Influence of deficiency in mevalonate kinase (MVK) on wide gene expression in Saccharomyces cerevisiae

Periodic fever syndrome is a disease that affects children mainly in the Mediterranean region, which has as one of the typical conditions of inflammation generalized by the body that can spontaneously regress. It has already been established that this pattern is presented by individuals carrying a mutant allele of the gene encoding the enzyme mevalonate kinase (MVK). This gene has an ortholog in the yeast Saccharomyces cerevisiae (ERG12) and mutations in this genes show phenotype that resemble those of the cells of these individuals. The present work aims to use this yeast as a biological model to analyze the role of the ERG12 gene (MVK) in the development of the disease. In this experiment, we used a wild type strain for the enzyme Erg12 (wt) and a genetically modified strain that decreases the activity of this enzyme (erg12-d) to 10%. The cells were cultured in synthetic medium containing glucose (reference medium) and each was re-inoculated and synthetic medium containing glucose or glycerol (erg12-d cells have growth deficiency in glycerol). By attaining D.O. value around 0.5 (600nm), the cells were collected for total RNA extraction and this was subjected to double-labeled microarray analysis in four combinations: erg12-d/wt and glycerol/glucose.周期性发热综合征（Periodic fever syndrome）是一种主要累及地中海地区儿童的疾病，其典型临床表现之一为可自发性消退的全身性炎症反应。现已明确，携带编码甲羟戊酸激酶（mevalonate kinase, MVK）基因突变等位基因的个体，会表现出该疾病的相关表型。该基因在酿酒酵母（Saccharomyces cerevisiae）中存在同源基因ERG12，且ERG12的突变所诱导的细胞表型，与上述患者体内细胞的表型高度相似。本研究旨在以该酿酒酵母作为生物模型，分析ERG12基因（对应人类MVK基因）在该疾病发生发展中的作用。本实验中，我们使用了两种酵母菌株：一种为Erg12酶野生型菌株（wt），另一种为经基因改造后该酶活性降至10%的敲减菌株（erg12-d）。菌株均培养于含葡萄糖的合成培养基（对照培养基）中，随后将其重新接种至含葡萄糖或甘油的合成培养基内（注：erg12-d菌株在甘油培养基中存在生长缺陷）。当细胞在600nm波长下的光密度（D.O.）值达到约0.5时，收集细胞并提取总RNA，随后针对总RNA开展双标记微阵列分析，共设置四组对比组合：分别为erg12-d与wt菌株、甘油培养基与葡萄糖培养基的两两配对组合。