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Análisis mediante RNAseq de la respuesta transcripcional de Saccharomyces cerevisiae en presencia de ácido acético en una cepa silvestre y en otra con pérdida de función del gen SSD1

Authors: Blanco Carmona, Enrique;

Análisis mediante RNAseq de la respuesta transcripcional de Saccharomyces cerevisiae en presencia de ácido acético en una cepa silvestre y en otra con pérdida de función del gen SSD1

Abstract

[ES] Una de las características distintivas (hallmark) de la mayoría de células cancerígenas es la relación pHi/pHo inversa respecto al resto de células somáticas, siendo más alcalino el medio intracelular que el extracelular. Este hecho es relevante debido a su aplicabilidad biomédica, puesto que esto deriva en una diferente regulación de eventos celulares como, por ejemplo, el ciclo celular y la tasa de crecimiento, entre otros. De igual manera, se ha demostrado que el suministro de ácidos débiles puede influir de forma negativa en dichos procesos. En este marco, SSD1 es un gen implicado en el progreso del ciclo celular, longevidad, patogenicidad, tolerancia a temperatura y morfogénesis celular en Saccharomyces cerevisiae. Muchas de sus funciones son aún desconocidas, entre ellas su implicación en la homeostasis del pH. Ssd1 es una proteína de unión al RNA y se cree responsable de la regulación transcripcional y post-transcripcional de genes específicos, entre ellos ciclinas del ciclo celular como CLN2. Esta interacción estabiliza mRNAs, aumentando así su vida media en la célula. SSD1 presenta dos alelos: SSD1-V y ssd1-d. El primero es silvestre y el otro presenta una mutación con pérdida de función. El alelo SSD1-V confiere resistencia a ácido acético en medio mínimo en contraposición al alelo ssd1-d. En el presente TFG se quiere ver la expresión génica diferencial de estos dos alelos en presencia y ausencia de ácido acético (45 mM). Esto se analiza mediante RNAseq, con el fin de individualizar genes responsables del crecimiento diferencial entre ambos alelos. Además, se caracteriza el perfil de crecimiento de ambos alelos mediante medición continua de absorbancia (Bioscreen). Los resultados del Bioscreen muestran lo evidenciado en experimentos anteriores, un perfil de crecimiento similar entre ambos alelos, tanto a pH 6 como a pH 4, pero un menor crecimiento de la cepa ssd1-d frente a la cepa SSD1-V en estrés por ácido acético. Asimismo, a la luz de los resultados preliminares del análisis de expresión diferencial, destaca el gen PCL2, que codifica para una ciclina del ciclo celular con función análoga a la ciclina CLN2. Estando ambas ciclinas presentes en la fase G1 y siendo responsables del progreso hacia el ciclo mitótico, PCL2 es el gen candidato para futuros experimentos de interacción entre SSD1- CLN2. [EN] One of the hallmarks of most cancer cells is the inverse pHi/pHo relation to the rest of somatic cells, the intracellular medium being more alkaline than the extracellular one. This fact is relevant due to its biomedical applicability, since this leads to a different regulation of cellular events such as the cell cycle and the growth rate, among others. Likewise, it has been demonstrated that the supply of weak acids can negatively influence said processes. In this framework, SSD1 is a gene involved in cell cycle progress, longevity, pathogenicity, temperature tolerance and cellular morphogenesis in Saccharomyces cerevisiae. Many of its functions are still unknown, including its relation in pH homeostasis. Ssd1 is an RNA binding protein. It is believed for Ssd1 to be responsible of the transcriptional and post-transcriptional regulation of specific genes, including cyclins such as CLN2. This interaction stabilizes mRNAs, thus increasing their half-life in the cell. SSD1 has two alleles: SSD1-V and ssd1-d. The first one is the wild type and the other one has a mutation with loss of function. The SSD1-V allele confers resistance to acetic acid in minimal medium. In the present dissertation we want to see the differential gene expression of these two alleles in the presence and absence of acetic acid (45 mM). This is analyzed by RNAseq, in order to identify genes responsible for differential growth between both alleles. In addition, the growth profile of both alleles is characterized by continuous absorbance measurement (Bioscreen). The results of the Bioscreen show what was evidenced in previous experiments, a similar growth profile between both alleles, both at pH 6 and at pH 4, but a lower growth of the strain ssd1-d compared to the strain SSD1-V in stress by acetic acid. Likewise, in light of the preliminary results of the differential expression analysis, the PCL2 gene, which codes for a cyclin of the cell cycle with analogous function to the cyclin CLN2, stands out. With both cyclines present in the G1 phase and being responsible for the progress towards the mitotic cycle, PCL2 is the candidate gene for future interaction experiments between SSD1-CLN2.

Keywords

regulación, growth, crecimiento, BIOQUIMICA Y BIOLOGIA MOLECULAR, Grado en Biotecnología-Grau en Biotecnologia, intracellular pH., regulation, Saccharomyces cerevisiae, RNAseq, SSD1, pH intracelular.

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