RIBOSOMAL-RNA GENES OF ENDOMYCES FIBULIGER - ISOLATION, SEQUENCING AND THE USE OF THE 26S RIBOSOMAL-RNA GENE IN INTEGRATIVE TRANSFORMATION OF SACCHAROMYCES-CEREVISIAE FOR EFFICIENT EXPRESSION OF THE ALPHA-AMYLASE GENE OF ENDOMYCES FIBULIGER

Endomyces fibuliger is a dimorphic yeast which is homothallic and exis ts predominantly in the diploid phase with a brief haploid phase. A re peat unit of the ribosomal RNA genes, or rDNA, from E. fibuliger 8014 met has been isolated, cloned and sequenced. In this report, the seque nces of the 17S, 5.8S and 26S rRNA genes are presented. Homology betwe en the sequenced rRNA genes and those of closely-related yeast strains , particularly Saccharomyes cerevisiae and Candida albicans, was obser ved. As a step towards the eventual development of a transformation sy stem for the yeast E. fibuliger, an integrative plasmid containing the 5.8S and a part of the 26S rRNA gene, a selectable marker conferring resistance to the G418 antibiotic and a reporter gene, the alpha-amyla se (ALP1) gene of E. fibuliger, was constructed. This plasmid was line arized at a unique restriction site within the 26S rRNA gene, and tran sformed into S. cerevisiae INVSC2 MATa his3 ura3 using the lithium ace tate method to test the functionality of the vector system. Transforma tion into S. cerevisiae INVSC2 MATa his3 ura3 was by virtue of the ext ensive homology between the sequenced 26S rRNA gene of E. Fibuliger 80 14 met and that of S. cerevisiae, so that homologous pairing and integ ration into the recipient chromosome was possible. The G418-resistant S. cerevisiae transformants produced halos on starch medium due to hyd rolysis by a-amylase, and they were further analysed by Southern hybri dization with the ALP1 gene and the gene encoding the aminoglycoside 3 '-phosphotransferase I enzyme which confers resistance to the G418 ant ibiotic. A band of 13.7 kb which corresponded to the linearized size o f the transforming plasmid DNA was obtained on the autoradiogram, sugg esting that tandem copies of the plasmid DNA are present in the chromo some. Finally, an assay of the alpha-amylase enzyme secreted extracell ularly was performed on the transformants.