Cloning and analysis of a yeast genomic DNA sequence capable of directing gene transcription in escherichia coli as well as in yeast, and construction of promoter-probe shuttle plasmid vectors between yeast and E. coil = 효모와 대장균의 이중 프로모터 활성을 지닌 신규 효모 DNA 의 분리 및 분자 생물학적 분석, 그리고 효모와 대장균에서의 프로모터 감지용 셔틀 백타 개발

A new promoter, designated as $\underline{YEP1}$ (Yeast and $\underline{E}$. $\underline{coil}$ dual Promoter), was isolated from yeast genomic DNA and characterized. A strategy was developed for isolating yeast genomic DNA fragments containing promoter activities of both $\underline{E}$. $\underline{coli}$ and yeast. The bacterial $Cm^R$ gene mediating the drug resistance to chloramphenicol by encoding an enzyme chloramphenicol acetyl transferase (CAT) was used as an indicator gene after removing its own promoter region. A promoter-probe plasmid vector, pK15 (7.6 kb), was constructed by the insertion of promoterless $Cm^R$ gene into a yeast 2 um-based shuttle plasmid between $\underline{E}$. $\underline{coil}$ and yeast. Another vector, pKW4 (5.9 kb), was constructed by removing a nonessential 1.7 kb-long region from pK15. The pK15 or pKW4 vector contains an unique BamHI restriction site immediately upstream of the ribosome-binding sequences of the promoterless $Cm^R$ gene. The constructed vectors, pK15 and pKW4, were proven to be useful for the promoter-probing in either organism of $\underline{E}$. $\underline{coil}$ and yeast. A well-known promoter of $\underline{E}$. $\underline{coli}$, $P_{tac}$, or yeast, $P_{ADHI}$, was inserted into the BamHI site upstream of the $Cm^R$ gene in pK15 or in pKW4. The $\underline{E}$. $\underline{coli}$ cells carrying the $P_{tac}$ -CAT-fusion plasmids or yeast cells carrying the $P_{ADHI}$ -CAT-fusion plasmids were able to grow in the presence of the drug, while the control $\underline{E}$. $\underline{coli}$ or yeast cells carrying the parental plasmids, pK15 or pKW4, were not. A yeast-$\underline{E}$. $\underline{coli}$ dual promoter ($\underline{YBP1}$), 2.2 kb in length, was isolated from Alul-Haelll-generated fragments of yeast genomic DNA by its ability of conferring the drug resistance in $\underline{E}$. $\underline{coli}$ and in yeast. The phenotypic selection for chloramphenicol resistance was sequentiall...
Yoo, Ook-JoonresearcherHan, Moon-Hiresearcher유욱준researcher한문희researcher
Issue Date
61169/325007 / 000805016

학위논문(박사) - 한국과학기술원 : 생물공학과, 1988.2, [ xi, 141 p. ]

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