Cloning of saccharomyces diastaticus URA 3 gene in saccharomyces cerevisiae

Abstract

The URA 3 gene of $\underline{Saccharomyces} \underline{diastaticus}$ coding for orotidine-5-phosphate carboxy-lyase was tried to clone in $\underline{S}$. $\underline{cerevisiae}$. $\underline{S}$. $\underline{diastaticus}$ is the only yeast which can utilize starch as substrate. The technique and knowledge obtained from this study may be applied for further genetical works such as transferring amylase gene from $\underline{S}$. $\underline{diastaticus}$ to $\underline{S}$. $\underline{cerevisiae}$ in the further. Experimental conditions for transferring $\underline{S}$. $\underline{cerevisiae}$ SHY3 using plasmid pMA 56 by competent cell method have been optimized. It was found that CsCl was as the best cation at the concentration of 0.1M, optimum incubation time of one hour, and favorate heat treating temperature of 46℃. Treatment with 70% polyethyleneglycol for one hour also showed better results. The optimum concentration of plasmid DNA was found to be 0.05 ug. When more DNA was used, the transformation efficiency decreased, although higher plate count was observed. Using the improves method, transformation frequency of $10^3-10^4$/ug plasmid, which is about 10 times higher than that of the conventional method, was obtained. The URA3 gene of $\underline{S}$. $\underline{diastaticus}$ was cloned on pMA56. The hybrid plasmid selected and designated as pSD42 was retransferred into $\underline{S}$. $\underline{cerevisiae}$ SHY3 under the conditions optimized as above, to confirm it including URA3 gene. The newly formed pSD42 has tital size of 22Kb which URA3 gene located within the 14kb fragment. The fragment contained sites for restriction enzyme of BglII, BamHI, HindIII, SmaI and SalI. A partial restriction map of pSD42 was constructed.