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GROWTH PHASE DEPENDENT EXPRESSION IN A TETRACYCLIN REGULATED SYSTEM IN SACCHAROMYCES CEREVISIAE Elizabeth R. M. Andrade,Mônica B. Rodriguez. Dep. Biologia Geral Instituto De Ciencias Biológicas, UFMG.

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GROWTH PHASE DEPENDENT EXPRESSION IN A TETRACYCLIN REGULATED SYSTEM IN SACCHAROMYCES CEREVISIAEElizabeth R. M. Andrade,Mônica B. Rodriguez. Dep. Biologia Geral Instituto De Ciencias Biológicas, UFMG

Reporter gene phenotype can be used to evaluate expression regulation of an uncharacterized promoter. A limitation of this approach is the reporter phenotype half-life, due to both mRNA and the protein stability. The quimeric tet0-CYC1 promoter system, active in the absence of tetracycline by the tetR-VP16 activator action, driven by a CMV promoter, and completely repressed by tetracycline addition, seems to be appropriated to evaluate the half life of a reporter gene phenotype in yeast. Several concentrations of tetracycline (up to 250ug/ml) were used, and no one showed any deleterious effect on Saccharomyces cerevisiae growth. At first we used LacZ as reporter sequence, and half-life analysis of its phenotype was done in logarithmic and stationary phases of growth, after the transcription of the reporter gene had been repressed by addition of tetracycline. It is known that the global gene expression is repressed in stationary phase of growth, although there are some genes that are activated or repressed in a specific phase of the yeast growth, and some genes have stable expression in all phases. With the tetracycline regulated system we determined that half-life of lacZ reporter gene phenotype, after transcriptional repression, was about of 40 minutes in logarithmic phase, and it was of 3 hours in stationary phase. The longer half-life in stationary phase could probably be due to low activity of transcription and translation in this phase, compromising protein turnover. Using the same system, but without transcriptional repression, we found initially a lacZ high expression in early logarithmic phase that is progressively reduced reaching a stable level in stationary phase. Therefore, our results show a growth phase dependent expression of tet0-CYC1/tetR-VP16 system, that may be related with CMV promoter regulation in yeast. Nevertheless, this system, once standardized with the half-life of B-galactosidase expression, can be used to evaluate any reporter genes expression phenotypes in yeast, if one considers the culture growth phase during the analysis.

Half-life analysis of a lacZ phenotype in a yeast logaritimic phase of growth.

Half-life analysis of a lacZ phenotype in a yeast stationary phase of growth.

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slide2

GROWTH PHASE DEPENDENT EXPRESSION IN A TETRACYCLIN REGULATED SYSTEM IN SACCHAROMYCES CEREVISIAEElizabeth R. M. Andrade,Mônica B. Rodriguez. Dep. Biologia Geral Instituto De Ciencias Biológicas, UFMG

Reporter gene phenotype can be used to evaluate expression regulation of an uncharacterized promoter. A limitation of this approach is the reporter phenotype half-life, due to both mRNA and the protein stability. The quimeric tet0-CYC1 promoter system, active in the absence of tetracycline by the tetR-VP16 activator action, driven by a CMV promoter, and completely repressed by tetracycline addition, seems to be appropriated to evaluate the half life of a reporter gene phenotype in yeast. Several concentrations of tetracycline (up to 250ug/ml) were used, and no one showed any deleterious effect on Saccharomyces cerevisiae growth. At first we used LacZ as reporter sequence, and half-life analysis of its phenotype was done in logarithmic and stationary phases of growth, after the transcription of the reporter gene had been repressed by addition of tetracycline. It is known that the global gene expression is repressed in stationary phase of growth, although there are some genes that are activated or repressed in a specific phase of the yeast growth, and some genes have stable expression in all phases. With the tetracycline regulated system we determined that half-life of lacZ reporter gene phenotype, after transcriptional repression, was about of 40 minutes in logarithmic phase, and it was of 3 hours in stationary phase. The longer half-life in stationary phase could probably be due to low activity of transcription and translation in this phase, compromising protein turnover. Using the same system, but without transcriptional repression, we found initially a lacZ high expression in early logarithmic phase that is progressively reduced reaching a stable level in stationary phase. Therefore, our results show a growth phase dependent expression of tet0-CYC1/tetR-VP16 system, that may be related with CMV promoter regulation in yeast. Nevertheless, this system, once standardized with the half-life of B-galactosidase expression, can be used to evaluate any reporter genes expression phenotypes in yeast, if one considers the culture growth phase during the analysis.

Half-life analysis of a lacZ phenotype in a yeast logaritimic phase of growth.

Half-life analysis of a lacZ phenotype in a yeast stationary phase of growth.

Supported by CAPES