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

Nda4是MCM家族的成员，参与DNA复制。为了分析Nda4蛋白的功能，分离了12个Nda4突变体的抑制突变体。这些菌株在相同的位点上有突变。我们将这个基因命名为sna41。预测sna41的氨基酸序列与Scerevisiae中CDC45的氨基酸序列同源。Sna41基因对生长和DNA复制至关重要。由于无法制备抗Sna41的抗体，因此用含有Sna41- flag基因的质粒转染细胞。将转化后的细胞固定，用抗flag抗体进行免疫荧光检测。Sna41蛋白位于G1期阻滞细胞的细胞核或细胞质中。在G2细胞中，未检测到Sna41蛋白。构建了sna41-3HA基因整合到染色体上的细胞。提取这些细胞并将其分为可溶部分和不溶部分。在两个组分中均检测到sna41蛋白。这表明Sna41蛋白的含量非常低，并且Sna41蛋白位于细胞核和细胞质中。对所有sna41抑制突变体的突变基因进行测序。一个基因有3个核苷酸的突变导致3个氨基酸的突变，其他11个基因只有一个核苷酸的突变导致一个氨基酸的突变。前者的氨基酸突变位点分别为110、146和507。突变位点分别为229、231、255、410、508和553。突变的sna41- 3ha基因不能替代sna41基因。这表明突变的Sna41蛋白是不稳定的，突变的Sna41- 3ha蛋白不能正常发挥功能。

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.