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.
Yeast의 gene cloning 에 요구되는, 간편하고 결과가 다시일에 나타나는 transformation method를 이용하기 위하여 competent cell method를 여러가지 요인에 대하여 optimization 했다.
$SHY_3$ strain을 host로, PMA56 plasmid를 donor DNA로 사용했을 경우, 최적조건은 다음과 같았다.
가장 효과가 좋은 ion은 0.1 M Cs+이고, optimum incubation time은 1시간, 적정 열처리 온도는 46℃였다. PEG는 PEG 4,000 70%를 1시간 처리한 경우 가장 높은 transformation frequency를 나타냈다. DNA 농도는 $0.05μg$ 일 때 가장 효과적이었으나, 형질 전환된 cell 수는 DNA를 많이 처리했을 때 더 많았다. 위의 결과를 이용하여 형질 전환을 실시한 경우 frequency를 기존의 $10^2 - 10^3 cells/μg$ DNA보다 10배 정도 높일 수 있었다.
위 방법과 일반적인 gene cloning 방법을 사용하여 Saccharomyces diastaticus 에서 URA3 gene을 S. cerevisiae에서 isolation 했다. 이때 vector는 yeast-E. coli shuttle vector인 PMA 56을 이용했다.
URA3 gene 을 포함한 fragment 는 13.7 kb 정도의 길이이며, 그 fragment 에는 BglII, BamHI, HindIII, SmaI, SalI site 등이 존재했다.
