Analysis of the relationship between Sna41 protein and pre-replication complex in fission yeast.

裂殖酵母Sna41蛋白与复制前复合体关系分析

• 批准号: 12680678
• 负责人: MIYAKE Sanae
• 金额: $ 2.37万
• 依托单位: TOHO-UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680678/
• 关键词: DNA replication; MCM; sna41; CDC45; pre-replication complex; DNA複製前複合体; 分裂酵母; cdc45

Nda4 is the member of MCM family and is involved in DNA replication. To analysis the function of Nda4 protein, the 12 suppressor mutants of nda4 mutant were isolated. These strains have the mutations in the same locus. We named this gene sna41. The predicted amino acids sequence of sna41 is homologous to that of CDC45 in Scerevisiae. Sna41 gene is essential for growth and DNA replication.Because the antibody against Sna41 could not be made, the cells were transformed with the plasmid which contains the Sna41-FLAG gene. The transformed cells were fixed and detected by immunofluorescence with anti-FLAG antibody. Sna41 protein located in nucleus or cytoplasm in arrested cells in G1. In G2 cells, no Sna41 protein was detected. The cell of which the sna41-3HA gene was integrated into the chromosome was constructed. These cells were extracted and were fractionated into the soluble fraction and insoluble fraction. The sna41 protein was detected in both fractions. This result suggested that the amount of Sna41 protein is very low and Sna41 protein locates in both nucleus and cytoplasm.All the mutated sna41 genes of the suppressor mutants were sequenced. One gene have the mutation in 3 nucleotides which caused mutations in 3 amino acids, the each of the other 11 genes has the mutation in only one nucleotide which caused mutations in one amino acid. The former case, the mutated positions of amino acids were 110, 146 and 507. The latter case,the mutated positions were 229, 231, 255, 410, 508 and 553. Themutatedsna41-3HA gene could not replace the sna41 gene. This result suggested that themutated Sna41 proteins are unstable and the mutated Sna41-3HA proteins could not function normally.

Nda 4是MCM家族成员，参与DNA复制。为了分析nda 4蛋白的功能，我们分离了12个nda 4突变体的抑制突变体。这些菌株在同一个位点上有突变。我们把这个基因命名为sna 41。sna 41的氨基酸序列与酿酒酵母中的CDC 45的氨基酸序列同源。Sna 41基因是细胞生长和DNA复制所必需的，由于不能制备抗Sna 41的抗体，因此用含有Sna 41-FLAG基因的质粒转化细胞。将转化细胞固定，用抗FLAG抗体进行免疫荧光检测。G1期阻滞细胞Sna 41蛋白定位于细胞核或细胞质。在G2细胞中未检测到Sna 41蛋白。构建了sna 41 - 3 HA基因整合到染色体上的细胞。提取这些细胞并将其分级为可溶性级分和不溶性级分。sna 41蛋白在两个级分中均被检测到。这一结果表明Sna 41蛋白的表达量很低，Sna 41蛋白定位于细胞核和细胞质中。其中1个基因发生了3个核苷酸的突变，导致3个氨基酸的突变，其他11个基因中的每一个都只发生了1个核苷酸的突变，导致1个氨基酸的突变。在前一种情况下，氨基酸的突变位置为110、146和507。在后一种情况下，突变位置为229、231、255、410、508和553。突变的sna 41 - 3 HA基因不能替代sna 41基因。这一结果表明突变的Sna 41蛋白是不稳定的，突变的Sna 41 - 3 HA蛋白不能正常发挥功能。