Rodríguez-Quiñones José F, Irizarry Rafael A, Díaz-Blanco Nitza L, Rivera-Molina Félix E, Gómez-Garzón Diana, Rodríguez-Medina José R
Department of Biochemistry, School of Medicine, Medical Sciences Campus, University of Puerto Rico, P.O. Box 365067, San Juan, Puerto Rico 00936-5067, USA.
BMC Genomics. 2008 Jan 23;9:34. doi: 10.1186/1471-2164-9-34.
The Saccharomyces cerevisiae MYO1 gene encodes the myosin II heavy chain (Myo1p), a protein required for normal cytokinesis in budding yeast. Myo1p deficiency in yeast (myo1Delta) causes a cell separation defect characterized by the formation of attached cells, yet it also causes abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, increased chitin synthesis, and hypersensitivity to the chitin synthase III inhibitor Nikkomycin Z. To determine how differential expression of genes is related to these diverse cell wall phenotypes, we analyzed the global mRNA expression profile of myo1Delta strains.
Global mRNA expression profiles of myo1Delta strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes (p < or = 0.01) were identified with 263 up regulated and 284 down regulated genes in the myo1Delta strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. The SLT2/MPK1 gene was up regulated in the microarray, and a myo1Deltaslt2Delta double mutant was non-viable. Overexpression of ribosomal protein genes RPL30 and RPS31 suppressed the hypersensitivity to Nikkomycin Z and increased the levels of phosphorylated Slt2p in myo1Delta strains. Increased levels of phosphorylated Slt2p were also observed in wild type strains under these conditions.
Following this analysis of global mRNA expression in yeast myo1Delta strains, we conclude that 547 genes were differentially regulated in myo1Delta strains and that the stress response and protein biosynthesis gene categories were coordinately regulated in this mutant. The SLT2/MPK1 gene was confirmed to be essential for myo1Delta strain viability, supporting that the up regulated stress response genes are regulated by the PKC1 cell integrity pathway. Suppression of Nikkomycin Z hypersensitivity together with Slt2p phosphorylation was caused by the overexpression of ribosomal protein genes RPL30 and RPS31. These ribosomal protein mRNAs were down regulated in the myo1Delta arrays, suggesting that down regulation of ribosomal biogenesis may affect cell integrity in myo1Delta strains.
酿酒酵母MYO1基因编码肌球蛋白II重链（Myo1p），这是芽殖酵母正常胞质分裂所需的一种蛋白质。酵母中Myo1p缺陷型（myo1Δ）会导致细胞分离缺陷，其特征是形成粘连细胞，但它也会导致异常出芽模式、形成增大和伸长的细胞、增加渗透敏感性、几丁质沉积错位、几丁质合成增加以及对几丁质合酶III抑制剂多氧霉素Z过敏。为了确定基因的差异表达如何与这些不同的细胞壁表型相关，我们分析了myo1Δ菌株的全局mRNA表达谱。
通过与酵母寡核苷酸微阵列杂交获得了myo1Δ菌株及其相应野生型对照的全局mRNA表达谱。选定基因的结果通过实时RT-PCR得到证实。在myo1Δ菌株中总共鉴定出547个差异表达基因（p≤0.01），其中263个基因上调，284个基因下调。基因集富集分析显示蛋白质生物合成和应激反应类别中的基因显著过度表达。微阵列中SLT2/MPK1基因上调，myo1Δslt2Δ双突变体无法存活。核糖体蛋白基因RPL×30和RPS31的过表达抑制了对多氧霉素Z的过敏反应，并增加了myo1Δ菌株中磷酸化Slt2p的水平。在这些条件下，野生型菌株中也观察到磷酸化Slt2p水平增加。
通过对酵母myo1Δ菌株全局mRNA表达的分析，我们得出结论，myo1Δ菌株中有547个基因受到差异调节，并且应激反应和蛋白质生物合成基因类别在该突变体中受到协同调节。证实SLT2/MPK1基因对myo1Δ菌株的活力至关重要，这支持上调的应激反应基因受PKC1细胞完整性途径调节。核糖体蛋白基因RPL30和RPS31的过表达导致多氧霉素Z过敏反应的抑制以及Slt2p磷酸化。这些核糖体蛋白mRNA在myo1Δ阵列中下调，表明核糖体生物合成的下调可能影响myo1Δ菌株中的细胞完整性。
