Extrachromosomal inheritance in Schizosaccharomyces pombe

Summary Primary and secondary spore clones were analyzed from two- and three-factor crosses involving the mitochondrial markers conferring resistance to antimycin (A ^R ), chloramphenicol (C ^R ), and erythromycin (E ^R ). As in zygote clones (Seitz-Mayr et al., 1978), transmission of markers is higher in two-factor trans-crosses than in cis-crosses. Except transmission of C ^R in the cross A ^R C ^R E ^R xA ^S C ^{S E S} , no significant differences between cis- and trans-configuration were observed in three-factor crosses. In contrast to zygote clones, in spore clones transmission rates of the two or three markers in a given cross are roughly equal. 18 out of 20 secondary spore clones of different mitochondrial phenotypes appeared to be homoplasmic, whereas 2 still continued to segregate. One of these spore isolates was analyzed, and segregation was found to continue for more than 150 generations after spore germination. Whereas up to more than 80% of zygote clones in certain crosses were uniform, only 2 out of 91 tetrads were uniform, i.e. all four spores were homoplasmic for the same mitochondrial genotype. Presence or absence of recombinant mitochondrial phenytypes among secondary spore clones from tetrads indicated, whether, cytoplasmic mixing had occurred in the original zygote or not. Within an ascus, the number of spores containing recombinant genotype(s) is a direct measure for the extent of cytoplasmic mixing in the zygote. In 82 tetrads analyzed, the number of tetrads with 0, 1, 2, 3, and 4 spores containing recombinant genotype(s) were 25, 37, 14, 5, and 1, respectively. In conclusion, the extent of cytoplasmic mixing at the cell stage before forespore membrane formation is highly variable.     

Nucleo-cytoplasmic interactions in the petite negative yeast Schizosaccharomyces pombe

Summary In the petite positive yeast, Saccharomyces cerevisiae, cycloheximide selectively inhibits protein synthesis on cytoplasmic ribosomes, and, as a consequence, nuclear DNA synthesis. Mitochondrial DNA, however, is synthesized for 4–6 h after cessation of protein synthesis. In this paper we show that in contrast to Saccharomyces cerevisiae, synthesis of mitochondrial and nuclear DNA is tightly coordinated in the petite negative yeast Schizosaccharomyces pombe, since inhibition of cytoplasmic protein synthesis leads immediately to cessation of both nuclear and mitochondrial DNA synthesis.Dedicated to Prof. Dr. F. Kaudewitz on occasion of his 60th birthday     

Synthesis of mitochondrial DNA during the cell cycle of the petite negative yeast Schizosaccharomyces pombe

Summary Synthesis of mitochondrial DNA (mitDNA) and nuclear DNA (nucDNA) during growth of synchronously dividing cultures of Schizosaccharomyces pombe (S. pombe) was followed by pulse labelling with radioactive adenine and determination of its rate of incorporation into total protoplast DNA and into the DNA of DNase-treated mitochondria at different stages of the cell cycle. It could be demonstrated that both mitDNA and nucDNA were synthesised discontinuously and at different points in the cell cycle.     

Extrachromosomal inheritance inSchizosaccharomyces pombe

Summary Spontaneous chloramphenicol (cap ^r)- and erythromycin (ery ^r)-resistant mutants were isolated from strain ade7–50 h ^- and the antimycin-resistant mutant ana ^r-8 ade 7–50 h^- of Schizosaccharomyces pombe (Sch. p.). By mitotic segregation analysis all 154 cap ^r- and 120 ery ^r-mutants derived from ade 7–50 h ^- proved to be recessive chromosomal, whereas all 108 cap ^r- and 200 ery ^r-mutants originating from ana ^r-8 were extrachromosomally inherited. The rate of spontaneous cap ^r- and ery ^r-mutants was about hundredfold in ana ^r-8 compared to ade 7–50 h ^-. Growth of cap ^r-and ery ^r-mutants was not inhibited by chloramphenicol or erythromycin, respectively, in glucose-medium and only slightly in glycerol-medium at concentrations which completely inhibited ana ^r-8. By mitotic segregation-, tetrad-, and mitotic haploidization-analysis the extrachromosomal inheritance of mutants derived from ana ^r-8 was established. Segregational patterns of cap ^r- and ery ^r-determinats during mitosis, meiosis, and mitotic haplidization of diploids are discussed.     

A cytoplasmic gene for partial suppression of a nuclear pleiotropic respiratory deficient mutant in the petite negative yeast Schizosaccharomyces pombe

Summary The nuclear pleiotropic respiratory-deficient mutant pet1 (previously M126) exhibits cytochromes aa₃ and b deficiencies accompanied by loss of the oligomycin-sensitivity of the mitochondrial ATPase. The mutant pet1, unable to grow on glycerol, exhibits in addition sensitivity of Antimycin A of the growth on glucose. The latter phenotypic trait symbolized by ANA^S-D, exhibits a high frequency (2 to 4×10^-5) of spontaneous suppression into Antimycin A-resistant strains. Mutagenesis with MnCl₂ increases by a factor of 10² the frequency of ANA^R-D derivatives. This suppression is partial since none of the suppressed strains is able to grow on glycerol even when respiratory functions and cytochromes activities are restored as in the pet1 [SUP2] strain. In the latter strain it is concluded that the extralocus suppressor gene [SUP2] is responsible for the ANA^R-D trait. Tetrad analysis in a cross homozygous for pet1 demonstrates a non-Mendelian segregation pattern for the SUP2 suppressor gene. In stable diploids, homozygous for pet1, the [SUP2] suppressor exhibits a mitotic segregation pattern. Furthermore the transmission of the [SUP2] gene is decreased by ethidium bromide treatment. Therefore, the [SUP2] suppressor gene responsible for partial suppression of the nuclear pleiotropic phenotype in mutant pet1 is of cytoplasmic heredity.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Anaesthetics delay and accelerate division in the fission yeast Schizosaccharomyces pombe

Pulse treatments with methoxyflurane in synchronous cultures of Schizosaccharomyces pombe produced an increasing division delay as they were applied later in the cycle, up to a transition point at 0.65 of the cycle. Pulse treatments with ether produced a delay when applied early in the cycle but an acceleration when applied between 0.3 and 0.65 of the cycle.     

Cell cycle regulation in Schizosaccharomyces pombe

Cdc2, a cyclin-dependent kinase, controls cell cycle progression in fission yeast. New details of Cdc2 regulation and function have been uncovered in recent studies. These studies involve cyclins that associate with Cdc2 in G1-phase and the proteins that regulate inhibitory phosphorylation of Cdc2 during S-phase and G2-phase. Recent investigations have also provided a better understanding of proteins that regulate DNA replication and that are directly or indirectly controlled by Cdc2.     

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.     

Subunit composition of RNA polymerase II from the fission yeast Schizosaccharomyces pombe

The subunit composition of RNA polymerase II (polII) was compared between the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. For this purpose, we partially purified the enzyme from S. pombe. Judging from the co-elution profiles in column chromatographies of both the RNA polymerase activity and the two large subunit polypeptides (subunit 1 (prokaryotic β' homologue) and subunit 2 (β homologue)), the minimum number of S. pombe polII-associated polypeptides was estimated to be ten, less than the proposed subunit number of the S. cerevisiae enzyme. These ten putative subunits of S. pombe polII correspond to subunits 1, 2, 3, 5, 6, 7, 8, 10, 11 and 12 of the S. cerevisiae counterparts     

Changes in phosphoprotein pattern in Schizosaccharomyces pombe

A variety of evidence suggests that protein phosphorylation (pp) may be important in cell-cycle control. Phosphorylated proteins from S. pombe have been examined for phosphorylation changes under several conditions: known triggers of the division control (low nitrogen, low phosphate), cell size mutants (WEE1 and CDC2 alleles) and cell cycle mutants (CDC2, CDC10, CDC17, CDC25 alleles). Three major phosphorylated proteins (pp38, pp45 and pp54) showed the greatest response to nutritional shifts. The changes in the phosphorylated states of these proteins correlated with growth rate. Some phosphorylations (e.g. pp53) occurred transiently following a stimulus to cell division suggesting a possible involvement with the division mechanism. An allele-specific alteration of charge was noted for pp45 suggesting that this protein is the product of the CDC2 gene. The wee1-6 phosphoprotein pattern is similar to wild-type indicating that this mutant cell line accurately senses its nutritional environment and that the mutation likely affects the transfer of this information to the division control. Cells blocked by various temperature-sensitive cell cycle mutants did not show an alteration of phosphoprotein pattern.     

The amiloride resistance gene, car1, of Schizosaccharomyces pombe

Amiloride, an inhibitor of various sodium transporters, is toxic to Schizosaccharomyces pombe at low concentration in minimal but not in rich media. Amiloride-resistant mutants were isolated and shown to represent a new locus (car1 for changed amiloride resistance) on chromosome I. The carl gene was cloned and sequenced. Sequence analysis revealed an open reading frame of 526 amino acids with a predicted molecular weight of 58 545 Da. It has 52% hydrophobic residues and belongs to the class of 12-transmembrane-domain transport proteins. Gene disruption of carl results in increased amiloride resistance. earl has sequence similarity to proteins from Candida associated with resistance to benomyl, methotrexate and cycloheximide. No single physiologically identifiable component of sodium transport appeared to be lost. We propose that earl serves an uptake function, perhaps as a symport with an unknown substrate and this carrier may transport amiloride into the cell. Further, we suggest that amiloride toxicity at low concentrations is not due to its effect on sodium transport but, rather, depends on intracellular interference with an unknown biosynthetic pathway.     

Functional expression of human P-glycoprotein in Schizosaccharomyces pombe

Human MDR1 cDNA was introduced into the human cultured cells KB-3-1 and Schizosaccharomyces pombe pmdI null mutant KN3. The drug sensitivity of KB-G2 and KN3/pgp, expressing human P-glycoprotein, was examined. KB-G2 was resistant to the peptide antibiotics valinomycin and gramicidin D as well as having a typical multidrug resistance (MDR) phenotype. KN3/pgp was resistant to valinomycin and actinomycin D, but not to adriamycin. The ATP-hydrolysis-deficient mutant did not confer KN3 resistance to these antibiotics. Human P-glycoprotein expressed in S. pombe seemed to lack N-glycosylation. The N-glycosylation-deficient mutant, however, conferred a typical MDR phenotype on KB-3-1. These results suggest that human P-glycoprotein functions as an efflux pump of valinomycin and actinomycin D in the membrane of S. pombe.     

A study of integrative transformation in Schizosaccharomyces pombe

Summary Organ-specific and constitutive expression of the Arabidosis HSP18.2 gene under normal growth conditions (22° C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for -glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.     

Indirect suppression of the wee1 mutant phenotype in Schizosaccharomyces pombe

For S. pombe cells mutations in the wee1 regulatory gene have been shown previously to allow cells to be smaller than normal at cell division, to endow the cell with a significantly long G1 cell cycle interval, and to alter the timing in the cell cycle of certain mutationally-defined cell cycle steps in G2. We show here that situations which lengthen S phase in proliferating wee1 mutant cells 'suppress' to varying degrees these wee1-mediated cell cycle alterations. Conditions chosen to protract S phase were use of cdc22.M45 mutant cells at semipermissive temperatures, and the presence of sub-arresting concentrations of the S phase inhibitors hydroxyurea or deoxyadenosine. Proliferation in the presence of each of these inhibitors was shown directly to result in protracted S phase. Residual cell division measurements were used to measure the cell cycle timing of G1 and G2 cell-cycle steps. The indirect suppression of the wee1 phenotype shown here can be understood in terms of the proposed role of the wee1+ gene product in coordinating cell division with cellular growth.     

Mutants of the fission yeast Schizosaccharomyces pombe which alter the shift between cell proliferation and sporulation

Summary Mutants of S. pombe have been isolated which undergo conjugation and sporulation in rich medium, conditions which are normally inhibitory for these processes. Two of these mutants are also able to sporulate from the haploid state in the absence of heterozygosity at the mating type locus. These recessive mutants define a single nuclear gene called ran1 which is unlinked to mating type. It is proposed that the ran1 gene codes for an inhibitor in the control of the initiation of conjugation and sporulation. In wild type cells the inhibitory effect is released by nutritional starvation and heterozygosity at the mating type locus. This allows the cells to proceed to sporulation. The ran1 mutants are unusual in that they attempt to undergo a reductional meiotic division from the haploid state. They are also genetically unstable and generate extragenic suppressors at high frequency.     

Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe

 New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts^-) or cold sensitive (cs^-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp ⁺ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression. Received: 15 April 1996/Accepted: 9 July 1996