A cDNA homologue of Schizosaccharomyces pombe cdc5(+) from the mushroom Lentinula edodes: characterization of the cDNA and its expressed product

Abstract

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) was isolated from the basidiomycete mushroom Lentinula edodes and it was named Le.cdc5 cDNA. The deduced Le.CDC5 (842 amino acid residues) possessed N-terminal amino acid sequence highly homologous to those of S. pombe cdc5(+) gene product (Sp.cdc5p) and Sp.cdc5p-related proteins (SPCDC5RPs). The N-terminal 185 amino acid peptide of Le.CDC5 (Le.CDC5(1-185) peptide) produced in Escherichia coli was subjected to random binding-site selection analysis, revealing that Le.CDC5(1-185) peptide binds to a 7-bp sequence with the consensus sequence of 5'GCAATGT3' (complementary; 5'ACATTGC3'). Genomic binding-site (GBS) cloning by using Le.CDC5(1-185) peptide resulted in an isolation of the DNA fragment that contained three sets of 7-bp consensus-like sequence and TATA box. The Le.CDC5 protein contained two putative phosphorylation sites of cAMP-dependent protein kinase (A kinase) in its C-terminus. There exists a possible leucine zipper between the two phosphorylation sites. The Le.CDC5 fragment containing the two phosphorylation sites was actually phosphorylated by commercially available A kinase. Yeast two-hybrid analysis suggested the homodimerization of Le.CDC5 protein probably through the leucine zipper. Northern blot analysis showed that Le.cdc5 gene is most actively transcribed in primordia and small immature fruiting bodies of L. edodes, implying that Le.cdc5 may play a role in the beginning and early stage of fruiting-body formation.