Induced recombination and reversion of theCDC8 gene in relation to the cell cycle of yeast

Summary The induction of mitotic recombination in theCDC8 locus was studied in a diploid strain heteroallelic forcdc8 mutations (cdc8-1/cdc8-3); mitotic reversion was studied in strainscdc8-1/cdc8-1 andcdc8-3/cdc8-3. Conversion and reversion did not occur in those cells blocked at the S stage of the cell cycle by exposure to a nonpermissive temperature. In stationary phase cells irradiated just prior to exposure to temperature stress, the induction of recombinants was rather low and the induction of revertants was minimal. Conversely, a significant induction ofcdc ⁺ occurred in logarithmic phase cells subjected to the same treatment. Irradiation of synchronously dividing cultures revealed that intragenic recombination occurs at all three stages of the cell cycle- G₁, S and G₂. It was also found that UV-induced gene reversion can occur during the S and G₂ stages, but not during the G₁ stage of the cell cycle.     

An abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe

Summary Adenine-requiring mutant strains of S. pombe enter the stationary phase after depleting a culture medium of adenine or its analogues. Stationary phase cells of six mutants defective at different stages of the purine nucleotide synthetic pathway were examined for cell volume and DNA content, and then compared in these respects with those of a prototrophic wild-type strain. The cell cycle of the wild-type strain was arrested in the G₂ phase (2C state) in the nitrogen rich medium, as is evident from DNA content per cell (0.0425 pg) and cell volume (47.7 m³). An AIR carboxylase-deficient (ade6) mutant strain was found to have an unusual cell volume (307.4 m³) and DNA content (0.1187 pg). By DAPI fluorescence microscopy, each mutant cell was seen to contain only one enlarged nucleus, which indicates the absence of cell populations containing cells in the 4C state of the S phase following nuclear division. It then follows that in ade6 mutant cells, DNA synthesis occurs in the absence of a completed nuclear division. Thus in S. pombe cells, the completion of nuclear division is not necessarily required for the next cycle initiation of DNA synthesis under certain physiological conditions.Abbreviation AIR aminoimidazole ribonucleotide - DAPI 4,6-diamidino-2-phenylindole - PCA perchloric acid - DABA 3,5-diaminobenzoic acid     

A 61-kb ring chromosome shows an ARS-dependent increase in its mitotic stability in the mcm2 mutant of yeast

We have studied the effects of ARS addition and deletion on the maintenance of a 61-kb ring derivative of chromosome III in a minichromosome maintenance mutant of yeast carrying the mcm2-1 mutation. When this ring chromosome, CIIIR, had either of its two strong origins deleted, the resultant chromosome showed a much greater instability in the mutant as compared to that of the wild-type strain. Integration of more ARSs improved the maintenance of CIIIR in the mutant but not in the wild-type strain. Increase in the size of CIIIR, without any ARS addition, did not improve the stability in either strain. A spontaneous revertant for improved growth at 35°C also co-reverted for minichromosome and CIIIR maintenance. The results suggest that ARS malfunctioning leads to minichromosome and chromosome loss from mutant cells, affecting their growth at higher temperatures.     

Fission yeast enters the stationary phase G0 state from either mitotic G1 or G2

Summary The cell cycle of Schizosaccharomyces pombe in continuous culture is controlled at two steps, one which limits the transition from G₁ to S phase and the other which determines the timing of cell division. We have investigated, by means of flow-cytofluorometry, the cell cycle characteristics of nutritionally starved cells in stationary phase. Cells were shown to become arrested in either G₁ or G₂, in ratios which depended on the composition of the growth medium. G₁ and G₂ stationary phase cells share certain properties. (1) They become relatively resistant to heat shock. (2) They can reenter the cell cycle after subculture into fresh medium. (3) The G₁ and G₂ arrested populations have equal long-term viability in stationary phase. (4) Both populations require the activity of the cdc2 ⁺ gene for reentry into the cell cycle. We suggest that cell cycle arrest in stationary phase is regulated by the activity of the same G₁ and G₂ controls which limit the rate of cell cycle progression in continuous culture. The data demonstrate that in fission yeast the transition from G₁ to S phase does not mark a point of commitment to the completion of the cell cycle.     

DNA polymerase III is required for DNA repair in Saccharomyces cerevisiae

We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 °C), but is not affected at the permissive temperature (23 °C). Under conditions where only a small number of lesions was introduced into DNA (80% survival), the repair of MMS-induced damage could also be observed in the mutant at the restrictive temperature, although with low efficiency. When the quantity of lesions increased (50% survival or less), the repair of single-strand breaks was blocked. At the same time we observed a high rate of reversion in the meth, his and trp loci of the cdc2-1 mutant under restrictive conditions. The results presented suggest that DNA polymerase III is involved in the repair of MMS-induced lesions in yeast DNA and that the cdc2-1 mutation affects the proofreading activity of this polymerase.     

Loss of <Emphasis Type="Italic">p16</Emphasis><Superscript><Emphasis Type="Italic">INK4A</Emphasis></Superscript> expression is associated with allelic imbalance/loss of heterozygosity of chromosome 9p21 in microdissected synovial sarcomas

Deletions of the short arm of chromosome 9 have been observed in many tumours and cell lines. This chromosomal region is frequently targeted during malignant transformation because it contains at least two known tumour suppressor genes: p16 ^INK4 and p15 ^INK4B . p16 ^INK4A acts as a negative cell cycle regulator by inhibiting G₁ cyclin-dependent kinases that phosphorylate the retinoblastoma protein and therefore block the progression of the cell cycle from G₁ to S phase. The role of p16 ^INK4A in the development of synovial sarcoma has not been comprehensively investigated. Ten samples of synovial sarcomas were examined for allelic imbalance/loss of heterozygosity (AI/LOH) of the 9p region and p16 protein expression. DNA was isolated from microdissected sections of normal and tumour cells, amplified by polymerase chain reaction and analysed for AI/LOH by using six microsatellite markers that map to the 9p region. Immunohistochemistry for p16 expression was done. AI/LOH with at least one microsatellite marker on 9p21 was detected in six of ten samples. The most frequent allelic deletions were observed within the coding sequence of p16 ^INK4A . Loss of p16 immunoreactivity was detected in eight samples, six of which showed evidence of alterations at 9p21 region. These findings suggest a possible role of loss of p16 ^INK4A in the development of synovial sarcoma.     

Transformation of Neurospora crassa with the trp-1 gene and the effect of host strain upon the fate of the transforming DNA

Summary Neurospora trp-1 ⁺ transformants, obtained by transforming a trp-1 inl strain with plasmid DNA containing the wild type trp1 ⁺ gene, were characterized by genetic and Southern blot analyses. The transforming trp-1 gene integrated at or near the resident site in all of the trp-1 ⁺ transformants obtained with circular DNA or DNA cut within the trp-1 coding region. The frequency of homologous integration decreased substantially when the donor DNA was cleaved outside the trp-1 coding region. The transformants were very stable mitotically and, in general, also showed meiotic stability. Analysis of trp-1 ⁺ transformants obtained with another recipient strain, trp-1 ⁺ ga-2 aro-9 inl, showed that homologous integration of donor DNA occurred in only 20% of the transformants, whether circular or linear DNA was used. Thus, the host strain employed for transformation appears to be a major factor in determining the fate of transforming DNA. Southern blot analysis of transformants showed that integration of the transforming DNA at the homologous site occurred by double crossover or gene conversion events rather than by insertion of the entire plasmid DNA. Multiple and apparently non functional integration events were observed in some transformants.     

Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase α: evidence for a multifunctional role of Mcl1 in DNA replication and repair

Schizosaccharomyces pombe rad2 is involved in Okazaki fragments processing during lagging-strand DNA replication. Previous studies identified several slr mutants that are co-lethal with rad2 and sensitive to methyl methanesulfonate as single mutants. One of these mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1 (mcl1-101) is allelic to mcl1⁺, which is required for chromosome replication, cohesion and segregation. mcl1-101 is temperature-sensitive for growth and is highly sensitive to DNA damage. mcl1 cells arrest with 2C DNA content and chromosomal DNA double-strand breaks accumulate at the restrictive temperature. Mcl1p, which belongs to the Ctf4p/SepBp family, interacts both genetically and physically with DNA polymerase . Mutations in rhp51 and dna2 enhance the growth defect of the mcl1-101 mutant. These results strongly suggest that Mcl1p is a functional homologue of Saccharomyces cerevisiae Ctf4p and plays a role in lagging-strand synthesis and Okazaki fragment processing, in addition to DNA repair.     

Exposure of cdc8 homozygous diploids of Saccharomyces cerevisiae to nonpermissive temperature leads to an increase in mitotic conversion frequencies

Summary In a diploid strain homozygous for the cdc8-1 mutation, a block in DNA synthesis caused by restrictive temperature resulted in a significant increase in the frequency of intragenic recombination at the HOM2 locus. Under restrictive conditions, incorporation of radioactivity into DNA was reduced to 2% of the control and alkaline sucrose gradient centrifugation revealed that only short DNA fragments were synthesized. There was no considerable fragmentation of template DNA during incubation of cdc8-1 strains under restrictive conditions.     

A role for the yeast cell cycle/splicing factor Cdc40 in the G<Subscript>1</Subscript>/S transition

The CDC40 (PRP17) gene of S. cerevisiae encodes a splicing factor required for multiple events in the mitotic and meiotic cell cycles, linking splicing with cell cycle control. cdc40 mutants exhibit a delayed G₁/S transition, progress slowly through S-phase and arrest at a restrictive temperature in the G₂ phase. In addition, they are hypersensitive to genotoxic agents such as methylmethane sulfonate (MMS) and Hydroxyurea (HU). CDC40 has been suggested to control cell cycle through splicing of intron-containing pre-mRNAs that encode proteins important for cell cycle progression. We screened a cDNA overexpression library and isolated cDNAs that specifically suppress the HU/MMS-sensitivity of cdc40 mutants. Most of these cDNAs surprisingly encode chaperones, translation initiation factors and glycolytic enzymes, and none of them is encoded by an intron-containing gene. Interestingly, the cDNAs suppress the G₁/S transition delay of cdc40 cells, which is exacerbated by HU, suggesting that cdc40 mutants are HU/MMS-sensitive due to their S-phase entry defect. A role of Cdc40p in passage through G₁/S (START) is further supported by the enhanced temperature sensitivity and G₁/S transition phenotype of a cdc40 strain lacking the G₁ cyclin, Cln2p. We provide evidence that the mechanism of suppression by the isolated cDNAs does not (at least solely) involve up-regulation of the known positive START regulators CLN2, CLN3, DCR2 and GID8, or of the large and small essential ribonucleotide reductase (RNR) subunits, RNR1 and RNR2. Finally, we discuss possible mechanisms of suppression by the cDNAs that imply cell cycle regulation by apparently unrelated processes, such as splicing, translation initiation and glycolysis.     

Evidence for a gene cluster involving trichothecene-pathway biosynthetic genes in Fusarium sporotrichioides

Two overlapping cosmid clones (Cos1-1 and Cos9-1) carrying the Tox5 gene were isolated from a library of F. sporotrichioides strain NRRL 3299 genomic DNA. These cosmids were used to transform three T-2 toxin-deficient mutants that are blocked at different steps in the trichothecene pathway. Both cosmids restored T-2 toxin production to Tox3-1 ^– or Tox4-1 ^– mutants but neither restored T-2 toxin production to a Tox1–2 ^– mutant. The production of T-2 toxin by the complemented Tox3-1 ^– and Tox4-1 ^– mutants, as well as the production of diacetoxyscirpenol by the cosmid-transformed Tox1-2 ^– mutant, were 2- to 10- fold higher than in strain NRRL 3299. In addition, those transformants carrying Cos9-1 produced significantly higher levels of trichothecenes than transformants carrying Cos1-1. Two different DNA fragments (FSC13-9 and FSC14-5), representing the region of overlap between the cosmid clones, were isolated. These fragments specifically complemented either the Tox3-1 ^– mutant (FSC14-5) or the Tox4-1 ^– mutant (FSC13-9). The trichothecene-production phenotype of these transformants was similar to NRRL 3299. These results suggest that two or more genes involved in the biosynthesis of trichothecenes are closely linked to Tox5.     

Suppression of a DNA polymerase δ mutation by the absence of the high mobility group protein Hmo1 in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The deletion of the gene encoding the high mobility group protein Hmo1 suppresses the growth retardation of the DNA pol δ mutation, pol3-14, at the restrictive temperature. pol3-14 mutant cells undergo cell cycle arrest, and hmo1Δ alleviates the arrest permitting continual division of the double mutant. Bypass of cell cycle control occurs with an increased rate of mutation. Both pol3-14 and hmo1Δ are mutators and their combination provokes a synergistic rate of CAN1 mutations. RAD18 controls branches of DNA repair pathways and its deletion also suppresses pol3 mutations. Comparing hmo1Δ and rad18Δ suppression of pol3-14 shows that while both require the presence of RAD52-mediated repair, their suppression is independent in that both can suppress in the presence of the other. We conclude that hmo1Δ suppression of pol3-14 occurs by a mechanism whereby normal controls on DNA integrity are breached and lesions flow into RAD52-mediated repair and error-prone pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.     

Physical characteristics of the genome of the phytopathogenic fungus Puccinia graminis

The physical characteristics of the genome of Puccinia graminis f. sp. tritici, the wheat stem rust fungus, were determined by reassociation kinetics. The results indicate that the haploid genome contains 67 Mb and consists of three classes of DNA sequences: (1) 64% unique; (2) 30% repetitive; and (3) 4% foldback. The repetitive sequences have a total complexity of 390 kb and are repeated an average of 52 times. The base composition was 45.3% G+C based on an analysis of the DNA melting temperature. The average amount of DNA per ungerminated urediniospore by diphenylamine assay, corrected for losses during extraction, was 435 fg. This was three times the expected value (147 fg) for dikaryotic spores with nuclei in the G₁ phase of the cell cycle, an indication that the spores were in G₂.     

Mcm2 is a target of regulation by Cdc7–Dbf4 during the initiation of DNA synthesis

The initiation of DNA synthesis is an important cell cycle event that defines the beginning of S phase. This critical event involves the participation of proteins whose functions are regulated by cyclin dependent protein kinases (Cdks). The Mcm2–7 proteins are a family of six conserved proteins that are essential for the initiation of DNA synthesis in all eukaryotes. In Saccharomyces cerevisiae, members of the Mcm2–7 family undergo cell cycle-specific phosphorylation. Phosphorylation of Mcm proteins at the beginning of S phase coincides with the removal of these proteins from chromatin and the onset of DNA synthesis. In this study, we identified DBF4, which encodes the regulatory subunit of a Cdk-like protein kinase Cdc7–Dbf4, in a screen for second site suppressors of mcm2-1. The dbf4 suppressor mutation restores competence to initiate DNA synthesis to the mcm2-1 mutant. Cdc7–Dbf4 interacts physically with Mcm2 and phosphorylates Mcm2 and three other members of the Mcm2–7 family in vitro. Blocking the kinase activity of Cdc7–Dbf4 at the G₁-to-S phase transition also blocks the phosphorylation of Mcm2 at this defined point of the cell cycle. Taken together, our data suggest that phosphorylation of Mcm2 and probably other members of the Mcm2–7 proteins by Cdc7–Dbf4 at the G₁-to-S phase transition is a critical step in the initiation of DNA synthesis at replication origins.     

A new enrichment approach identifies genes that alter cell cycle progression in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mechanisms that coordinate cell growth with division are thought to determine the timing of initiation of cell division and to limit overall cell proliferation. To identify genes involved in this process in Saccharomyces cerevisiae, we describe a method that does not rely on cell size alterations or resistance to pheromone. Instead, our approach was based on the cell surface deposition of the Flo1p protein in cells having passed START. We found that over-expression of HXT11 (which encodes a plasma membrane transporter), PPE1 (coding for a protein methyl esterase), or SIK1 (which encodes a protein involved in rRNA processing) shortened the duration of the G1 phase of the cell cycle, prior to the initiation of DNA replication. In addition, we found that, although SIK1 was not part of a mitotic checkpoint, SIK1 over-expression caused spindle orientation defects and sensitized G2/M checkpoint mutant cells. Thus, unlike HXT11 and PPE1, SIK1 over-expression is also associated with mitotic functions. Overall, we used a novel enrichment approach and identified genes that were not previously associated with cell cycle progression. This approach can be extended to other organisms.     

Effects of topical corticosteroids on inflammatory mediator–induced eicosanoid release by human airway epithelial cells

Background: Airway epithelial cells are among the first cells to come in contact with aerosolized corticosteroids. However, the relative potencies and time course of action of the several commonly used aerosolized corticosteroids on eicosanoid production by airway epithelial cells are unknown. Objectives: This study compared the effects of fluticasone, budesonide, and triamcinolone on eicosanoid output by human airway epithelial cells in vitro. We also determined the spectrum of eicosanoids affected and the mechanism for corticosteroid action. Methods: Cultured BEAS-2B airway epithelial cells (a transformed cell line) were exposed to corticosteroids (1 nmol/L to 1 μmol/L) for 2 to 48 hours and then assayed for basal- and bradykinin (BK)-stimulated eicosanoid output. The eicosanoid profile was identified by HPLC in tritiated arachidonic acid prelabelled cells, and PGE₂, the major eicosanoid product, was quantitated by RIA. The effect of corticosteroids on the immunoreactivity of key proteins involved in eicosanoid metabolism (ie, cyclooxygenase [COX], phospholipase A2 [PLA₂], and Clara cell protein, a PLA₂ inhibitor) was determined by Western blotting. Results: Eicosanoid output was largely confined to prostaglandins with values of 5 ± 2 and 82 ± 35 ng PGE₂/10⁶ cells for basal- and BK stimulation, respectively (n = 8). All 3 corticosteroids inhibited basal- and BK-induced PGE₂ output in a dose- and time-dependent manner. Fluticasone and budesonide completely eliminated PGE₂ output in nanomolar concentrations in contrast to triamcinolone, which required micromolar concentration. The rank order of potency was: fluticasone = budesonide > triamcinolone. The time course of action for PGE₂ inhibition also differed, with budesonide acting more slowly than the other 2 corticosteroids (P = .04). All 3 corticosteroids markedly reduced COX2 with little effect on COX1, cPLA₂ (Type IV), or iPLA₂ (Type VI) immunoreactivity or their relative distribution in cytosol versus membrane fractions. Clara cell protein immunoreactivity was undetectable in control and corticosteroid-treated cell lysates. Conclusion: These results show that in a human airway epithelial cell line, the 3 inhaled corticosteroids commonly used to treat asthma differ in onsets of action as inhibitors of prostaglandin synthesis and vary considerably in potency. All 3 corticosteroids act mechanistically in similar fashion by inhibiting COX2 synthesis. (J Allergy Clin Immunol 1999;103:1081-91.)     

Isolation and characterization of Chlamydomonas temperature-sensitive mutants affecting gametic differentiation under nitrogen-starved conditions

Summary Two conditional mutants of Chlamydomonas reinhardtii, dif-1 and dif-2, affecting gametic differentiation under conditions of nitrogen (N)-starvation, have been isolated. These mutant cells remain vegetative at the restrictive temperature (35°C) in — N medium, as defined by assays of cell body-agglutinin and cell wall lytic enzyme activities in the soluble fractions of cell homogenates. Moreover, the mutants fail to form mating structures at the restrictive temperature, but do so at the permissive temperature (25°C). Temperature-shift experiments show that mutant cells which have differentiated into gametes at 25°C dedifferentiate into vegetative cells under N-starvation conditions after transfer to 35°C, but differentiate again into gametes at 25°C. Genetic analyses indicate that the dif-1 and dif-2 genes are recessive and unlinked to each other or to the matingtype locus; the dif-1 phenotype cosegregates with a conditional flagellales phenotype expressed in both +N and-N medium at the restrictive temperature.     

RAN1 + controls the transition from mitotic division to meiosis in fission yeast

Summary We have investigated the genetic and physiological control of meiosis in fission yeast. Nutritionally depleted h ⁺/h ^– diploid cells become irreversibly commited to meiosis immediately prior to the initiation of premeiotic S phase. Premeiotic DNA synthesis requires matP ⁺, matM ⁺, mei2 ⁺ and mei3 ⁺ but not the mitotic cell cycle control gene, cdc2 ⁺, ran1 ⁺ is an essential gene, loss of which provokes sexual conjugation, premeiotic DNA synthesis, pseudo-meiosis and the sporulation of haploid cells. Our experiments suggest that sexual differentiation is achieved physiologically by the inhibition of ran1 ⁺ activity in a two-step process. In the first step, partial inhibition of ran1 ⁺ in starved haploid cells, leads to cell cycle arrest in G₁ followed by sexual conjugation. In the second step, a pathway requiring the matP ⁺, matM ⁺ and mei3 ⁺ genes of the newly-formed zygote, further inhibits ran1 ⁺ and thereby commits the cell to meiosis. mei2 ⁺ is required for meiotic commitment after full inhibition of ran1 ⁺. ran1 ⁺ is normally essential for vegetative cell reproduction but is inessential in cells which have abnormally high levels of cAMP-dependent protein kinase. We propose that the ran1 ⁺ gene encodes a highly controlled protein kinase which shares key substrates with cAMP-dependent protein kinase.     

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys⁺ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys ^- Lys⁺ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.