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.     

The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe

Summary The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h ⁹⁰) strains of Schizosaccharomyces pombe. The MT region of h ⁹⁰ comprises three cassette genes: the expression site mat1: 1 and two silent loci, mat2: 2 and mat3: 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1: 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2: 2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.     

A Drosophila gene encoding a DEAD box RNA helicase can suppress loss of wee1/mik1 function in Schizosaccharomyces pombe

We describe a screen to isolate cDNAs encoding Drosophila mitosis inhibitors capable of suppressing the mitotic catastrophe phenotype resulting in Schizosaccharomyces pombe from the combination of the weel-50 mutation with either a deletion allele of mil1, or with overexpression of cdc25 ⁺. One plasmid was isolated which could suppress the temperature sensitive lethality of both these strains. The cDNA in this plasmid encodes a protein highly homologous to the DEAD-box family of ATP-dependent RNA helicases, rather than to protein kinases as might be expected. It is possible that the RNA helicase described here may regulate entry into mitosis by down regulating the expression of other genes whose activity may be rate-limiting for entry into mitosis.     

Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis

Summary The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9 192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 by long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.     

The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is only expressed in M cells and the gene product is responsible for the secretion of the mating pheromone, M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue. The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone. We show that STE6 can also mediate secretion of M-factor in S. pombe. Received: 20 December 1996 / Accepted: 29 January 1997     

The Hermes transposon of Musca domestica and its use as a mutagen of Schizosaccharomyces pombe

Transposon mutagenesis allows for the discovery and characterization of genes by creating mutations that can be easily mapped and sequenced. Moreover, this method allows for a relatively unbiased approach to isolating genes of interest. Recently, a system of transposon based mutagenesis for Schizosaccharomyces pombe became available. This mutagenesis relies on Hermes, a DNA transposon from the house fly that readily integrates into the chromosomes of S. pombe. The Hermes system is distinct from the retrotransposons of S. pombe because it efficiently integrates into open reading frames. To mutagenize S. pombe, cells are transformed with a plasmid that contains a drug resistance marker flanked by the terminal inverted repeats of Hermes. The Hermes transposase expressed from a second plasmid excises the resistance marker with the inverted repeats and inserts this DNA into chromosomal sites. After S. pombe with these two plasmids grow 25 generations, approximately 2% of the cells contain insertions. Of the cells with insertions, 68% contain single integration events. The protocols listed here provide the detailed information necessary to mutagenize a strain of interest, screen for specific phenotypes, and sequence the positions of insertion.     

Mitotic recombination between dispersed but related rRNA genes of Schizosaccharomyces pombe generates a reciprocal translocation

Summary Recombination between dispersed yet related serine tRNA genes of Schizosaccharomyces pombe does occur during mitosis but it is approximately three orders of magnitude less frequent than in meiosis. Two mitotic events have been studied in detail. In the first, a sequence of at least 18 nucleotides has been transferred from the donor sup3 gene on the right arm of chromosome I to the related acceptor gene sup12 on the left arm of the same chromosome, thereby leading to the simultaneous change of 8 bp in the acceptor gene. This event must be explained in terms of recombination rather than mutation. It is assumed that it represents mitotic gene conversion, although it was not possible to demonstrate that the donor gene had emerged unchanged from the event. The second case reflects an interaction between sup9 on chromosome III and sup3 on chromosome I. Genetic and physical analysis allows this event to be described as mitotic gene conversion associated with crossingover. The result of this event is a reciprocal translocation. No further chromosomal aberrations were found among an additional 700 potential intergenic convertants tested. Thus intergenic conversion is much less frequently associated with crossingover than allelic conversion. However, the rare intergenic conversion events associated with crossingover provide a molecular mechanism for chromosomal rearrangements.     

prp4 from Schizosaccharomyces pombe, a mutant deficient in pre-mRNA splicing isolated using genes containing artificial introns

Summary We have generated a bank of temperature-sensitive (ts) Schizosaccharomyces pombe mutant strains. About 150 of these mutants were transformed with a ura4 gene containing an artificial intron. We screened these is mutants for mutants deficient in splicing of the ura4 intron. With this approach three mutants were isolated which have a general defect in the splicing process. Two of these mutants fall into the prp1 complementation group and one defines a new complementation group, prp4.     

Isolation and initial characterization of a Schizosaccharomyces pombe mutant exhibiting temperature-dependent radiation sensitivity due to a mutation in a previously unidentified rad locus

Summary We have isolated a mutant of the yeast Schizosaccharomyces pombe which exhibits sensitivity to UV light when grown at either 30° or 37°C, as compared to the parental wild-type strain. This increased sensitivity is more pronounced when cells are grown at 37°C. The mutant is also sensitive to 18 MeV electrons at the high temperature. Tetrad analysis of spores generated by crossing the mutant and a Rad⁺ strain revealed that sensitivity to both types of radiation cosegregate 2:2, relative to wild-type resistance, indicating that a single altered chromosomal locus is responsible for the radiation sensitivities observed. In addition, analysis of spores resulting from crosses between the mutant and all other known S. pombe rad mutants indicates that the temperature-dependent sensitivity described in this report is mediated by a mutation in a previously unidentified rad locus.     

Cloning by functional complementation, and inactivation, of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc ₁ complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1 ^– mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc ₁ complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.     

Identification of the DNA damage-responsive elements of therhp51+ gene, arecA andRAD51 homolog from the fission yeastSchizosaccharomyces pombe

TheSchizosaccharomyces pombe rhp51 ⁺ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and theSaccharomyces cerevisiae RAD51 protein. Levels ofrhp51 ⁺ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. Anrhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulatingrhp51 ⁺ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (fordamage-responsiveelement), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes fromS. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility ofrhp51 ⁺ expression.     

Hyperspeckled mutants of Schizosaccharomyces pombe: frequent mating-type switching without detectable double-strand breaks

Mating-type (MT) switching in homothallic (h> ⁹⁰ ) strains of Schizosaccharomyces pombe is initiated by a DNA double-strand break (DSB) at the distal end of the expression cassette mat1. The cis-acting smt-s1 mutation C13-P11 reduces the frequency of MT switching. It is a small deletion mapping approximately 50 by distal to the site of the DSB. From the h ⁹⁰ smt-s1 strain we isolated 13 mutants with a hyperspeckled iodine reaction. In these mutants the frequency of MT switching is increased. The mutations define nine different hsp genes, none of which maps in or close to the MT region. We tested one mutant of each gene for the presence of DSBs at mat1. Curiously, in none of the h ⁹⁰ smt-s1 hsp strains could DSBs be detected, although some sporulate nearly as efficiently as the h ⁹⁰ smt-n wild type. The hsp mutations show no effect in smt-0 strains; the smt-0 deletion abolishes MT switching completely. Furthermore, we tested the interaction of hsp1-1 with swi1, swi2 and swi7 mutations. hsp1-1 has no effect in swi2 strains, whereas it increases MT switching in swi7 and, to a lesser degree, in swi1 mutants.     

Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.     

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1 ⁺ gene as a multi-copy suppressor of snm1. The pac1 ⁺ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1 ⁺ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1 ⁺ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.     

Theste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeastSchizosaccharomyces pombe

When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13 ⁺ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13 ⁺ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13 ⁺ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13 ⁺ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.