啤酒酵母DNA修复基因RAD24温度敏感突变株的分离及其特性的初步研究

Isolation and Characterization of the Temperature Sensitive Mutant of DNA Repair Gene RAD24 of Saccharomyces cerevisiae

以ＹＣｐｌａｃ系列带Ｔｒｐ、Ｈｉｓ和Ｕｒａ标志基因的载体为骨架构建含野生型和经羟胺处理的突变型的啤酒酵母ＲＡＤ２４基因质粒，用质粒替换方法分离ＲＡＤ２４基因温度敏感突变株（ｒａｄ２４－ｔｓ３）．紫外生存试验发现，ｒａｄ２４－ｔｓ３对紫外线敏感；同位素（３Ｈ－ＴｄＲ，３Ｈ－ＵＲ，３Ｈ－Ｌｅｕ）参入试验表明，该突变株ＤＮＡ、ＲＮＡ及蛋白质合成均较野生型明显降低．

The RAD24 gene of Saccharomyces cerevisiae is involved in nucleotide excision repair.It encodes a protein of 268 amino acids.Deletion of this gene in genome is lethal.To study the function of RAD24 gene,the temperature sensitive mutant of the gene was isolated by plasmid shuffling.First,the full length of wild type RAD24 gene was cloned into the YCplac33 boned plasmid pRB363 which contains Ura3 gene,named pTS1 and transformed yeast HY684.Next,the Rad24 gene in genome of HY684 was deleted by one step gene disruption in which the His3 gene marker was used.Then,the wild type RAD24 gene was cloned into YCplac22 boned plasmid pRB364 containing Trp1 gene and treated with hydroxylamine to mutate the gene of interest and transformed HY684 containing pTS1 plasmid.Trp +Ura + transformants were tested for temperature sensitive growth prior to transfer to medium containing 5 FOA,which was selected for Ura cells,which lost the non mutagenized RAD24 gene on the pRB364.This procedure eliminated any non rad24 ts mutations.Finally,viability of the 5 FOA resistant Ura Trp + cells could only be due to the presence of the mutagenized pRB364 plasmid and a rad24 ts3 mutant was obtained by screening replicating plates.UV survival test indicates that the mutant was highly sensitive to UV,and its synthesis of DNA,RNA and protein was much decreased compared with the wild type strain.