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 vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

A mutant ofLactobacillus acidophilus with temperature-sensitive regulation of DNA synthesis

Among other temperature-sensitive mutants ofLactobacillus acidophilus the mutant “ts 9” with temperature-sensitive initiation of DNA synthesis was isolated. In this mutant, the course of DNA synthesis under non-permissive conditions proceeds in two phases. During the first 90–120 min, a slight increase (20–50%) of DNA content takes place. Then during further incubation at 40°C, the capacity for initiation of further DNA synthesis increases and a second round of DNA synthesis starts after 3–4h of incubation. The initiation of DNA synthesis is prevented by chloramphenicol and the preceding lag is temperature-dependent. It is concluded that an accumulation of an initiation factor is required for the onset of a new cycle of DNA synthesis and that in thets 9 mutant this accumulation is inhibited at non-permissive temperature.     

Production of infectious particles at the nonpermissive temperature by a temperature-sensitive mutant of bacteriophage SH-133 specific forPseudomonas facilis

The preliminary characterization of a unique temperature-sensitive (ts) mutant of bacteriophage SH-133, designatedts18, is reported. The mutant showed a substantial reduction in the ability to form plaques at the nonpermissive temperature (32°C) when compared with its plaqueforming ability at the permissive temperature (27°C). However, the supernatant fromts18-infected cells grown at 32°C exhibited significant infectivity when assayed at 27°C, which indicates that the reduced titer ofts18 at 32°C is not due to its inability to form phage particles at that temperature. Phage particles produced at 32°C, but not at 27°C, were thermolabile when tested at 32°C. The thermolability of phage yields from cells mixedly infected at 32°C with increasing wild-type/ts18 input ratios was independent of the quantity of wild-type gene product per cell. Thermostable phage particles were yielded byts18-infected cells that received short pulses of permissive temperature during the latter part of the latent period. These data indicate that the defect of the mutant is due to the production of a nonstructural assembly protein that misfunctions when viral maturation proceeds at the nonpermissive temperature.     

Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase.

A cell extract prepared from the lig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin E1 plasmid at 25 °C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from the thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25 °C to 32 °C, whereas that in the lig⁺ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the lig-ts extract, even at 25 °C.The DNA pulse-labelled with the lig-ts extract for 30 seconds at 32 °C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular molecules upon chasing with a lig⁺ extract. DNA-DNA hybridization experiments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5′-to-3′ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component.The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plasmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compound does not interfere with elongation of progeny strands. Chain elongation in the lig-ts extract at 25 °C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the lig-ts extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin E1 plasmid DNA replicate discontinuously.     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Preliminary characterization of the temperature-sensitive defect in DNA replication in a mutant mouse L cell

Temperature-sensitive ts A1S9 mouse L cells synthesize DNA apparently normally for 6–8 hr upon incubation at 38.5°C. Thereafter, these cells are able to perform limited polydeoxyribonucleotide chain synthesis at the high temperature, but are unable to convert newly replicated small single-strand segments of DNA (of the order of molecular weight 10⁶ daltons) to large molecular weight chromosomal DNA. Data obtained are compatible with a model which suggests that ts A1S9 cells are able to carry out most individual reactions of DNA synthesis at the high temperature, but are temperature-sensitive in a protein which participates in the joining of small DNA segments to make chromosomal DNA strands. When cells are reincubated at a permissive temperature, after the temperature-sensitive lesion has been established, they recover the latter capability several hours before they are able once again to synthesize DNA at normal rates.     

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.     

Temperature-sensitive mutants of Corynebacterium ammoniagenes ATCC 6872 with a defective large subunit of the manganese-containing ribonucleotide reductase

Chemical mutagenesis of the nucleotide-producing strain Corynebacterium ammoniagenes ATCC 6872 with N-methyl-N-nitro-N-nitrosoguanidine followed by an enrichment protocol yielded 46 temperature-sensitive (ts) clones. A rapid assay for the allosterically regulated Mn-ribonucleotide reductase (RRase) was developed with nucleotide-permeable cells of C. ammoniagenes in order to screen for possible defects in DNA precursor biosynthesis at elevated temperature. Three mutants (CH 31, CH 32, and CH 33) grew well at 30° C but did not proliferate at 40° C because they did not reduce ribonucleotides to 2′-deoxyribonucleotides. They were designated nrd ^ts (nucleotide reduction defective). When the cultures were shifted from 30 to 40° C, the nrd ^ts mutants immediately ceased to incorporate radiolabeled nucleic acid precursors into the DNA fraction, while DNA chain elongation was barely affected. Thus, exhaustion of the deoxyribonucleotide pool ultimately inhibited cell division, leading to a filamentous growth morphology. In contrast to the wild-type, all three nrd ^ts mutants displayed a distinctly enhanced sensitivity of ribonucleotide reduction towards hydroxyurea (in permeabilized cells and in vitro) at 30° C. The results from assays for biochemical complementation of heat-inactivated (2 min, 37° C) mutant enzyme with either the small or the large subunit of wild-type Mn-RRase located the mutational defect on the large subunit. Received: 28 December 1995 / Revision received: 22 January 1997 / Accepted: 29 January 1997     

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion.     

Overexpression ofapc10 + in fission yeast can suppress the temperature sensitivity ofnuc2-663 mutant but not its sterility

One of the key cell cycle regulators, the anaphase promoting complex (APC) or cyclosome, plays a dual role during mitotic exit. By destroying anaphase inhibitors it promotes sister chromatid separation, and by destroying B-type cyclins it promotes cytokinesis and removes the replication block. Under unfavorable growth conditions, most eukaryotic cells, including the fission yeastSchizosaccharomyces pombe exit mitosis normally but are arrested in G₁ and do not enter the S phase. InS. pombe, mutations in two APC/cyclosome subunits,nuc2-663 andapc10 ^ts, cause mitotic defects at 36°C, and under nitrogen starvation at 25°C they lead to inability of stopping in G₁ and hence to sterility. To gain more insight into the mechanisms regulating APC/cyclosome activity during normal growth and under nitrogen starvation, we screened a genomic library to identify high-copy suppressors of the temperature sensitivity ofnuc2-663. Here we show that overexpression ofapc10 ⁺ allows this strain to grow at 32°C and rescues it from sensitivity to the protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone at 25°C. These observations are consistent with the proposed role for Apc10p as a positive regulator of the APC/cyclosome. However,apc10 ⁺ does not suppress the sterility ofnuc2-663 mutant cells, suggesting that it plays a specific role in APC regulation (e.g., in substrate recognition) rather than in general APC activation.     

A system for temperature-controlled expression of a foreign gene with dual mode in Saccharomyces cerevisiae

A temperature-controlled expression system for a foreign gene in Saccharomyces cerevisiae was constructed. In this system, a MATa hmlα2-102 HMRa sir3–8^ts double mutant was used as host, and a DNA fragment bearing the promoter and pre- or pre-pro regions of the MFα1 gene encoding the α-factor of S. cerevisiae was used as a promoter for expression of a foreign gene cloned on a vector. When the host cells were incubated at a restrictive temperature for the sir3–8^ts mutation (30°C to 35°C) they showed the α mating type and a PHO5 DNA fragment of S. cerevisiae, encoding repressible acid phosphatase, connected downstream of the MFα1 promoter was expressed. But when they were incubated at permissive lower temperature (25°C), at which they have the a mating type, the PHO5 DNA was not expressed. Acid phosphatase activity was increased 30-fold by shifting the incubation temperature from 25°C to 30°C. In this system it may also be possible to express a foreign gene at lower temperature but shut off its expression at higher temperature by connecting the gene to a promoter DNA of an a-specific gene.     

Host--virus interactions in the control of T4 prereplicative transcription. I. tabC (rho) mutants.

In this paper we describe properties of old (Takahashi, 1978) and new tabC^ts and tabC^cs bacterial mutants. We find that under non-permissive conditions they differently inhibit the synthesis of specific T4 prereplicative gene products. Among such products, that we have been able to identify, are P43 and PrIIA. In contrast, P32 and PrIIB are not affected.Inhibition of P43 (T4 DNA polymerase) synthesis is sufficient to account for depressed DNA synthesis in tabC (Takahashi, 1978).In heterodiploids: (1) all tabC mutants are recessive; (2) all tabC mutants do not complement with each other; (3) at least one, tabC^ts-5521, becomes dominant at 42.6 °C if rho mutant ts15 (Tab⁺) (Das et al., 1976) is situated in trans; (4) tabC^ts-5521 also becomes dominant at 42.6 °C if tabC^cs-110 and tabC^cs-18 are situated in trans (42.6 °C is non-permissive for T4 development on tabC^cs-5521 and permissive for T4 development on tabC^cs mutants).We discuss the possibility that in tabC mutants rho protein is altered and insensitive to T4-specific anti-termination functions. We also discuss a model that accounts for the differential effect of tabC mutants on the synthesis of T4 prereplicative proteins.     

The genetics of dopa decarboxylase in Drosophila melanogaster. III. Effects of a temperature sensitive dopa decarboxylase deficient mutation on female fertility

The mutation Ddc^ts1 effects female sterility when homozygous, hemizygous, or heterozygous over a series of Ddc null alleles (Ddc^x) indicating that some aspect of Ddc gene function is necessary for female fertility. Ovary transplant experiments demonstrate that the female sterility phenotype is ovary autonomous. Two to 3% of the total DDC activity measurable in newly hatched females is localized in their previtellogenic ovaries. The degree to which females heterozygous for Ddc^ts1 over different Ddc null alleles are fertile at 22°C reflects a continuous spectrum of allelic complementation similar to that observed for the effects of these genotypes on viability at 30°C. Fertility of all the Ddc^ts1/Ddc^x females tested is significantly depressed at 30 vis-a-vis 22°C providing evidence that it is the DDC enzyme activity itself which is required for female fertility. Ddc^ts1/Ddc^ts1 homozygous and Ddc^ts1/Df hemizygous females are nonconditionally, completely sterile at 18, 20, 22, 25, and 30°C. Although all homo- and hemizygous females do lay some eggs, no evidence of embryogenesis or fertilization has ever been detected. The absolute, nonconditional sterility of Ddc^ts1 homo- and hemizygous females stands in stark contrast to the conventional temperature dependent effects of these same genotypes on viability and to the temperature sensitive effects of Ddc^ts1/Ddc^x heterozygous females on both fertility and viability. Reasons for these tissue-specific and genotypic differences are discussed.     

Temperature-sensitive mutants of B. subtilis defective in deoxyribonucleotide synthesis

Summary Cessation of DNA synthesis in the temperature sensitive mutant 167 tsA 13 of Bacillus subtilis is correlated with the disappearance of dCTP and dATP pools at the nonpermissive temperature; dGTP and dTTP residual pools are stable. In the presence of AdR and CdR at 45°C, the dCTP and dATP pools remain normal and the cells continue to synthesise DNA and grow. It is inferred that in 167 tsA 13 AdR and CdR kinases exist, that the deoxynucleotide kinases function normally and the ribonucleotide reduction is deficient. B. subtilis strains have a hydroxyurea sensitive reductase and the drug inhibition can be reversed by exogenous deoxynucleosides. Evidence that the tsA 13 mutation is in the structural gene of the ribonucleotide reductase is discussed.     

Physiological suppression of the temperature-sensitive sporulation defect in a Bacillus subtilis RNA polymerase mutant

Summary Five hundred putative RNA polymerase mutants of Bacillus subtilis were isolated by selecting for resistance to the RNA polymerase inhibitors rifampin (Rif^r), streptovaricin (Str^r) or streptolydigan (Std^r). This collection was screened for mutants that were unable to sporulate at the non-permissive temperature of 46°C, yet which sporulated well at 37°C and had normal vegetative growth (Spo^ts phenotype). Nearly one half of the Rif^r and one quarter of the Stv^r mutants were Spo^ts, whereas none of the Std^r mutants had this phenotype.The streptovaricin resistant strain stv84 was studied in detail. The stv84 mutation maps between cysA14 and strA39 on the B. subtilis chromosome, and the Stv^r and Spo^ts phenotypes cotransform at a frequency of 100%. The Spo^ts phenotype of stv84 could be physiologically corrected by supplementing the growth medium with inhibitors of RNA synthesis such as rifampin or azauracil, with carbohydrates such as ribose, mannose or glycerol, or with lipids such as Tween 40 or fatty acids native to Bacillus subtilis membranes. A Spo^ts phenotype resembling that of stv84 was produced in wild type B. subtilis by adding cerulenin, an inhibitor of fatty acid biosynthesis, to the growth medium. This cerulenin-induced sporulation defect was reversed by the same treatments that correct the temperature-sensitive genetic defect of stv84. These data indicate that the Spo^ts phenotype of strain stv84 is not due to an intrinsic inability of the mutant RNA polymerase to transcribe developmentally-specific genes at the nonpermissive temperature. Rather, the data suggest that the stv84 lesion causes a physiological imbalance which disrupts membrane structure or function in sporulating cells.     

Nuclear and mitochondr1al DNA synthesis during yeast sporulation

Nuclear and mitochondrial DNA synthesis during sporulation of Saccharomyces cerevisiae has been studied in a wild-type (aα) strain and 3 sporulation deficient strains. We find that in a strain carrying a dominant mutation which prevents sporulation, nuclear DNA synthesis is initiated but not completed; mitochondrial DNA synthesis, on the other hand, does take place. In aa and αα diploids no initiation of nuclear DNA synthesis is seen to occur, and only a very low level of mitochondrial DNA synthesis is observed. We conclude that mitochondrial DNA synthesis in sporulation medium is uncoupled from nuclear DNA synthesis. In addition, the steps at which the sporulation process is arrested in aa and αα cells and in the dominant mutant can be ordered in time as being before and after the initiation of nuclear DNA synthesis.     

Fruit-body production of two ectomycorrhizal fungi in the genusHebeloma in pure culture

Fruit-body production of two ectomycorrhizal fungi,Hebeloma radicosum andhebeloma sp. (nagaenosugitakedamashi in Japanese), in pure culture was examined. First, nutrients that promote mycelial growth of the fungi when added to the basal medium consisting of barley grains and sawdust were determined. Then the fungi were cultivated to produce fruit-bodies in larger-scale media containing additional nutrients selected for each fungus. Mature fruit-bodies bearing basidiospores were formed after incubation at 22°C for 35–42 d, followed by incubation at 17°C for 21–32 d.