Horie S, Watanabe Y, Tanaka K, Nishiwaki S, Fujioka H, Abe H, Yamamoto M, Shimoda C
Department of Biology, Faculty of Science, Osaka City University, Osaka, Japan.
Mol Cell Biol. 1998 Apr;18(4):2118-29. doi: 10.1128/MCB.18.4.2118.
The mei4+ gene of the fission yeast Schizosaccharomyces pombe was cloned by functional complementation. The mei4 disruptant failed to complete meiosis-I but could proliferate normally. mei4+ was transcribed only in meiosis-proficient diploid cells after premeiotic DNA replication. The mei4+ open reading frame encodes a 57-kDa serine-rich protein comprised of 517 amino acids with a forkhead/HNF3 DNA-binding domain in the amino-terminal region. Transcription of spo6+, a gene required for sporulation, was dependent on the mei4+ function. Two copies of the GTAAAYA consensus sequence, proposed as the binding site for human forkhead proteins, were found in the promoter region of spo6+. A gel mobility shift assay demonstrated the sequence-dependent binding of the GST-Mei4 forkhead domain fusion protein to DNA fragments with one of the consensus elements. Deletion of this consensus element from the spo6 promoter abolished the transcription of spo6+ and resulted in a sporulation deficiency. One-hybrid assay of Mei4 which was fused to the Gal4 DNA-binding domain localized the transcriptional activation domain in the C-terminal 140 amino acids of Mei4. These results indicate that Mei4 functions as a meiosis-specific transcription factor of S. pombe.
通过功能互补克隆了裂殖酵母粟酒裂殖酵母的mei4⁺基因。mei4破坏株无法完成减数分裂I,但能正常增殖。mei4⁺仅在减数分裂前DNA复制后的减数分裂能力正常的二倍体细胞中转录。mei4⁺开放阅读框编码一种57 kDa的富含丝氨酸的蛋白质,由517个氨基酸组成,在氨基末端区域具有一个叉头/HNF3 DNA结合结构域。spo6⁺是孢子形成所需的基因,其转录依赖于mei4⁺的功能。在spo6⁺的启动子区域发现了两个GTAAAYA共有序列拷贝,该序列被认为是人类叉头蛋白的结合位点。凝胶迁移率变动分析表明,GST-Mei4叉头结构域融合蛋白与具有其中一个共有元件的DNA片段发生序列依赖性结合。从spo6启动子中删除该共有元件会消除spo6⁺的转录并导致孢子形成缺陷。与Gal4 DNA结合结构域融合的Mei4的单杂交分析将转录激活结构域定位在Mei4的C末端140个氨基酸中。这些结果表明,Mei4作为粟酒裂殖酵母的减数分裂特异性转录因子发挥作用。