The cdc 22 mutation by Schizosaccharomyces pombe is a temperature-sensitive defect in nucleoside diphosphokinase

A number of temperature-sensitive cdc- mutants of Schizosaccharomyces pombe that are affected in DNA replication, were screened for the absence of deoxynucleoside triphosphate(s) when blocked at their restrictive temperature. The preliminary screening simply involved analysis of perchloric acid-soluble cell extracts by two-dimensional thin-layer chromatography on poly(ethyleneimine)-impregnated cellulose. One mutant strain, cdc 22-M45, was found which apparently lacked dTTP. Pulse-labelling of intracellular nucleotides revealed that not only did dTTP become depleted, but that dTDP accumulated when this mutant was blocked by a temperature shift-up, indicating a defective nucleoside diphosphokinase. Nucleoside diphosphokinase from cdc 22-M45 was less active than that from wild-type strain 972 when assayed at high temperatures. The nucleoside diphosphokinase of the mutant also has an altered Km for dTDP at both permissive (25 degrees C), and at the restrictive (36.8 degrees C) temperatures. At the restrictive temperature the Km for dTDP of the mutant enzyme is more than 11-times greater than that of the wild type. Characterisation of the biochemical basis of the defect in this cdc- mutant has shown that in S. pombe, despite its having an apparently complex system of genetic control over progression through S-phase, one factor at least is merely availability of a nucleoside triphosphate precursor to DNA synthesis.     

Cold-sensitive mutant with defective growth at 5 degrees C from a psychrotrophic bacterium

A cold-sensitive (CS) mutant of the psychrotroph, Bacillus psychrophilus, was obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenization and penicillin counterselection. In the presence of citrate, the wild-type grew well at both 5 and 20 degrees C whereas the CS mutant grew well at 20 degrees C (the permissive temperature) but, at 5 degrees C (the restrictive temperature), grew at a reduced rate for two to three generations followed by a complete plateau in growth. Upon return of the CS mutant to 20 degrees C, after a delay of about 40 h, growth resumed at the appropriate rate. The CS mutant exhibited growth rates similar to parental rates on a wide variety of carbon sources at 5 degrees C, but when Krebs cycle intermediates were used as substrates and in the presence of an equimolar amount of citrate, the typical cold-sensitive growth pattern occurred. Comparison of oxidative phosphorylation in the parent and CS mutant indicated that no phosphorylation occurred at 5 degrees C in the CS mutant during the plateau in growth. Examination of the effect of temperature on ATPase activity showed that at 5 degrees C the specific activity of ATPase isolated from the CS mutant grows at 5 degrees C was 15-fold less than the ATPases isolated from wild-type cells grown at either 5 or 20 degrees C and 10.5-fold lower than ATPase from CS mutant cells grown at 20 degrees C. The large reduction in CS mutant ATPase activity at 5 degrees C appears to be at least partly due to an effect on synthesis since citrate did not inhibit preformed ATPase.     

Physiological studies of a temperature-sensitive sporulation mutant of Bacillus cereus T.

Growth of temperature-sensitive mutant Bacillus cereus T JS22-C occurred normally at the restrictive temperature (37 degrees C), but sporulation was blocked at stage 0. The production of extracellular and intracellular proteases and of alkaline phosphatase occurred at 37 degrees C, but the expression of a functional tricarboxylic acid cycle did not. At the permissive temperature (26 degrees C), the mutant sporulated at a slightly lower frequency (60%) and at a lower rate than the parent strain. The oxidation of organic acids, which accumulate in the growth medium began at T0 in cultures of the parent strain but was delayed until about T3 in cultures of the mutant. Later events in sporulation were also delayed in the mutant by about 3 h. Experiments in which the temperature of growth was shifted from 37 to 26 degrees C or from 26 to 37 degrees C at various times showed that the temperature-sensitive event began approximately 1 h after the end of exponential growth and ended when the cells reached the end of stage II (septum formation). The absence of a functional tricarboxylic acid cycle in cells of the mutant grown at 37 degrees C or shifted from 26 to 37 degrees C before T1 did not appear to be due to a lesion in one of the structural genes of the tricarboxylic acid cycle but was more likely due to the inability of the cells to derepress the synthesis of some of the enzymes of that cycle.     

Production of Bacteriophage by Temperature-Sensitive Sporulation Mutants of Bacillus cereus T

Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permissive temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction is accompanied, in some of the mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage with those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 10(3) plaque-forming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 x 10(9) PFU/ml at 37 C and 7 x 10(8) PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase.     

Consequences of Ca2+ deficiency on macromolecular synthesis and adenylate energy charge in Yersinia pestis.

A 37 but not 26 degrees C virulent Yersinia pestis is known to require at least 2.5 mM Ca2+ for growth; this requirement is potentiated by Mg2+. After shift of log-phase cells (doubling time of 2 h) from 26 to 37 degrees C in Ca2+-deficient medium, shutoff of net ribonucleic acid synthesis preceded that of protein and cell mass. With 2.5 mM Mg2+, about two doublings in cell mass and number occurred before restriction with synthesis of sufficient deoxyribonucleic acid to account for initiation and termination of two postshift rounds of chromosome replication. Temperature shift with 20 mMMg2+ resulted in a single doubling of cell mass and number with one round of chromosome replication. Subsequent to shutoff of ribonucleic acid accumulation, ribonucleoside but not deoxyribonucleoside triphosphate pools became reduced to about 50% of normal values and the adenylate energy change fell from about 0.8, typical of growing cells, to about 0.6. Excretion of significant concentrations of adenine nucleotides under both permissive and restrictive conditions was observed. Only trace levels (less than 0.01 microM ol/g [dry weight]) of guaninosine 5'-diphosphate 3'-diphosphate accumulated under restrictive or permissive conditions; guanosine 5'-triphosphate 3'-diphosphate was not detected. Return of fully restricted cells from 37 to 26 degrees C with Ca2+ resulted in prompt growth, whereas addition of Ca2+ at 37 degrees C was ineffective. This finding indicates that the observed temperature-sensitive lesion in ribonucleic acid synthesis that results in restriction can be prevented but not reversed by cultivation with Ca2+.     

cdc9 ligase-defective mutants of Saccharomyces cerevisiae exhibit lowered resistance to lethal effects of bleomycin

Conditional ligase-deficient mutants of Saccharomyces cerevisiae were more sensitive than their parental (CDC9) strain to dose-dependent killing by bleomycin, even when mutant cells were pregrown and exposed to the antibiotic at permissive temperatures. Pretreatment incubation at the restrictive temperature (37 degrees C) under growing or nongrowing conditions enhanced bleomycin killing of both cdc9-1 and cdc9-9 mutants. This sensitization could be relieved by incubation at the permissive temperature before treatment.     

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.     

Cell-free transfer of the vesicular stomatitis virus G protein from an endoplasmic reticulum compartment of baby hamster kidney cells to a rat liver Golgi apparatus compartment for Man8-9 to Man5 processing.

We report the reconstitution of the transfer of a membrane glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reticulum to Golgi apparatus and its subsequent Man8-9GlcNAc2 to Man5GlcNAc2 processing in a completely cell-free system. The acceptor was Golgi apparatus from rat liver immobilized on nitrocellulose. The endoplasmic reticulum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside triphosphate plus cytosol-dependent transfer and processing of radiolabeled VSV-G protein was observed with donor from BHK cells infected at 37 degrees C with wild-type VSV or at the permissive temperature of 34 degrees C with the ts045 mutant. With Golgi apparatus as acceptor, specific transfer at 37 degrees C in the presence of nucleoside triphosphate was eightfold that at 4 degrees C or in the absence of ATP. About 40% of the VSV-G protein transferred was processed to the Man5GlcNAc2 form. Processing was specific for cis Golgi apparatus fractions purified by preparative free-flow electrophoresis. Fractions derived from the trans Golgi apparatus were inactive in processing. With the ts045 temperature-sensitive mutant, transfer and processing were much reduced even in the complete system when microsomes were from cells infected with mutant virus and incubated at the restrictive temperature of 39.5 degrees C but were able to proceed at the permissive temperature of 34 degrees C. Thus, Man8-9GlcNAc2 to Man5GlcNAc2 processing of VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae as the acceptor and shows temperature sensitivity, donor specificity, requirement for ATP, and response to inhibitors similar to those exhibited by transfer and processing of VSV-G protein in vivo.     

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.     

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.     

SwoHp, a nucleoside diphosphate kinase, is essential in Aspergillus nidulans

The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature. The swoH gene was mapped to chromosome II and cloned by complementation of the temperature-sensitive phenotype. The sequence showed that swoH encodes a homologue of nucleoside diphosphate kinases (NDKs) from other organisms. Deletion experiments showed that the swoH gene is essential. A hemagglutinin-SwoHp fusion complemented the mutant phenotype, and the purified fusion protein possessed phosphate transferase activity in thin-layer chromatography assays. Sequencing of the mutant allele showed a predicted V83F change. Structural modeling suggested that the swoH1 mutation would lead to perturbation of the NDK active site. Crude cell extracts from the swoH1 mutant grown at the permissive temperature had ~20% of the NDK activity seen in the wild type and did not show any decrease in activity when assayed at higher temperatures. Though the data are not conclusive, the lack of temperature-sensitive NDK activity in the swoH1 mutant raises the intriguing possibility that the SwoH NDK is required for growth at elevated temperatures rather than for polarity maintenance.     

Differential In Vivo Regulation of Steroid Hormone Receptor Activation by Cdc37p

The CDC37 gene is essential for the activity of p60^v-src when expressed in yeast cells. Since the activation pathway for p60^v-src and steroid hormone receptors is similar, the present study analyzed the hormone-dependent transactivation by androgen receptors and glucocorticoid receptors in yeast cells expressing a mutant version of the CDC37 gene. In this mutant, hormone-dependent transactivation by androgen receptors was defective at both permissive and restrictive temperatures, although transactivation by glucocorticoid receptors was mildly defective only at the restrictive temperature. Cdc37p appears to function via the androgen receptor ligand-binding domain, although it does not influence receptor hormone-binding affinity. Models for Cdc37p regulation of steroid hormone receptors are discussed.     

Characterization of T antigen in cells infected with a temperature-sensitive mutant of simian virus 40.

T antigen induced in African green monkey kidney cells by a temperature-sensitive mutant of simian virus 40, defective in a function required for cell transformation, was characterized. The number of T antigen-positive cells estimated by an immunofluorescent techniques was almost equal at permissive (32.5 C) and restrictive (38.5 C) temperatures, but was slightly reduced when the infected cells were incubated at a higher temperature (40.5 C). However, a complement fixation test indicated that the amount of T antigen induced by the mutant is not significantly different from that induced by wild-type virus at 40.5 C. These results suggest that the T antigen-inducing ability of the mutant is not defective. Two distinct molecular species of T antigen were induced by the mutant at the permissive temperature, whereas only one form was observed at the restrictive temperature. The larger molecular form (14 to 15S) induced by the mutant at the permissive temperature was more heat labile than that induced by wild-type virus, suggesting that the mutated gene product is a component of the larger molecular form.     

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.     

Thermosensitive respiratory deficiency in yeast associated with specific effects on particulate cytochrome oxidase.

A mutant of Saccharomyces cerevisiae, unable to grow at the expense of non fermentable carbon sources at 37 degrees C, has been selected; at 25 degrees C the mutant strain behaves like the parental wild strain. Evaluations of respiration rates during aerobic growth at restrictive temperature on one hand, enzymatic and/or spectral evaluations of the individual components of the respiratory chain on the other hand show that the respiratory deficiency is specifically correlated with a reduced level of cytochrome oxidase. The decrease of enzyme activity is the direct consequence of a lowering of hemoprotein (a,a3) concentration. Temperature-activity relationship of cytochrome oxidase elaborated at the permissive temperature by the mutant strain is modified as far as the particulate enzyme is concerned, but no difference is observed after partial solubilization of the enzyme by non ionic surfactant. Genetic analysis shows that the mutant phenotype results from a nuclear gene mutation.     

Genetic characterization of Escherichia coli type 1 pilus adhesin mutants and identification of a novel binding phenotype

Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C. The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.     

The effect of temperature onshibire ts cell clones in the compound eye ofDrosophila melanogaster

Summary We have analysed the effect of temperature on both developing and adult eye cell clones homozygous forshi ^ST139, a temperature-sensitive mutant ofDrosophila melanogaster. The mutant gene, autonomous in its cellular expression, causes structural modifications of ommatidial cells when adult clones of cells are exposed to the restrictive temperature (29°C) for several days. However, the mutant phenotype reverses to normal within 4 days at the permissive temperature (20°C). The results of pulse, shift-up and shift-down experiments show that the temperaturesensitive period for developing compound eye cells is from the late second instar up to the early pupa. Cytodifferentiation of compound eye cells is blocked by restrictive temperature treatment during this period, whereas cell proliferation does not seem to be directly affected. These results are discussed with regard to the other known aspects of the phenotype observed in mutant individuals.     

Arginine Auxotrophic Phenotype of Mutations in pyrA of Salmonella typhimurium: Role of N-Acetylornithine in the Maturation of Mutant Carbamylphosphate Synthetase

Mutations in pyrA that abolish catalytic activity of carbamylphosphate synthetase cause auxotrophy for both arginine and a pyrimidine. Eight pyrA mutants auxotrophic only for arginine (AUX) were isolated by the mutagenized phage technique; three of these required arginine only at low temperature (20 degrees C). Explanations of the AUX phenotype based on bradytrophy were eliminated by the discovery that blocking the utilization of carbamylphosphate for pyrimidine biosynthesis by insertion of an additional mutation in pyrB (encoding aspartic transcarbamylase) did not reduce the requirement for arginine. In contrast, mutational blocks in the arginine biosynthetic pathway before N-acetylornithine (argB, argC, argG, or argH) did suppress the mutation in pyrA. This suggests that exogenous arginine permits growth of the AUX mutants by inhibiting the first step in the arginine pathway, thereby preventing accumulation of an intermediate that antagonizes mutant pyrA function. A mutation in argA (N-acetylornithinase) failed to suppress AUX, indicating that N-acetylornithine was the inhibitory intermediate. This intermediate had no effect on the catalytic or regulatory properties of carbamylphosphate synthetase from mutant cells grown under permissive conditions (37 degrees C). However, the regulatory properties of carbamylphosphate synthetase synthesized under restrictive conditions (20 degrees C) were demonstrably defective (insensitive to activation by ornithine); the enzyme synthesized under permissive conditions was activated by ornithine. A strain carrying an additional mutation (argC), which prevents the accumulation of N-acetylornithine, produced an ornithine-activatable enzyme at both growth temperatures. These results suggest that N-acetylornithine antagonizes the proper preconditioning or maturation of the mutant carbamylphosphate synthetase.     

Characterization of a temperature-sensitive mutant of Saccharomyces cerevisiae that undergoes uncontrolled protein synthesis.

A mutant of Saccharomyces cerevisiae, DW137, isolated after treatment of a wild-type strain with ICR-170. The mutant was respiration-deficient and showed abnormal cell division when grown at 30 degrees C. In addition, the mutant was temperature-sensitive and underwent lysis when grown at 37 degrees C. Random spore analysis, induced reversion profiles, and complementation analysis indicated that the abnormal phenotypes were under the control of a single recessive mutation caused by a base-pair substitution in a nuclear gene. Macromolecular analysis of the mutant at permissive and restrictive temperatures showed that at restrictive temperatures the mutant cannot synthesize DNA. Surprisingly, at restrictive temperatures, protein synthesis in the mutant continued at a rate greater than that observed at permissive temperatures. Cell death and lysis of the mutant could be prevented by treatment of cultures with cycloheximide, an inhibitor of protein synthesis. The data suggest that the abnormally high rate of protein synthesis and the inability to synthesize DNA are jointly responsible for death of the cells, and most probably play and integrating role in the incipient cell lysis.     

Temperature sensitive mutant of Trichoderma reesei defective in secretion of cellulase

Summary Temperature sensitive mutants of Trichoderma reesei derived from hypersecretory strain RL-P37 were isolated and characterized. Compared to the parent strain, one mutant (LU-ts 1) grew well in the mycelial phase at both permissive (25°C) and non-permissive (37°C) temperatures. However, the secretion of overall protein and active cellulases was significantly reduced in the mutant at the higher temperature. No accumulation of active cellulases or intracellular proteins was observed in the mycelia of LU-ts 1 at 37°C. The inhibitory effects of temperature on cellulase secretion in LU-ts 1 were reversible. Isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses confirmed that the secretion of the major cellulases was greatly reduced in LU-ts 1 at 37°C. Molecular characterization of the various temperature sensitive secretion mutants of T. reesei should help elucidate the crucial aspects of the secretory pathway of this cellulolytic fungus.