Masayuki Yamamoto

Research Associate:
Hidetoshi lida

Research Associate:
Yoshiyuki Imai

NIBB Post-doctoral Fellow:
Masuo Goto

Graduate Student:
Tomoko Ono (from The University of Tokyo)

Visiting Scientist:
Makiko S. Okumura

Meiosis is a crucial step in gamete formation and is essential for sexual reproduction. Meiosis is highly conserved among eukaryotic genera, from yeast to mammals. This Division aims to explore the regulation of meiosis in higher organisms. The major strategy currently taken is as follows. In the fission yeast Schizosaccharomyces pombe, which is a unicellular eukaryotic microorganism, many genes involved in control of meiosis have been cloned and characterized. Mutant cells that have a defect in these genes have been obtained either by classical genetics or by chromosome manipulation. We thus rationalize that it is possible to isolate homologs of these genes from animals and plants, by using either similarity in nucleotide sequences or functional complementation of the mutants. The main project of this Division has been performed along this line. We have also carried out two additional projects. One of them was to characterize the mating pheromone of the fission yeast and examine its potential to affect cell growth and sexual development of this yeast. The other was to study the effect of calcium on cell proliferation in the budding yeast. The results obtained in these three projects are described below.

I. Screening for mammalian and plant genes that can replace genes essential for meiosis in the fission yeast.

We prepared cDNA libraries from mouse testis and Arabidopsis thaliana. The vector carries a promoter functional in the fission yeast and thus the cloned genes can be expressed in fission yeast cells. We transformed two meiotic mutants of S. pombe with these libraries. One is the sme2 mutant that cannot perform the first' meiotic division. The sme2 gene product has been shown to function as an RNA molecule. The other mutant is mes1, which cannot perform the second meiotic division. We obtained five mouse genes and four Arabidopsis genes that can rescue sme2. The Arabidopsis genes include those encoding a novel protein kinase family. We isolated a fission yeast homolog of this kinase family and have shown that it also has the ability to rescue sme2. So far we obtained only one mouse gene that can rescue mes1. This mouse gene generates a specific transcript in testis that is not detectable in other tissues tested. Further characterization of the isolated genes is in progress.

II. Characterization of the fission yeast mating pheromone P-factor.

S. pombe h+ cells secrete a diffusible mating pheromone called P-factor. Although M-factor secreted by h cells was previously characterized, the identity of P-factor was not known. We now have shown that the map2 gene, a defect of which confers mating deficiency only in h+ cells, encodes the precursor of P-factor. We purified P-factor from cells overexpressing map2 and determined its amino acid sequence. P-factor is a peptide of 23 residues, with the sequence Thr-Tyr-Ala-Asp-Phe-Leu-Arg-Ala-Tyr-Gln-Ser-Trp-Asn-Thr-Phe-Val-Asn-Pro-Asp-Arg-Pro-Asn-Leu. We synthesized a peptide of this sequence. It has the same specific activity and chromatographic profile as the purified P-factor, suggesting that P-factor is unmodified. By using mutant cells that are derepressed for sexual development in rich medium, we could show that P-factor can induce not only responses towards mating but also arrest of the cell cycle at the G1 phase in h cells (Figure 1). This proves that the S. pombe mating pheromone has the ability to arrest cell cycle progression, which has previously been obscured by the usual requirement for mating of nutritional starvation and subsequent growth arrest (Y. Imai and M. Yamamoto, Genes Dev., 8, 328-338, 1994).

III. Genetic analysis of the role of calcium during sexual differentiation in the budding yeast.

We isolated Saccharomyces cerevisiae mutants apparently defective in the Ca2+ signalling, on the basis of our discovery that Ca2+ is essential for maintaining viability of the cells destined for mating. S. cerevisiae cells do not survive if they cannot take up enough Ca2+ when they are exposed to the mating pheromone. The mutants, named mid (mating pheromone-induced death), die even in the presence of a sufficient amount of Ca2+ if they are exposed to the mating pheromone. The MIDI and MID2 genes have been cloned. Both genes apparently encode membrane-associated proteins. Characterization of these and other MID genes and the functional relationship among them are currently under study.

Publication List:

Gotoh, Y. Nishida, E., Shimanuki, M., Toda, T., Imai, Y. and Yamamoto, M. (1993) Schizosaccharomyces pombe Spk 1 is a tyrosine-phosphorylated protein functionally related to Xenopus mitogen-activated protein kinase. MoL. CelL. Biol. 13, 6427-6434.

Tabata, S., Sato, S., Watanabe, Y., Yamamoto, M. and Hotta, Y. (1993) Evidence of meiosis-specific regulation of gene expression in lily microsporocytes. Plant Sci. 89, 31-41.

Tanaka, K., Davey, J., Imai, Y. and Yamamoto, M. (1993) Schizosaccharomyces pombe map3+ encodes the putative M-factor receptor. Mol. Cell. Biol. 13, 80-88.
Last update Oct21, 1994