Eri TSURUMAKI

Isolation of the Actin Gene of the Yeast Cryptococcus humicola to Develop of a Dominant Selection System in the Yeast.

Ryo-hei YAMADA, Yoshio KERA, Shouji TAKAHASHI

D-Aspartate oxidase, which catalyzes the oxidative deamination of acidic D-amino acids, is produced in the basidiomycetous yeast Cryptococcus humicola UJ1 only when D-aspartate is present in the culture medium, suggesting that a D-aspartate-sensing mechanism is working in this yeast. The study of this mechanism at the molecular level requires a host-vector system. We thus isolated the actin gene from the yeast (ChACT) to construct a dominant selection system using its promoter and a heterologous drug resistance gene.
Two phage clones including a part of ChACT gene were previously isolated from the yeast genomic DNA library. We isolated and sequenced the ChACT gene fragments. The ChACT gene has an ORF of 1,509 bp interrupted by six introns that encodes a protein of 375 amino acids. The deduced amino acids sequence exhibited the highest homology to the actin of C. neoformans (96.3%). Additionally, the ChACT had 96.0 and 94.7 % homology to the actin of Phaffia rhodozyma and Schizophyllum commune, respectively. The ChACT formed a cluster with actins of basidiomycota in phylogenetic analysis.
We designed tree specific primers to amplify the promoter region of the ChACT gene. The promoter region of 1,296 bp was successfully amplified by TAIL-PCR using the primers.