  National Insitute for Basic Biology

DIVISION OF CELL PROLIFERATION

(ADJUNCT)

Professor:: Masayuki Yamamoto

Research Associates:: Hidetoshi Iida; Yoshiyuki Imai

NIBB Post-doctoral Fellows:: Masuo Goto; Makiko Kawagishi-Kobayashi

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

Visiting Scientist:: Makiko S. Okumura

This Division aims to explore the regulation of meiosis in higher organisms. Meiosis is a crucial step in gamete formation and is essential for sexual reproduction. Meiotic steps are highly conserved among eukaryotic species. The major strategy taken by us to isolate genes that may be relevant to the regulation of meiosis in animals or plants depends upon trans-complementation between heterologous organisms. In the fission yeast Schizosaccharomyces pombe, which is a unicellular eukaryotic microorganism, genes involved in control of meiosis have been well characterized and many of them are cloned. Mutants defective in these genes, isolated either by classical genetics or by gene disruption and chromosome manipulation, are available. We have thus set out to isolate homologs of these S. pombe genes from animals and a plant, by using either similarity in nucleotide sequences or functional complementation of the mutants. To facilitate this strategy, we also paid efforts to elucidation of the regulatory mechanisms of meiosis in the fission yeast. In addition to the above, a project to characterize the effect of calcium ion on cell proliferation of the budding yeast has been performed.

I. Animal and plant genes that trans-complement meiotic defects in the fission yeast

Using cDNA Iibraries prepared from mouse testis, Xenopus oocyte and Arabidopsis thaliana, we screened for genes that can rescue loss of function of the S. pombe sme2 gene, which encodes an RNA product essential for the promotion of meiosis I (see section II). Several genes have been isolated from mouse and Arabidopsis in this screening. Some of the mouse genes thus obtained apparently encode RNA binding proteins, and one of the Arabidopsis genes encodes a putative single strand DNA-binding protein. We also characterized three Arabidopsis cDNA clones that could rescue the meiosis-deficient phenotype of the S. pombe pdel mutant, which lacks cAMP phosphodiesterase. The first clone apparently encodes protein phosphatase 2C (PP2C). The second encodes a homolog of human Dr1. Dr1 is a protein that interacts with TATA-binding protein (TBP) and blocks its ability to activate transcription. The third encodes Arabidopsis TBP itself. Furthermore, we obtained mouse and Xenopus cDNA clones that can suppress a defect in meiosis 11 in the fission yeast. Further characterization of these isolated genes is in progress and their relevance to the regulation of meiosis in the original organisms is to be examined.

II. Identification of an RNA-protein complex as a critical regulator of meiosis in the fission yeast

S. pombe mei2 has been shown to be essential for the initiation of premeiotic DNA synthesis. We demonstrated that a temperature-sensitive mei2 mutant per-forms premeiotic DNA synthesis but does not undergo meiotic divisions, suggesting that Mei2 is required also for meiosis I. We screened for high copy suppressors of this mei2 ts defect, and isolated a gene as an efficient suppressor. Surprisingly, this gene, named sme2, does not encode a protein product. It encodes a novel, polyadenylated RNA species of 0.5-kb in length (meiRNA), which specifically binds to Mei2 both in vivo and in vitro. Cells in which the sme2 gene is deleted perform premeiotic DNA synthesis but cannot undergo meiosis I. Therefore the meiRNA-Mei2 complex is apparently required for promotion of meiosis I but not for premeiotic DNA synthesis. However, mutations introduced in the RNA-binding motifs in Mei2 blocked premeiotic DNA synthesis. This suggests that Mei2 is likely to couple with another RNA species to promote premeiotic DNA synthesis (Figure 1).

Figure 1.
A model for the regulation of meiosis by Mei2-RNA complexes in the fission yeast The function of Mei2 is required both prior to premeiotic DNA synthesis and prior to meiosis I. Mei2 complexes with meiRNA to promote meiosis l. This RNA is essential for the promotion, but it is not required for premeiotic DNA synthesis. Mei2 is likely to couple with another RNA partner to fulfill its function prior to premeiotic DNA synthesis, because the RNA-binding ability of Mei2 is indispensable to perform this function. (Adopted from ref. Watanabe and Yamamoto, 1994)

III. Analysis of budding yeast genes involved in Ca²⁺ signaling

The mid1 and mid2 mutants were isolated as those defective in Ca²⁺ signaling, on the basis that Ca²⁺ is essential for maintaining viability in Saccharomyces cerevisiae cells which are exposed to the mating pheromone released from the mating partner. The MIDI and MID2 genes were cloned and their nucleotide sequences determined. The deduced MID1 gene product is a protein of 548 amino acid residues, which is localized in the plasma membrane. This protein is essential for Ca²⁺ influx and mating but not for vegetative cell growth. The MID2 gene product is a putative integral membrane protein of 376 amino acids residues. It carries a potential transmembrane domain and a conserved Ca²⁺- binding domain in its C-terminal half. This protein is not essential for vegetative cell growth but is required to keep viability of the cell in the presence of the mating pheromone. Moreover, expression of MID2 was found to be stimulated three fold by the mating pheromone signaling.

Publication List:

Davey, J., Davis, K., Imai, Y., Yamamoto, M. and Matthews, G. (1994) Isolation and characterization of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe. EMBO J. 13, 5910-5921.

Hughes, D. A., Yabana, N. and Yamamoto, M. (1994) Transcriptional regulation of a Ras nucleotide-exchange factor gene by extracellular signals in fission yeast. J. Cell Sci. 107, 3635-3642.

Iida, H., Nakamura, H., Ono, T., Okumura, M. S. and Anraku, Y. (1994) MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca²⁺ influx and mating. Mol Cell Biol. 14, 8259-8271.

Imai, Y. and Yamamoto, M. (1994) The fission yeast mating pheromone P-factor: Its molecular structure, gene structure, and ability to induce gene expression and G1 arrest in the mating partner. Genes Dev. 8, 328-338.

Kuromori, T. and Yamamoto, M. (1994) Cloning of cDNAs from Arabidopsis thaliana that encode putative protein phosphatase 2C and a human Dr1-like protein by transformation of a fission yeast mutant. Nucl. Acids Res. 22, 5296-5301.

Maeda, T., Watanabe, Y., Kunitomo, H. and Yamamoto, M. (1994) Cloning of the pkal gene encoding the catalytic subunit of the cAMP-dependent protein kinase in Schizosaccharomyces pombe. J Biol. Chem. 269, 9632-9637.

Mineyuki, Y., Iida, H. and Anraku, Y. (1994) Loss of microtubules in the interphase cells of onion root tips from the cell cortex and their appearance in the cytoplasm after treatment with cycloheximide. Plant Physiol. 104, 281-284.

Nakajima-Shimada, J., Iida, H., Anraku, Y. and Aoki, T. (1994) Determination of intracellular calcium concentration of individual host cells infected with Trypanosoma cruzi using microfluorimetry in conjunction with digital image processing. Jpn. J. Parasitol. 43, 136-139.

Nocero, M., Isshiki, T., Yamamoto, M. and Hoffman, C. S. (1994) Glucose repression of fbp1 transcription in Schizosaccharomyces pombe is partially regulated by adenylate cyclase activation by a G protein a subunit encoded by gpa2 (git8). Genetics 138, 39-45.

Ono, T., Suzuki, T., Anraku, Y. and Iida, H. (1994) The MID2 gene encodes a putative integral membrane protein with a Ca²⁺-binding domain and shows mating-pheromone stimulated expression in Saccharomyces cerevisiae. Gene 151, 203-208.

Saran, S., Nakao, H., Tasaka, M., Iida, H., Tsuji, F. I., Nanjundiah, V. and Takeuchi, I. (1994) Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA. FEBS Lett. 337, 43-47.