaC locus is found. dnaC mutants are considered to be defective in DNA initiation. Some of the data are consistent with the view that the dna-325 mutation is temperature sensitive in the process of DNA initiation rather than DNA chain elongation: (1) more than two cell divisions occur after a shift to 40 degrees C; (2) upon a shift down to 30 degrees C, cell division occurs again only after the DNA content of the cells has doubled; (3) 80% more DNA is made at 30 degrees C in the presence of chloramphenicol after prior inhibition of DNA synthesis at 40 degrees C. These three observations indicate that rounds of DNA replication were completed at 40 degrees C. Also (4) infective lambda particles can be made at 40 degrees C long after bacterial DNA replication has ceased. It appears however that some DNA initiation can occur at 40 degrees C since (1) a limited amount of DNA synthesis does occur at 40 degrees C after prior alignment of the chromosomes by amino acid starvation at 30 degrees C, and (2) after incubation in bromouracil at the restrictive temperature, heavy DNA is found with both strands containing bromouracil.     

Further characterization of recessive suppression in yeast. Isolation of the low-temperature sensitive mutant of Saccharomyces cerevisiae defective in the assembly of 60 S ribosomal subunit

It has been shown that recessive suppressor mutations in the yeast Saccharomyces cerevisiae may cause sensitivity towards low temperatures (very slow growth or lack of growth at 10 degrees C). One of the sup 1 low temperature sensitive (Lts-) mutants, 26-125A-P-2156, was studied in detail. After a prolonged period of incubation (70 h) under restrictive conditions the protein synthesis apparatus in the mutant cells was irreversibly damaged. In addition, Lts- cells incubated under restrictive conditions synthesize unequal amounts of ribosomal subunits, the level of 60 S subunit being reduced. It has been suggested that the recessive suppression is mediated by a mutation in the gene coding for 60 S subunit component, probably a ribosomal protein. The mutation leads simultaneously to a defect in the assembly of 60 S subunit and to low-temperature sensitive growth of the mutant.     

Biosynthesis and secretion of several enzymes in Escherichia coli dnaK and dnaJ mutants

Escherichia coli null dnaJ and dnaKdnaJ mutants were defective in the biosynthesis and secretion of several enzymes. The synthesis of beta-galactosidase induced in delta dnaJ and delta dnaKdnaJ mutants was abolished at 42 degrees C and significantly decreased at 30 and 37 degrees C. The activity of alkaline phosphatase in the periplasm in both mutant strains at high temperature was lower than in the wild-type strain. The synthesis of b-type cytochromes was defective in two deletion mutants while the synthesis of nitrate reductase-A at 42 degrees C was influenced by dnaK mutation only. The lack of DnaK and DnaJ does not impair the activity of catechol 2,3-dioxygenase irrespective of growth temperature.     

Mutants of Escherichia coli Unable to Make Protein at 42 C

Members of a collection of mutants of Escherichia coli unable to form colonies on nutrient agar at 42 C have been characterized on the basis of their growth response to a shift from 32 to 42 C in liquid medium. Forty-four mutants, which show an abrupt, nonlethal cessation of growth when moved to the restrictive temperature, have been characterized with respect to the effect of the mutation responsible for temperature sensitivity on deoxyribonucleic acid, ribonucleic acid, and protein synthesis. In 12 mutants, the mutation causing temperature sensitivity of growth primarily affects protein synthesis, in each case through an altered aminoacyl-transfer ribonucleic acid synthetase. Mutants with temperature-sensitive glutamyl-, phenylalanyl-, and valyl-transfer ribonucleic acid synthetases have been obtained, and the genes specifying these enzymes have been mapped by conjugation and transduction. Another mutant has been shown to possess a temperature-sensitive tryptophanyl-transfer ribonucleic acid synthetase, but this is not responsible for inability to grow at 42 C on media containing tryptophan.     

CDC37 is required for p60v-src activity in yeast.

Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C. However, p60v-src levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form.     

Accumulation of cyclic GMP in filaments of Escherichia coli BUG6

Experiments with Escherichia coli BUG6, a temperature-sensitive cell division mutant, have shown that at the restrictive temperature (42 degrees C) the loss of cell division potential (filamentation) was accompanied by an unusual increase in intracellular cyclic GMP (cGMP). At the permissive temperature (30 degrees C), cell division proceeded normally, and cGMP did not accumulate. Increasing the osmotic strength of the medium with NaCl suppressed filamentation in BUG6 at 42 degrees C and also suppressed the temperature-sensitive accumulation of cGMP. The addition of nalidixic acid to BUG6 at 30 degrees C induced filamentation but failed to cause cGMP accumulation. A similar accumulation of cGMP has not been observed in other E. coli strains.     

Protein synthesis in yeast. Identification of an altered elongation factor in thermolabile mutants of the yeast Saccharomyces cerevisiae

Cell-free extracts from the wild type yeast strain (A364A) and from a group of noncomplementing mutants that are conditionally defective in translation were preincubated at a restrictive temperature prior to incubation at a permissive temperature for protein synthesis. Results of these experiments showed that upon exposure to the restrictive temperature (39 degrees C), all five of the noncomplementing mutants lost ability to incorporate amino acid into protein. The wild type parent strain retained better than 80% of the activity under identical conditions of heat treatment. Mutant extracts could be revived to incorporate amino acid by the addition of the purified yeast elongation factor 3. Factors 1 and 2 had no effect. The heat-treated extract from one mutant did not supplement the activity of the other mutant. Although all five of the mutants were inactivated by preincubation at 39 degrees C, each showed a variable rate and extent of thermolability. Heat-treated mutant extracts were fully active in polyphenylalanine synthesis with liver ribosomes but not with the yeast ribosomes. Since liver ribosomes do not require factor 3, this assay then confirms that factor 3 is the thermolabile component in this group of noncomplementing mutants.     

Mutant alleles of Arabidopsis RADIALLY SWOLLEN 4 and 7 reduce growth anisotropy without altering the transverse orientation of cortical microtubules or cellulose microfibrils

The anisotropic growth of plant cells depends on cell walls having anisotropic mechanical properties, which are hypothesized to arise from aligned cellulose microfibrils. To test this hypothesis and to identify genes involved in controlling plant shape, we isolated mutants in Arabidopsis thaliana in which the degree of anisotropic expansion of the root is reduced. We report here the characterization of mutants at two new loci, RADIALLY SWOLLEN 4 (RSW4) and RSW7. The radial swelling phenotype is temperature sensitive, being moderate (rsw7) or negligible (rsw4) at the permissive temperature, 19 degrees C, and pronounced at the restrictive temperature, 30 degrees C. After transfer to 30 degrees C, the primary root's elongation rate decreases and diameter increases, with all tissues swelling radially. Swelling is accompanied by ectopic cell production but swelling is not reduced when the extra cell production is eliminated chemically. A double mutant was generated, whose roots swell constitutively and more than either parent. Based on analytical determination of acid-insoluble glucose, the amount of cellulose was normal in rsw4 and slightly elevated in rsw7. The orientation of cortical microtubules was examined with immunofluorescence in whole mounts and in semi-thin plastic sections, and the orientation of microfibrils was examined with field-emission scanning electron microscopy and quantitative polarized-light microscopy. In the swollen regions of both mutants, cortical microtubules and cellulose microfibrils are neither depleted nor disoriented. Thus, oriented microtubules and microfibrils themselves are insufficient to limit radial expansion; to build a wall with high mechanical anisotropy, additional factors are required, supplied in part by RSW4 and RSW7.