Estudo da regulação do gene STL1 codificando o sistema de simporte H+/glicerol em Saccharomyces cerevisiae : protocolos

Protocolos das aulas práticas de Biologia Molecular - Expressão Génica, Mestrado em Genética Molecular, ano lectivo de 2004/2005

Disciplina de biociências: módulos de biologia celular, conceitos de genética e conceitos de biologia molecular e genómica

Protocolos dos trabalhos laboratoriais da disciplina de Biociências do Mestrado em Supervisão Pedagógica do Ensino da(s) Ciências da Natureza/Biologia e Geologia, ano lectivo de 2004/2005

Ferramentas de Engenharia Genética 2008/2009: protocolos das aulas práticas

Publicações pedagógica

Expressão dos genes GUP1 e GUP2 de Saccharomyces cerevisiae: influência da fonte de carbono e do stresse salino

Comunicação apresentada nas "9as Jornadas de Biologia de Leveduras Professor Nicolau van Uden", realizadas em Alcochete, Portugal, em 2001.Dois genes com elevado grau de homologia relacionados com um fenótipo de resistência a stress salino foram identificados em Saccharomyces cerevisiae1. A análise da actividade transportadora com glicerol radioactivo, permitiu estabelecer a função de transporte transmembranar de glicerol para estes dois genes: GUP1 e GUP2 (glycerol uptake). Em células cultivadas em etanol, a actividade de simporte com protões foi atribuida a Gup1p, enquanto que em células cultivadas em glucose nenhuma estirpe exibiu transporte de glicerol. O mutante duplo gpd1gpd2, assim como outros mutantes afectados em pelo menos um gene GPD, uma vez cultivados em glucose na presença de NaCl, exibiu uma actividade transportadora inesperadamente elevada2. Uma mutação adicional nestas estirpes no gene GUP1 reduziu esta actividade para cerca de 50%. A actividade restante foi atribuida a Gup2p. A expressão destes dois genes foi estudada por RT-PCR na estirpe selvagem e na estirpe gpd1gpd2. Os resultados revelaram níveis significativos de mRNA para GUP1 e GUP2 em células desreprimidas e em células cultivadas em glucose. Os níveis de mRNA de GUP1 aumentam em maior grau em estirpes afectadas nos genes GPD, particularmente em células cultivadas em glucose com NaCl (1M) e glicerol (15mM), enquanto que os maiores níveis de mRNA de GUP2 foram observados em células cultivadas em sal sem glicerol. Estes resultados sugerem mecanismos de regulação diferentes para a transcrição de GUP1 e GUP2. Por outro lado, os resultados também indicam a existência de mecanismos póstranscrição de regulação da actividade transportadora de glicerol. 1 - HØlst, B. et al. (2000) Mol. Microbiol, 37:98-107 2 – Lages, F. and Lucas, C. (1997). Bioch. Biophys Acta, 1322: 8-1

Saccharomyces cerevisiae genomic library screening in search for the gene responsible for inductive active glycerol uptake

Comunicação apresentada no "18th International Conference on Yeast Genetics and Molecular Biology", realizado em Stellenbosch, África do Sul em 1997.In Saccharomyces cerevisiae, physiological response to osmotic stress is done, mainly, by increased synthesis and intracellular accumulation of glycerol as compatible solute. Previous studies revealed the existence of a glycerol/H+ symport, inducible by growth under gluconeogenic conditions (Lages and Lucas) and independent of the Fps1 channel for glycerol (Sutherland et al.). In order to isolate the gene encoding for glycerol specific carrier, an isogenic strain to W303- 1A, strain YSH642, carrying gpd1 gpd2 mutations unable to synthesize glycerol, was studied for further screening of a S. cerevisiae genomic library. Physiological assays consisting on detection of extracellular alcalinization of cell suspensions upon addition of glycerol and determination of intracellular accumulation of [14C]glycerol, were performed on glucose-grown cells (repressed cells) and on ethanol-grown cells (derepressed cells). No significant differences were found between the results obtained with either YSH642 and W303-1A strains, from which we concluded that disruptions of GPD1 and GPD2 genes do not interfere with regulation of active glycerol uptake. To choose selection conditions, we assumed that the derepressed activity of the glycerol symporter will contribute to increased halotolerance in gpd1 gpd2 genetic background, provided the presence of extracellular glycerol. Thus, selective medium was designed according with previous phenotypic characterization of salt stress tolerance. Screening of a genomic library of S. cerevisiae in the multicopy plasmid YEp13 with inserts of 8-10Kb at BamHI restriction site, is underway by electroporation of strain YSH642. A field strength of 1500V and resistance of 200_ is being employed giving 0.073% viability and an efficiency of 1.1x104 trf/μgDNA, using, as selective medium, mineral medium supplemented with convenient auxotrophic requirements for both yeast strain and plasmid YEp13, glucose 2% (w/v), NaCl 1.4M and glycerol 50mM. Clones able to grow on this medium are being further characterized for osmotic tolerance and, for glycerol transport activity under conditions of repression.European Community Project - BIOTECH PL 95016

Expression studies of GUP1 and GUP2, genes involved in glycerol active transport in Saccharomyces cerevisiae, using semi-quantitative RT-PCR

Glycerol active uptake in Saccharomyces cerevisiae, characterised physiologically as a H+/symport, was previously described as repressed by glucose, induced by growth on non-fermentable carbon sources and unresponsive to growth under salt stress. GUP1 and GUP2 were identified and characterised as genes involved in glycerol active uptake. Using SQ-RT-PCR, GUP1 and GUP2 transcription was measured. Unlike active transport activity determined previously, this was shown to be constitutive and not affected by either glucose repression or growth under salt stress. Furthermore, transcription of GUP1 and GUP2 was still not affected in the gpd1gpd2 mutant strain grown under salt stress in the presence of small amounts of glycerol, in which case a very high Vmax of glycerol uptake has been reported. Intracellular compounds were determined. Glycerol, acetate and trehalose were found to be the major compounds accumulated. Surprisingly, gpd1gpd2 mutant was shown to produce significant amounts of glycerol. Yet, results do not evidence a correlation between the amount of each compound and glycerol transport activity in any of the strains

Expression of GUP1 and GUP2, Saccharomyces cerevisiae glycerol active transport genes

Poster apresentado no "2nd International Meeting on Cellular Responses to Oxidative and Osmotic Stress. Sensing, Signalling and Gene Expression", realizado na Póvoa de Varzim, Portugal de 7 a 10 de Novembro de 2001