Isolation of the Phenol-Utilizing Yeast Candida tropicalis URA3 Gene and Its Application

• Main Authors: Ryh-Shin Shiah, 夏日新
• Other Authors: Ming-Cheng Chang
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/26004951416091016102

碩士 === 國立成功大學 === 生物化學研究所 === 82 === Candida tropicalis 是一株具有酚類資化(phenol-utilizing)能力的不 完全酵母菌,此菌不僅能夠在17mM高濃度的酚類碳源培養基裡生長,更可用 烷類或脂肪酸誘發peroxisome和.belta.-oxidation pathway酵素大量增 加,因此無論就探討酚類代謝機制,或是增生之調節作用,開發其宿主載體 系統有其必要性。有關宿主載體系統方面,將已構築含有C.tropicalis 6.0kb URA3(orotidine-5'-monophosphate decarboxylase)標誌基因之質 體pCU1進行限制酵素圖譜分析,藉以完成次選殖工怍。最後選殖到1.4kb HindⅢ片段仍能互補C.tropicalis U-6及Saccharomyces cerevisiae SHY3兩株ura3突變株,並構築於修飾過之pGEM-7Zf載體上,命名為pCU2,完 成C.tropicalis插入型質體之系統。接著利用ExonucleaseⅢ unidirectional deletion method,及雙去氧鏈終止反應方法完成此1.4 kb HindⅢ URA3基因片段之DNA序列,共有1382bp,經電腦PC/GENE軟體之分 析,其開放閱讀架具有804個bp ,解譯268個胺基酸,預測分子量為29.7kDa 。在其 5'-非解譯區域,找到真核生物之TATA box啟動子,亦發現其有類似 E.coli-35(TTGACA)和-10(TATAAT)之啟動子區域。將 C.tropicalis URA3 基因與所解譯之ODCase(orotidine- 5'-monophosphate decarboxylase) 蛋白質和其他已發表之菌 株相比較,發現Candida菌屬之間不論是DNA或胺 基酸序列之相似性均遠高於其他菌株,所謂親水性、厭水性序列幾乎一樣, 支持分類學之定位。利用本實驗室 minicell system測定C.tropicalis URA3基因解譯ODCase蛋白質之分子量為30kDa左右,與DNA序列分析相符合 。利用C.tropicalis代謝酚類基因缺失之P-17突變株進行第二次突變,並 利用5-FOA的幫助,進行phenol-和ura3-雙重變異株之篩選,做為phenol hydroxylase基因選殖宿主。令人意外的,雖然得到三株穩定的ura3-突變 株,但phenol-卻回復突變為野生種相若。C. tropicalis複製型質體仍未 開發成功,所以利用基因拯救法策略來進行 phenol hydroxylase基因之選 殖。從以YEp-13構築之基因庫已找到8個可能帶有phenol hydroxylase基 因之質體,目前暫命名為pSL1~pSL8。需進一步實驗方可知曉是否真正選殖 到C.tropicalis phenol hydroxylase 基因o Candida tropicalis is an asporogenous phenol utilizing yeast. It can metabolize 17mM phenol and acts as a model organism for studying on peroxisome biogenesis.The host vector system may be valuable in elucidating the mechanism of phenol metabolism and the induction mechanism of peroxisome biogenesis in C. tropicalis. A plasmid named pCU1,contained a 6kb DNA fragment of C.tropicalis ,which was capable of complementing the pyrF- mutation in Echerichi coli(AT3143)and Saccharomyces cerevisiae ura3-deficient host(SHY3),had been isolated in our laboratory.C. tropicalis URA3 gene was subcloned into pGEM7Zf,along with a 2um circle capable of giving plasmids the ability to replicate autonomously in SHY3. The results of subcloning of various restriction fragments indicated that URA3 gene was located on the 1.4kb HindⅢ fragment ,and was further subcloned into pBCSK phagemid for determining the DNA sequence.Nucleotide sequence analysis predicted a possible open reading frame composed of 804bp encoded a 268 amino acids with a molecular weight 29.7kDa, whcih is in reasonable agreement with the size drived from minicell system analysis.The C.tropicalis URA3 gene has high DNA and protein homology with the ODCase(orotidine-5'- monophosphate decarboxylase)genes of budding yeasts.The compared results may disclose information on the taxonomy or the evolution of the methylotrophic yeast,C. tropicalis.The TATA box promoter was found at the position -125 upstream of the initiation codon ATG.We also found sequences similiar to the E.coli promoter consensus sequences(-35,-10) regions.We attempt to utilize gene rescue method to clone C. tropicalis phenol hydroxylase gene.We have found phenol hydroxylase- carrying candidates from the gene bank constructed by YEp13 vector.However,futher studies should be proceeded to confirm whether there is a true C.tropicalis phenol hydroxylase gene in our clones.