Shunsuke Narita

Isolation of the actin gene of the yeast Cryptococcus humicolus UJ1

Ryouhei Yamada, Yoshio Kera, Shouji Takahashi

The yeast Cryptococcus humicolus UJ1 produces a significant amount of D-aspartate oxidase only when it grows on D-aspartate as a sole nitrogen source, which suggests that there is a D-aspartate-specific induction mechanism in the yeast. To investigate this mechanism, we have already constructed a host-vector system for the yeast by using an auxotrophic marker gene. However, another selective marker is necessary to reveal the detailed mechanism. In this study, I tried to isolate the actin gene including its promoter and termiator sequences of the yeast to construct a new host-vector system by using a drug resistance gene as a selective marker.
A 1.0 kbp DNA fragment was obtained by PCR with the genomic DNA of the yeast as a template using degenerate oligonucleotide primers which were designed from highly conserved regions among actin protein of other yeasts. The amino acid sequence deduced from the nucleotide sequence of the DNA fragment was strongly homologous to those of the actin proteins of other fungi, confirming that the DNA fragment codes for a part of the actin protein of the yeast. Southern blot analysis of the genomic DNA, digested with several restriction endonucleases, using the DNA fragment as a probe indicated the existence of a single genomic copy of the actin gene, and also showed the restriction sites around the gene in the chromosomal DNA. To obtain the entire actin gene including both of its promoter and terminator sequences, I isolated 10 candidate phage clones from the phage genomic DNA library of the yeast by plaque hybridization technique. Although there was no clone which had the gene including both of the sequences in the isolated clones, it was suggested that it could be generated by fusion of the two overlapping DNA fragments from two different phage clones.