Mapping of the ras1 gene of Schizosaccharomyces pombe

Summary The ras1 gene, an oncogene homologue, is known to be essential for recognition of the mating pheromone and hence for conjugation but not for vegetative growth in Schizosaccharomyces pombe. To facilitate further characterization and genetic manipulation of this gene, we have mapped it by using S. pombe strains which carry the Saccharomyces cerevisiae LEU2 gene inserted next to ras1 on the chromosome. Crosses with tester strains revealed that ras1 is tightly linked to pro2 on chromosome I. Furthermore, we have shown that ras1 is allelic with ste5, one of the sterility genes described by O. Girgsdies. The map position previously reported for ste5 eventually turned out to be false.     

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe

Background

HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability.

Results

A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8.

Conclusions

This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.     

d-Amino Acid-Induced Expression of d-Amino Acid Oxidase in the Yeast Schizosaccharomyces pombe

We investigated d-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E.?coli displayed oxidase activity to neutral and basic d-amino acids, but not to an l-amino acid or acidic d-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without d-amino acid, and was approximately doubled by adding d-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. l-Alanine also induced the activity, but only by about half of that induced by d-alanine. The induction by d-alanine reached a maximum level at 2?h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was d-alanine, followed by d-proline and then d-serine. Not effective were N-carbamoyl-d,l-alanine (a better inducer of DAO than d-alanine in the yeast Trigonopsis variabilis), and both basic and acidic d-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.     

Schizosaccharomyces pombe encodes a mutated AP endonuclease 1

Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are among the most frequent lesions in DNA. Repair of AP sites is initiated by AP endonucleases and most organisms possess two or more of these enzymes. Saccharomyces cerevisiae has AP endonuclease 1 (Apn1) as the major enzymatic activity with AP endonuclease 2 (Apn2) being an important backup. Schizosaccharomyces pombe also encodes two potential AP endonucleases, and Apn2 has been found to be the main repair activity, while Apn1 has no, or only a limited role in AP site repair. Here we have identified a new 5' exon (exon 1) in the apn1 gene and show that the inactivity of S. pombe Apn1 is due to a nonsense mutation in the fifth codon of this new exon. Reversion of this mutation restored the AP endonuclease activity of S. pombe Apn1. Interestingly, the apn1 nonsense mutation was only found in laboratory strains derived from L972 h(-) and not in unrelated isolates of S. pombe. Since all S. pombe laboratory strains originate from L972 h(-), it appears that all experiments involving S. pombe have been conducted in an apn1(-) mutant strain with a corresponding DNA repair deficiency. These observations have implications both for future research in S. pombe and for the interpretation of previously conducted epistatis analysis.     

裂殖酵母Cnb1参与胞质分裂过程

丝/苏氨酸特异性钙调磷酸酶(Calcineurin, CN)是一种在真核生物中广泛存在的蛋白, 是参与转录调控的重要分子。裂殖酵母中的CN是由催化亚基Ppb1和调节亚基Cnb1组成的异源二聚体。文章报道了裂殖酵母中cnb1+的缺失引起细胞生长速度缓慢, 产生多隔膜现象, 胞质分裂受阻滞。胞质分裂过程中, Cnb1与Ppb1组成CN复合物, 与收缩环在分裂平面上共定位, 并与收缩环一起收缩。cnb1Δ菌株的隔膜成熟过程存在缺陷, 微管出现纵穿隔膜的现象。上述结果说明Cnb1可能参与隔膜的成熟过程。此外, 还检测了cnb1D菌株中胞裂蛋白的信号。胞裂蛋白包括Spn1、Spn2、Spn3和Spn4, 它们是引导隔膜降解的重要分子。结果显示, 在cnb1D菌株中, 80%左右的细胞在隔膜处缺失Spn2和Spn3的信号, 20%左右的细胞缺失Spn1和Spn4的信号。由于胞裂蛋白的蛋白表达量在cnb1D中没有降低, 因此胞裂蛋白信号的消失不是转录缺陷引起的, 这暗示Cnb1可能采用了不依赖转录的方式来调控胞裂蛋白环的稳定性。以上结果提示, Cnb1可能通过影响隔膜的成熟及胞裂蛋白环的稳定性参与调节裂殖酵母的胞质分裂过程。     

Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe

Recombinant plasmids were constructed that direct the synthesis of human antithrombin III in baker's yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe. The signal sequence of antithrombin III was recognized by both yeast species, and antithrombin III was secreted into the medium. When the signal sequence was replaced by a sequence of ten arbitrary amino acids, the product expressed from such a construct stayed inside the cell. Antithrombin III was glycosylated by the baker's and fission yeast and was immunologically identical to antithrombin III isolated from human plasma. Antithrombin III isolated from the culture media of recombinant yeasts was biologically active, as could be shown by progressive inhibitor activity and heparin cofactor activity.     

Checkpoint controls in Schizosaccharomyces pombe: rad1.

'Checkpoint' controls ensure that the events of the cell cycle are completed in an orderly fashion. For example, such controls delay mitosis until DNA synthesis and repair of radiation-induced DNA damage are complete. The rad series of radiosensitive fission yeast mutants was examined to identify strains deficient for the DNA damage-responsive checkpoint control. Five were identified. A characterization of one (rad1-1) and the wild-type is presented. The rad1-1 mutant does not arrest after irradiation, is sensitive to killing by radiation and is not arrested by hydroxyurea, and thus is also deficient for the DNA synthesis-responsive checkpoint control. The radiosensitivity of the rad1-1 mutant was greatly reduced when irradiated and maintained for 6 h in a non-dividing (density inhibited) state, demonstrating that rad1-1 is repair proficient and radiosensitive only through failure to delay. The checkpoint controls for which rad1 is required appear to regulate G2-M progression through the activity of cdc2, here implicated in this role by the coincidence of the radiation transition point and the cdc2 execution point.     

Essential Role for Schizosaccharomyces pombe pik1 in Septation

Background

Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4^G107S.

Principal Findings

Gene deletion revealed that pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-of-function allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25°C. At 36°C, cells arrested with abnormally thick, misplaced or supernumerary septa, indicating a defect late in septation. In addition to being Golgi associated, ectopically expressed GFP-tagged Pik1 was observed at the medial cell plane late in cytokinesis. New alleles, created by site-directed mutagenesis, were expressed ectopically. Lipid kinase and Cdc4-binding activity assays were performed. Pik1^D709A was kinase-dead, but bound Cdc4. Pik1^R838A did not bind Cdc4, but was an active kinase. Genomic integration of these substitutions in S. pombe and complementation studies in Saccharomyces cerevisiae pik1-101 cells revealed that D709 is essential in both cases while R838 is dispensable. In S. pombe, ectopic expression of pik1 was dominantly lethal; while, pik1^D709A,R838A was innocuous, pik1^R838A was almost innocuous, and pik1^D709A produced partial lethality and septation defects. The pik1 ectopic expression lethal phenotype was suppressed in cdc4^G107S. Thus, D709 is essential for kinase activity and septation.

Conclusions

Pik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4.     

Expression of the Saccharomyces cerevisiae gene YME1 in the petite-negative yeast Schizosaccharomyces pombe converts it to petite-positive

Organisms that can grow without mitochondrial DNA are referred to as "petite-positive" and those that are inviable in the absence of mitochondrial DNA are termed "petite-negative." The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the alpha- and gamma-subunits of mitochondrial ATP synthase. These mutations are similar or identical to those occurring in the same subunits of the same enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1 in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera directed against Yme1p. Mutations that block the formation of the F(1) component of mitochondrial ATP synthase are also petite-negative. Thus, the F(1) complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.     

Radiation resistance in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe serves as an excellent alternative and complementary model system for the analysis of genes and gene products involved in DNA repair. This brief review outlines the advantages of S. pombe and describes the radiation-sensitive mutants available for the analysis of DNA repair and recombination mechanisms in this organism. The progress in the cloning and characterization of representative genes is also described.