The Schizosaccharomyces pombe Gene Encoding ${gamma}-Glutamyl$ Transpeptidase I Is Regulated by Non-fermentable Carbon Sources and Nitrogen Starvation원문보기
In our previous study, the first structural gene (GGTI) encoding ${\gamma}-glutamyl$ transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the trans...
In our previous study, the first structural gene (GGTI) encoding ${\gamma}-glutamyl$ transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of ${beta}-galactosidase$ from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.
In our previous study, the first structural gene (GGTI) encoding ${\gamma}-glutamyl$ transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of ${beta}-galactosidase$ from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.
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제안 방법
To identify the upstream region responsible for the induction of the GGTI gene by non-fermentable carbon sources and nitrogen starvation, the upstream region was serially deleted from the original fusion plasmid pGT98 by PCR (Fig. 4). Appropriately synthesized primers were used for the PCR amplification by Ex Taq DNA poly merase (TaKaRa, Shuzo Co.
이론/모형
The yeast cells were grown at 30℃, with shaking, and the cell growth was monitored by the absorbance at 600 nm. The P-galactosidase activity in the extracts was measured by a spectrophotometric method, using o-nitrophenyl P-D- galactopyranoside (ONPG) as a substrate (Guarente, 1983). The protein content of the extracts was measured by the Bradford method (Bradford, 1976), using bovine serum albumin as a standard.
The P-galactosidase activity in the extracts was measured by a spectrophotometric method, using o-nitrophenyl P-D- galactopyranoside (ONPG) as a substrate (Guarente, 1983). The protein content of the extracts was measured by the Bradford method (Bradford, 1976), using bovine serum albumin as a standard.
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