A Genomic Approach for the Identification and Classification of Genes Involved in Cell Wall Formation and its Regulation in Saccharomyces Cerevisiae

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of β1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect β1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation

Búsqueda de genes regulados por el receptor de hormona tiroidea

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 05-10-1995En este trabajo se ha desarrollado una mueva metodología para la búsqueda de genes regulados por el receptor de hormona tiroidea (TR) in vivo . En un primer paso se seleccionaron los fragmentos del DNA genómico unidos por el receptor in vitro. En el segundo paso se seleccionaron los fragmentos que contienen-sitios-de-unión-cercanos-a-genes-activamente-transcritos-en-el-hígadomediante una hibridación con cDNA. El análisis de la expresión de estos genes mostró que mas del 90% estaban regulados in vivo por hormona tiroidea. Este resultado, junto con la presencia de sitios de unión del TR, sugiere que los fragmentos seleccionados pueden contener elementos de respuesta a hormona tiroidea funcionales. Esta aproximación podría ser de utilidad para la búsqueda de genes regulados por otros factores de transcripción.A new methodology for the identification of genes modulated by the thyroid hormone receptor (TR) in vivo is described. Mouse genomic DNA fragments bound by the TR were selected and amplified in vitro. Subsequent hybridisation with biotinylated cDNA allowed the selection of those DNA fragments containing binding sites for TR that corresponded to transcribed DNA. Expression analysis of the corresponding genes showed that more than 90% are indeed modulated by thyroid hormones in vivo in the liver. Together with the presence of consensus binding sites for TR this result suggests that the selected DNA fragments may contain TR transcriptional regulatory elements. This method might be useful to other transcription factors with slight modifications

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae. Comp. Funct. Genomics 2:124–142

Abstract Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of b1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect b1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation