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

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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.