Identification of ten genes that control ribosome formation in yeast

Summary Twenty-three temperature-sensitive mutants of Saccharomyces cerevisiae, all of which undergo a rapid cessation of net RNA accumulation following a shift from the permissive (23°) to the restrictive temperature (36°), have been characterized. Genetic studies demonstrate that these mutants belong to ten different complementation groups and that, in most cases, their properties are the result of a single, recessive mutation in a nuclear gene. Although the mutants were isolated for heat sensitivity, mutants from 2 of the complementation groups are cold sensitive (at 13°) as well. The mutants continue to synthesize protein, including an enzyme, alkaline phosphatase, for two to four hours following a shift from 23° to 36°, suggesting that they are capable of messenger RNA synthesis and the translation of messenger RNA with fidelity at the restrictive temperature. The small amount of RNA that is synthesized in these mutants at the restrictive temperature has been examined on sucrose gradients and by acrylmide gel electrophoresis; in addition, the RNA components in polyribosomes have been fractionated by a new technique that separates messenger RNA from ribosomal RNA. As a result of these analyses we conclude that these mutants are strongly inhibited in the accumulation of 5S, 7S, 17S, and 25S RNA components but are only slight if at all inhibited in the synthesis of messenger RNA and 4S RNA. The results reported here define ten genes, designated rna 2 through rna 11, that play an essential role in the formation or maturation of ribosomes in yeast.     

Isolation and characterization of temperature-sensitive mutants of Streptomyces coelicolor A3(2) blocked in macromolecular synthesis.

A collection of temperature-sensitive mutants of Streptomyces coelicolor A3(2) was isolated. The majority of the mutants showed an osmotically remedial phenotype. Mutants defective in macromolecular synthesis were identified and characterized further. Four mutants were found in which DNA replication was defective, but which continued to synthesize RNA and protein at the restrictive temperature (39 degrees C). The kinetics of cessation of DNA synthesis allowed a tentative identification of slow (initiation) and fast (elongation) stop dna mutants. The inhibition of DNA replication in the four mutants was found to be reversible on returning to the permissive temperature (30 degrees C), but only after a delay of about 2 h. Three other mutants were identified which showed not only cessation of DNA replication at the restrictive temperature, but also defects in other macromolecular synthesis events.     

Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe

Summary Twenty seven recessive temperature sensitive mutants have been isolated in Schizosaccharomyces pombe which are unable to complete the cell division cycle at the restrictive temperature. These mutants define 14 unlinked genes which are involved in DNA synthesis, nuclear division and cell plate formation. The products from most of these genes complete their function just before the cell cycle event in which they are involved. Physiological characterisation of the mutants has shown that DNA synthesis and nuclear division form a cycle of mutually dependent events which can operate in the absence of cell plate formation. Cell plate formation itself is usually dependent upon the completion of nuclear division.     

Temperature sensitive radiosensitive mutants of the yeast Saccharomyces paradoxus

Summary The properties of four radiation sensitive ts mutants of the yeast Saccharomyces paradoxus, txs ₁, txs ₂, txs ₄and txs ₅,were studied. Genetic analysis proves the mutants to be non-allelic. The mutants are sensitive to X-rays and ultraviolet. Holding at 37°C in a complete medium but not in water sharply decreases the viability. With txs ₂this effect could also be observed in water. The mutants formed filaments and showed abnormal cytokinesis under restrictive conditions, this effect being the most pronounced with txs ₄.The frequency of mitotic recombiantion induced by X-rays and UV is much lower in the mutants (except txs ₂)as compared to the wild type. Restrictive temperature somewhat increased the mitotic recombination in the mutants (excepts txs ₁).Shift of the mutants into restrictive temperature leads to immediate, inhibition of DNA synthesis; the extent of this inhibition correlates with the levels of radiation and temperature sensitivity, both being higher in txs ₂and txs ₄.Elevated temperature also decreased RNA and protein synthesis in txs ₂.It is suggested that mutation txs ₂affects a function participating at the last stages in postirradiation repair of DNA, probably DNA ligase, txs ₄seemed to affect the process of nuclear division.     

pet 18: A chromosomal gene required for cell growth and for the maintenance of mitochondrial DNA and the killer plasmid of yeast

Summary Mutations in the pet18 gene of Saccharomyces cerevisiae (formerly denoted pets) confer three phenotypes on mutant strains: (i) inability to respire (petite), (ii) inability to maintain the double-stranded RNA killer plasmid (sensitive), and (iii) temperature sensitivity for growth. We find that pet18 mutants lack mitochondrial DNA. However, despite their inability to maintain the killer RNA plasmid and mitochondrial DNA, pet18 mutants still can carry the other yeast plasmids, [URE3-1], [PSI], and 2-micron DNA. The temperature sensitivity of the pet18 mutants is not expressed as a selective defect in total DNA, RNA, or protein synthesis.     

Temperature sensitive mutations affecting RNA synthesis in Escherichia coli

Summary A streptomycin method has been used for the isolation of mutants with RNA synthesis inhibited at elevated temperature. The method is based on the observation that streptomycin kills bacteria with normal RNA synthesis and does not affect the cells with inhibited synthesis of RNA. This selection method increases the yield of temperature sensitive mutants by a factor 10–20, the amount of mutants with disturbed RNA synthesis is increased 3–5 fold as compared with the method of replicas.Several types of mutants were found among the temperature sensitive strains: those possessing temperature sensitivity of one, two or three types of cellular macromolecules DNA, RNA and protein. The screening among the mutants with affected RNA synthesis revealed a strain ts-19 showing low RNA polymerase activity in cell extracts and partially purified RNA polymerase preparations. The presented evidence suggests that ts-19 mutation affects the structural gene of one of the RNA polymerase subunits.The mapping of the corresponding locus indicated that it was located between the str and thy loci in E. coli K 12 chromosome at a distance of about 20 recombination units from the first locus.     

Derepression of single-stranded DNA-binding protein genes on plasmids derepressed for conjugation, and complementation of an E. coli ssb- mutation by these genes

Summary Plasmid single-stranded DNA-binding protein genes complement the E. coli ssb-1 mutation, and partially restore capacity for DNA synthesis, DNA repair (direct role as well as role in SOS induction) and general recombination. Plasmid mutants derepressed for fertility derived from R1, R64 and R222 show a higher level of complementation compared to the parental repressed plasmids. Derepressed mutants of R222 synthesize more RNA which hybridizes with the ssb gene of the F factor than does the original R222 plasmid. This indicates that plasmid ssb genes are regulated coordinately with fertility genes.     

Conditional Mutants of Meiosis in Yeast

Three temperature-sensitive mutants, spo1-1, spo2-1, and spo3-1, were characterized with respect to their behavior in sporulation medium at a restrictive temperature. The time of expression of the functions defective in the mutants was determined by temperature-shift experiments during the sporulation process. In addition, each mutant was examined for the following: (i) its ability to undergo the nuclear divisions of meiosis; (ii) deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis; (iii) protein turnover; and (iv) colony-forming ability after exposure to sporulation medium. Mutant spo1-1 is defective in a function which confers a temperature-sensitive period which extends over 32% of the sporulation cycle. The temperature-sensitive period of mutant spo2-1 occupies 34% of the cycle, whereas the temperature-sensitive period of mutant spo3-1 extends over 2% of the sporulation cycle. Cytological evidence indicates that all three mutants initiate but do not complete the meiotic nuclear divisions. The DNA content of sporulation cultures of mutants spo1-1 and spo3-1 did not increase to the wild-type level; DNA synthesis in spo2-1 was normal. All three strains exhibit a loss of colony-forming ability during incubation in sporulation medium at the restrictive temperature. RNA and protein synthesis and protein turnover occur in the mutants.     

Characterization of thermosensitive mutants of Physarum polycephalum

Summary Amoebal thermosensitive mutants of Physarum polycephalum have been isolated after mutagenesis of the amoebal form by nitrosoguanidine treatment. About 70% of the independent thermosensitive amoebal mutants obtained were also thermosensitive in the plasmodial form. Two basic screening methods were applied at the same time to thermosensitive microplasmodia in order to detect strains defective in premitotic events, mitosis or chromosomal DNA synthesis. The first method consists in the determination of increase in protein. RNA and DNA with incubation time at the non-permissive temperature. It allowed the detection of four independent thermosensitive mutant strains, showing an early arrest in DNA synthesis. The second one is the quantification of the variations of the different nuclear types at the restrictive temperature. Two mutant strains presented very large nuclei, uni- or multinucleolate, very similar to those obtained after methyl benzimidazole carbamate treatment, suggesting a defect in one of the mitotic processes. One of these two mutant strains showed an early arrest in DNA synthesis at the restrictive temperature. These two screening procedures were completed by electron microscopic observation. This technique allowed the detection of intra-nuclear macrotubular crystal-like structures in a thermosensitive mutant showing a reduced DNA synthesis at the non-permissive temperature.     

In vitro replicative arrest in dnaAts mutants.

Toluene-treated Escherichia coli, conditionally defective in initiation of DNA replication, have been studied. Toluenized mutants of the dnaA_ts, class rapidly stop DNA synthesis at the restrictive temperature. This is in contrast to the slow arrest of replicative synthesis noted in vivo in these strains. The in vitro cessation of replicative DNA synthesis can be prevented by the presence of the detergent Triton X-100. Our results suggest a role in elongation of DNA by the dnaA gene product during replicative synthesis in vitro.     

Macromolecular synthesis in chromosome initiation mutants of Bacillus subtilis.

Summary Inactivation of the dna B or dna D gene product in Bacillus subtilis stimulates RNA and protein synthesis. Strains containing ts dna B and D mutations have been constructed by introducing the mutations by transformation into a thymine requiring strain which does not lyse during thymine starvation. The consequences of inactivation of these gene products have been assessed by comparing RNA and protein synthesis during thymine starvation at the restrictive temperature with the recipient strain. In the ts ⁺ strain, there is a doubling in rate of RNA synthesis during thymine starvation. In the ts dna B and D mutations at the restrictive temperature the rate of RNA synthesis increases four fold. By preincubating the mutants in the absence of thymine for one generation at the permissive temperature the two fold increase in rate of RNA synthesis associated with inactivation of the initiation complex can be demonstrated under conditions where the ts ⁺ strain shows a decrease in rate of RNA synthesis. The rate of protein synthesis observed largely reflects the rate of RNA synthesis in all strains. Completion of the chromosome at the restictive temperature has no significant effect on the rate of RNA synthesis. It is suggested that inactivation of the initiation complex after chromosome initiation could play an important role in control of RNA synthesis in relation to the cell cycle.     

Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants ofUstilago maydis

Summary The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungusUstilago maydis after incubation at the restrictive temperature (32° C) for eight hours. Mutantsts-220,ts-207,ts-432 andts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutantsts-20,tsd 1-1,ts-84 andpol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutantpol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutantts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32°C.tsd 1-1 andts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis with correlates to increasing UV sensitivity of these strains on incubation at 32° C. Apol 1-1ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.     

The isolation of new DNA synthesis mutants in the yeast Saccharomyces cerevisiae

Summary Sixty-eight new conditional cell cycle mutants have been isolated on the basis of their terminal cellular morphology (dumbbells). Fifteen mutants falling into nine complementation groups, were grossly defective in DNA replication and have been assigned the provisional gene symboldbf (fordumbbellformer). Dbf1 and2 stop DNA synthesis immediately on transfer to 37°C and are presumably defective in enzymes required for polymerization. Neither, however, possess a thermolabile DNA polymerase A or B.Dbf3 and4 show a pattern of synthesis consistent with their being deficient in initiation of DNA synthesis. This is confirmed in the accompanying paper.The remaining mutants are deficient in the synthesis of RNA as well as DNA. Indeed the four members of one complementation group are allelic withrna3, one of the group of mutants originally isolated as defective in RNA synthesis, and which do not exhibit a cell cycle phenotype. A re-examination of this group of mutants however, showed the bulk of them also to be defective in DNA synthesis. Furthermore, in preliminary experimentsrna3 and our four new alleles of it, together withrna6 anddbf5 and6, showed enhanced spontaneous mutation frequency.     

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.     

A procedure for isolation of spontaneous mutants with temperature sensitive synthesis of RNA and/or protein

Summary A procedure for the isolation of spontaneous temperature sensitive mutants of Escherichia coli has been developed. They are selected as survivors at high temperature against the combined killing effects exerted by a temperature inducible lambda prophage and either streptomycin plus ampillicin or ampicillin plus cycloserine. The mutants so obtained are blocked in vivo in the synthesis of RNA or protein or both at restrictive temperature.     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitive mutants of HK/68 influenza virus.

Specific single stranded DNA probes have been obtained for both influenza virion RNA (vRNA) and complementary RNA (cRNA) by cloning a hemagglutinin gene fragment in the single stranded DNA phase M13. These probes were used for hybridization with the total labeled RNA from cytoplasmic extracts of infected cells. MDCK cells were infected with temperature-sensitive mutants of influenza HK/68 and the production of the virus specific RNA species was analysed at both permissive and restrictive temperatures. Results show that two NP mutants which undergo intracistronic complementation exhibit two different phenotypes at the non permissive temperature: ts2C is poly A cRNA and vRNA negative whereas ts463 is RNA positive. Two mutants of P genes were also analysed and we discuss the relationship existing between the synthesis of the three RNA species especially between poly A and non poly A cRNA.     

Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColE1-like plasmids in Escherichia coli

Summary Thermal inactivation of the dnaA gene product leads to a considerable decrease in the rate of replication of ColE1-like plasmids. To test the possiblity that the dnaA protein may affect synthesis of RNA I, which is an inhibitor of primer formation, or synthesis of RNA II, which is the primer precursor for replication of ColE1 (Tomizawa and Itoh 1982), the effect of the dnaA46 mutation on the efficiency of the RNA I and the RNA II promoters was examined. It appears that thermal inactivation of the dnaA protein results in a considerable increase in the activity of the RNA I promoter. We suggest that overproduction of RNA I in dnaA mutants grown at the restrictive temperature is responsible for the reduced replication of ColE1-like plasmids.It has been found that addition of rifampicin to cultures of the dnaA46 or the dna ⁺ strain grown at 42°C results in a dramatic increase in the rate of replication of ColE1-like plasmids. We show that the activity of the RNA II promoter at 42°C is exceptionally resistant to rifampicin. In the presence of the drug, this leads, to an altered ratio of RNA I to RNA II, in favor of the latter RNA species.