Cloning, sequence analysis and expression of the basidiomycete Lentinus edodes gene uck1, encoding UMP-CMP kinase, the homologue of Saccharomyces cerevisae URA6 gene.

ABSTRACT

Sequence analysis of the downstream region of the basidiomycete Lentinus edodes priB gene encoding a protein with a 'Zn(II)2Cys6 zinc cluster' DNA-binding motif (Endo, H., Kajiwara, S., Tunoka, O., Shishido, K., 1994. A novel cDNA, priBc, encoding a protein with a Zn(II)2Cys6 zinc cluster DNA-binding motif, derived from the basidiomycete Lentinus edodes. Gene 139, 117-121) suggested the presence of a Saccharomyces cerevisiae URA6 gene homologue encoding UMP kinase. We isolated a corresponding cDNA from a mature fruiting-body cDNA library of L. edodes. The nucleotide sequence of this was determined and compared with that of the genomic DNA, revealing that the URA6 gene homologue encodes 227 amino acids (aa) and is interrupted by four small introns. The deduced aa sequence showed an overall identity of 51.1% to that of the S. cerevisiae URA6 gene product. The URA6 homologue protein produced in Escherichia coli using the glutathione S-transferase gene fusion system was found to catalyze the phosphoryl transfer from ATP to UMP and CMP efficiently and also to AMP and dCMP with lower efficiencies. Thus, the URA6 gene homologue was designated uck1 and its product UMP-CMP kinase. Northern-blot analysis showed that the uck1 is actively transcribed in the gill tissue of mature fruiting bodies of L. edodes, implying that uck1 may play a role during the formation of basidiospores occurs in the gill tissue.