Analysis of the interaction between HTL1 and HTL1-related genes

• Main Authors: Ya-Chi Kuo, 郭雅琪
• Other Authors: Ming-Yuan Cheng
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/16997352462423581881

碩士 === 國立陽明大學 === 遺傳學研究所 === 87 === HTL1 gene is essential for high temperature growth in yeast. Five HTL1-interacting genes were identified. By using yeast two-hybrid system, RSC8 was screened. shl1, shl2, shl3/sth1 motations were discovered to have synthetic lethality effect with htl1 null mutation. YDR303c was screened from the multi-copy suppress scheme. To understand the functional residues of Htl1p, PCR mutagenesis was used to generate htl1 mutant genes. Seven htl1 mutant genes exhibiting formamide-sensitive (fs) and temperature-sensitive (ts) phenotypes in htl1 deletion strain were isolated. They were designated htl1-1 to htl1-7. All htl1 mutant genes contain multiple mutations. To dissect the functional residues of Htl1p, the N-termini (aa.1-35) and C-termini (aa.36-78) of three htl1 mutant genes were swapped with wild-type HTL1. The domain-swapped htl1 mutant genes were introduced into the htl1 deletion strain and three HTL1-synthetic lethality strains for phenotypical observation. The amino acid changes in C-termini of Htl1p-3, Htl1p-6 and Htl1p-7 cause the fs/ts phenotypes in htl1 deletion strain and synthetic lethality in shl1, shl3. With the exception of the N termini of htl1-6 in shl1, all other strains containing mutant N-termini exhibit wild type phenotypes. Q58P and F27L mutations were chosen to proceed site-direct mutagenesis to examine whether they affect the function of Htl1p. Only Q58P mutation has fewer sectors in shl3/sth1. Phenotypes of the others are similar to those of wild type HTL1 in htl1 deletion strain and HTL1-synthetic lethality strains. The C-termini of htl1 mutants and the Q58P mutant were subjects for two-hybrid assay with RSC8. The interaction between all htl1 mutants and RSC8 were diminished. These results indicate that the amino acid changes in C-termini of Htl1p cause the growth defect in htl1 deletion strain, non-sectoring in shl1 and shl3. Moreover, the amino acid changes in C-termini of Htl1p could affect the interaction affinity between Htl1p and Rsc8p.