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KMID : 0364819910290010016
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Korean Journal of Microbiology
1991 Volume.29 No. 1 p.16 ~ p.24
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Molecular Cloning, Chromosomal Integration and Expression of the Homoserine Kinase Gene THR1 of Saccharomyces cerevisiae
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Choi Myoung-Sook
Lea Ho-Zoo
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Abstract
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1
The yeast gene THRI encodes the homoserine kinase (EC 2.7.1.39: HKase) which catalyses the first step of the threonine specific arm at the end of the common pathway for methionine and threonine biosynthesis. A recombinant plasmid pMC3 (12.6 kilobase pairs, vector YCp5O) has been cloned into E. coli HB101 from a yeast genomic library through its complementing activity of a thrl mutation in a yeast recipient strain M39-1D. When subcloned into pMC32 (8.6 kbp, vector YRp7) and pMC35 (8.3 kbp, vector YIp5), the Hindlll fragment (2.7 kbp) of pMC3 insert was positive in the thrl complementing activity in both yeast and E. coli auxotrophic strains. The linearized pMC35 was introduced into the original recipient yeast strain and the mitotically stable chromosomal integrant was identified among the transformants. Through the tetrad analysis, the integration site of the pMC35 was localized to the region of THRI structural gene at an expected genetic distance of approximately 11.1 cM from the ARG4 locus on the right arm of the yeast chromosome VIII. When episomically introduced into the auxotrophic cells and cultured in Thr omission liquid medium, the cloned gene overexpressed the HKase in the order of thirteen to fifteenfold, as compared with a wildtype. HKase levels are repressed by addition of threonine at the amount of 300 mg/1 and 1,190 mg/1 for pMC32 and pMC3, respectively. Data from genetic analysis and HKase response thus support that the cloned Hindif yeast DNA fragment contains the yeast thrl structural gene, along with necessary regulatory components for control of its proper expression.
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KEYWORD
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