| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1994: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Transcription of PHO5 (encoding repressible acid phosphatase) , PHO8 (encoding repressible alkaline phosphatase) , PHO84 (encoding P_i-transporter) , and PHO81 (encoding one of the regulatory genes) of yeast are repressed by P_i in the medium. The P_i signals are conveyed to these genes through a system composed of five proteins, Pho2p, Pho4p, Pho80p, Pho81p, and Pho85p. In low-P_i medium, Pho4p, in combination with Pho2p, activates transcription of PHO5 and PHO84 by binding at upstream activation sites (USA) having a 6-bp CACGT (G/T) motif in their promoters. When cells were cultivated in high-P_i medium, a Pho80p-Pho85p complex inhibits the Pho4p activity by phosphorylation. Then, the transcription of PHO5 and PHO84 were severely repressed. In low-P_i medium, Pho81p inhibits the Pho80p-Pho85p complex and allows Pho4p in active. Transcription of PHO81 encoding Pho81p is also under the regulation of Pho4p, indicating that the PHO regulatory system forms a closed feedback circuit. The s
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ame regulatory system regulates PHO8 transcription, but without contribution of Pho2p, and that of PHO81 is under the relaxd regulation of the same system. Ratio of PHO5 expression in low-P_i and high-P_i media is about 500 and that of PHO84 is 80, while that of PHO81 is 10 and that of PHO8 is 2 to 3, with a high basal level of expression. In comparison with the USA sequence of the PHO genes, a similar USA sequence, RTCACRTG (R=purine) , was shown to be distributed in centromeres and in the promoters of the MET genes for binding of Cpf1p protein. We performed targeted mutagenesis on various 36-bp fragments bearing the native 6-bp motif and flanking sequences of the PHO promoters. The modified oligonucleotides were inserted into a CYC1-lacZ fusion gene on a single copy yeast plasmid and determined their activity for transcription in the yeast host. Results showed that specific sequences for binding of Pho4p but discriminate Cpflp are GCACGTGGG and GCACGTTTT and suggested that specific transcriptional mode of each PHO gene is conferred by a combination of these two 9-bp motifs and a short A/T segment in the promoter. Less
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