Schixosaccharomyces pombe Involved in Thiamine-Regulated Gene Expression

Mutants from Schizosaccharomyces pombe deficient in the regulation of thiamine-repressible acid phosphatase have been isolated. Mutants expressing derepressed levels of the enzyme in the presence and absence of thiamine map in three genes, tnrl, tnr2 and tnr3. mRNA levels of the pho4 gene (coding for thiamine repressible acid phosphatase) and another thiamine-regulatable gene, thi3 (coding for a thiamine biosynthetic enzyme and corresponding to nmtl) are constitutively synthesized in the mutants. The mutants also exhibit constitutive thiamine transport which is thiamine repressible in wild type. The tnr3 mutants reveal a 10-20-fold higher intracellular thiamine level than tnrl and tnr2 mutants and wild type. Mutants expressing repressed levels of thiamine-repressible acid phosphatase map in gene thil. No or little amounts of pho4and nmtl-specific mRNA can be detected. These mutants are impaired in thiamine uptake and are thiamine auxotrophic due to the inability to synthesize the thiazole moiety of the thiamine molecule. All tested tnr and thil alleles are recessive, and thil mutations are epistatic over tnr mutations. We assume that the thil and tnr genes are involved in thiamine-mediated transcription control. T HIAMINE (vitamin BI) regulates gene expression in yeast. In Schizosaccharomyces pombe it represses mRNA synthesis of phol-coded acid phosphatase and of a gene product coded for by gene nmtl (M. E. SCHWEINGRUBER et al. 1986; MAUNDRELL 1990). Both genes control thiamine metabolism. Nmt l corresponds to gene thi3 and is responsible for the synthesis of the pyrimidine moiety of the thiamine molecule (SCHWEINGRUBER et al. 199 1). Thiaminerepressible acid phosphatase is a N-glycosylated cell wall protein and is believed to dephosphorylate thiamine phosphates which may occur as natural substrates in growth media (M.E. SCHWEINGRUBER et al. 1986; SCHWEINGRUBER et al. 199 1). We also observed that thiamine represses thiamine transport and mating in fission yeast (SCHWEINGRUBER et al. 199 1 ; SCHWEINGRUBER and EDENHARTER 1990). Whether this is achieved by regulated gene expression still has to be shown. For Saccharomyces cerevisiae repression of a gene pho3 which corresponds to thiamine-repressible acid phosphatase in S. pombe has been observed by M. E. SCHWEINGRUBER et al. (1986) and confirmed by NOSAKA et al. (1 988). The role of vitamin B1 as coenzyme (in form of its diphosphate) in different reactions of the carbohySciences, Banacha 12-16, PL-90-237 Lodz, Poland. Genetics 130 445-449 (March. 1992) I Present address: Microbiology and Virology Centre. Polish Academy of drate metabolism has been known for a long time (for review see FRIEDRICH 1987). The first observation that thiamine can also regulate gene expression has been reported only a few years ago (M. E. SCHWEINGRUBER et al. 1986), and the role of the vitamin as regulator of gene expression has not yet been explored. As a step toward this goal we report in this communication isolation and characterization of mutants from S. pombe defective in thiamine-regulated gene expression. MATERIALS AND METHODS Strains and media: S. pombe wild-type strains containing the mat2 and nutritional markers and acid phosphatasedeficient mutants are from our collection; strain phol-44 has the entire phol gene coding for phosphate-repressible acid phosphatase deleted (ELLIOT et al. 1986). Strains were grown in supplemented or unsupplemented liquid (MM) or solid (MMA) minimal medium as described (SCHWEINGRUBER and EDENHARTER 1990) or in liquid (YE) or solid (YEA) yeast extract medium. Determination of acid phosphatase activity: Activity was tested on plates with a-naphtyl phosphate and Fast Blue (SCHWEINGRUBER, SCHWEINCRUBER and SCHUEPACH 1982) and in liquid cultures by p-nitrophenyl phosphate (A. M. SCHWEINGRUBER et al. 1986). Isolation and mapping of tnr and thi mutants: Equal amounts of cells from strains phoI-44h+ and phoI-44hwere mixed and mutagenized with nitronitrosoguanidine (NNG) as described by DHAMIJA, FLURI and SCHWEINGRUBER (1 986). Cells were sporulated and the spore suspension was used as the source to isolate thi and tnr mutants. tnr mutants were obtained by staining colonies on YEA plates for acid phosphatase activity. Red colonies (acid phosphatase activity is not repressed) were picked. The nmol thi mutants were isolated on MMA plates containing 40 nm thiamine per liter. They stained only very weakly with the acid phosphatase plate assay. Out of roughly 40,000 tested colonies 20 tnr and 8 thi mutants could be isolated. The mutants were back-crossed and subsequently tested for allelism by crossing them with each other and by counting wild-type recombinants essentially as described by SCHWEINGRUBER et al. Construction and analysis of diploids and double mutants: Standard genetical methods were used (GUTZ et al. 1974). For the construction of stable diploids heterozygous or homozygous at the tnr or thil loci haploid tnr and thil strains in a phol-44 ura4-Dl8 background were crossed with a phol-44ade7-JiOmat2-102 strain containing the mutant or wild-type allele of the genes in question. The mat2-102 allele allows the maintenance of stable diploids (EGEL 1984). Diploids were tested for acid phosphatase activity on plates and in liquid cultures. thil diploids were also tested on plates and in liquid medium for thiamine auxotrophy. To obtain tnrthil double mutants, tnr mutants were crossed with thil strains and double mutants were isolated from asci of the recombinant ditype. They were assayed for acid phosphatase activity and thiamine auxotrophy as the diploid strains. Growth experiments with thi mutants: Requirement of thi mutants for thiamine or intermediates of thiamine synthesis was tested in growth experiments as described before (SCHWEINGRUBER et al. 199 1). 4-Amino-5-hydroxymethyl2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole (later referred to as pyrimidine and thiazole moieties) were kindly supplied by G . MOINE from Hoffmann-La Roche and Co. AG, Basel. Extraction and determination of thiamine and thiamine phosphates: Thiamine and its phosphates were extracted in HCI and determined by high performance liquid chromatography as described previously (SCHWEINGRUBER et al. Determination of thiamine uptake: Cells were grown in MM or MM containing thiamine to an optical density of 24 at 530 nm, washed and tested for thiamine uptake as described by SCHWEINGRUBER et al. (1991). Northern blot hybridization: Strains were grown in MM, MM containing 1 PM thiamine or YE to a density of 1-2 X IO' cells and RNA was extracted as described by GRIMM et al. (1991). RNA was separated on 1.2% agarose/glyoxal gels, transferred to Genescreen membranes (NEN) with a vacuum blotter (Pharmacia) and hybridized to '*P-labeled ura4, pho4 and nmtl probes by the dextran sulfate method. Glyoxal gels and hybridization were done according to the GeneScreen manual. The ura4 signal is weaker than the nmtl and pho4 signals and required longer exposure time. (1991).