CONSTRUCTION OF A UMP SYNTHASE EXPRESSION CASSETTE FROM DICTYOSTELIUM-DISCOIDEUM

We have prepared a DNA cassette containing the UMP synthase (UMPS)-enc oding gene (PYR5-6) from Dictyostelium discoideum. This gene contains no introns and can be used for expression of the UMPS protein. Due to the high percentage of AT in the flanking regions, useful restriction sites were absent, therefore the PYR5-6 was subcloned as three separat e parts, manipulated, and religated to make a full-length clone. After reconstructing the coding region, we examined its functionality by in troducing this gene under the control of the yeast GAL1 promoter into several uracil-requiring mutants of Saccharomyces cerevisiae. These st udies demonstrated that the reconstructed PYR5-6 gene was functional a nd could complement independent ura3 and ura5 mutations in yeast.