Yuichi OHSHIRO

Construction of an Autonomously Replicating Vector for the Yeast Cryptococcus humicolus UJ1

Ryohei YAMADA, Yoshio KERA, Shyoji TAKAHASHI


Cryptococcus humicolus UJ1 is a yeast which can utilize D-aspartate as a sole source of nitrogen. The yeast grown under this condition produces a significant amount of D-aspartate oxidase, whereas other amino acids are ineffective in inducing the enzyme production. For more efficient production of the enzyme, information on the induction mechanism seems to be essential. To investigate the mechanism, we have already constructed the integrative transformation system using the integrative vector, pICUG, and the uracil auxotrophic mutant, UM3 strain. However, since this system has some disadvantages, another system has been required. In this study, I therefore attempted to isolate an autonomously replicating sequence (ARS) of the yeast to construct a transformation system using an autonomously replicating vector.

For the isolation of ARS from the genomic DNA of the yeast, a partial genomic DNA library was constructed by insertion of the 1.0- 6.0 kbp genomic DNA fragments into pICUG. This library was introduced to UM3 strain by an electroporation method to select the plasmids carrying ARS from the library. Through this selection, 10 different kinds of plasmids, which might have ARS, were obtained. Among them, the plasmid named as pUAH11 had the highest transformation efficiency ( /ƒÊg-DNA ), although the efficiency was very low compared to those of other yeasts. Southern blot analysis of total DNA from UM3 strain transformed with the plasmid showed that the plasmid existed extrachromosomally in the cells, suggesting that the plasmid has ARS. The plasmid had a 7.8 kbp genomic DNA insert. The transformation analysis of the fragments of the insert indicated the possibility that a 1.0 kbp fragment of the insert has ARS function.