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

[Carta a Ignacio Hernando de Larramendi y Montiano]

Información adicional de autor de la carta: Presidente, MAPFRE GuanartemeFotografía número: 32

Identification of the mitochondrial NADH dehydrogenase subunit 3 (ND3) as a thyroid hormone regulated gene by whole genome PCR analysis

We previously described a modification of the whole genome PCR method which allowed us to characterize several genes whose expression is regulated by thyroid hormone in the mouse liver. Following this procedure, we now report the identification of the mitochondrial NADH dehydrogenase subunit 3 (ND3) gene as target of thyroid hormone. ND3 gene expression is regulated by thyroid hormone in rat brain and heart. Sequencing and electrophoretic mobility shift assays confirmed the presence of a thyroid hormone receptor (TR)/c-erbA specific binding site in the mitochondrial ND3 gene. Hypothyroidism decreases ND3 mRNA levels in several brain areas such as cortex and hippocampus during the early postnatal development. In line with the recent findings showing the presence of TR/c-erbA α and β proteins inside the mitochondria, our results suggest the possibility of direct transcriptional regulation of mitochondrial genes by thyroid hormone.This work was supported by grants from the CICYT (Plan NAcional I+D, SAF92/0396) and Fundación Ramón Areces.Peer Reviewe

Thyroid hormone-dependent transcriptional repression of neural cell adhesion molecule during brain maturation

Thyroid hormone (T3) is a main regulator of brain development acting as a transcriptional modulator. However, only a few T3-regulated brain genes are known. Using an improved whole genome PCR approach, we have isolated seven clones encoding sequences expressed in neonatal rat brain which are under the transcriptional control of T3. Six of them, including the neural cell adhesion molecule NCAM, α-tubulin and four other unidentified sequences (RBA3, RBA4, RBB3 and RBB5) were found to be upregulated in the hypothyroid brain, whereas another (RBE7) was downregulated. Binding sites for the T3 receptor (T3R/c-erbA) were identified in the isolated clones by gel-shift and footprinting assays. Sites in the NCAM (in an intron), α-tubulin (in an exon) and RBA4 clones mediated transcriptional regulation by T3 when inserted upstream of a reporter construct. However, no effect of the NCAM clone was found when located downstream of another reporter gene. Northern blotting and in situ hybridization studies showed a higher expression of NCAM in the brain of postnatal hypothyroid rats. Since NCAM is an important morphoregulatory molecule, abnormal NCAM expression is likely to contribute to the alterations present in the brain of thyroid-deficient humans and experimental animals.This work has been supported with Grants from the CICYT, Plan Nacional I+D (SAF92-0396 and SAF95-0738) and FIS94/0273, Fundación Ramón Areces and the European Union (HCM, Laboratory Networks CHRX-CT93-0185).Peer Reviewe

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