Profiling of Rgt1-regulated gene transcripts (37º C) in the fungal pathogen Candida albicans

C. albicans glucose sensing and signaling is important for full virulence in the mammalian host. To better understand this phenomenon, we investigated the Hgt4 glucose-sensing pathway, focusing on the downstream transcriptional repressor CaRgt1. CaRgt1-regulated gene transcripts were identified by comparing total RNA extracted from wild type and rgt1 mutant strains. Cells were grown at 37 degrees C in the presence of glycerol, conditions under which CaRgt1 was expected to repress gene transcription. Keywords: cell type comparison A C. albicans genespecific microarray consisting of 70-mer oligonucleotides representing 6,346 of the 6,354 predicted ORFs in the annotated C. albicans genome assembly 19 was used. Each ORF was represented by a specific oligonucleotide, and one genome equivalent of this oligo was spotted three times per slide. To identify Rgt1-regulated transcripts, C. albicans cells from the wild type strain BWP17 or the rgt1 mutant strain CM18 were cultured in synthetic medium containing 5% glycerol at 37º C. Cells were harvested and RNA was extracted, labeled, and hybridized to the arrays. Each experiment consisted of a pair-wise competetive hybridization of total RNA samples derived from the wild type and mutant strains and included a reciprocal dye-flip replicate. Since two biological duplicates were tested for each strain, a total of four DNA microarray slides were used. Slides were scanned immediately after hybridization on a ScanArray express HT scanner (Perkin-Elmer). The scanner photomultiplier tube (PMT) values were set for optimal intensity with minimal background. An additional scan was done for each slide (low PMT) with the PMT such that <1% of the elements were saturated in order to characterise spots which were saturated at the higher PMT setting.

白色念珠菌（Candida albicans, C. albicans）的葡萄糖感知与信号转导，对于其在哺乳动物宿主中的完整毒力至关重要。为更好地阐释这一生物学现象，我们针对Hgt4葡萄糖感知通路展开研究，重点关注其下游的转录阻遏蛋白CaRgt1。CaRgt1调控的基因转录本，通过对比野生型与rgt1突变菌株提取的总RNA得以鉴定。实验于37℃下以甘油为唯一碳源培养细胞，该培养条件下CaRgt1预期会抑制靶基因的转录。关键词：细胞类型对比。本研究使用了一款白色念珠菌基因特异性微阵列，该阵列采用70聚寡核苷酸作为探针，可覆盖注释版白色念珠菌基因组组装版本19中6354个预测开放阅读框（Open Reading Frame, ORF）里的6346个。每个开放阅读框均由一条特异性寡核苷酸对应，且每张玻片上该寡核苷酸的基因组当量点样三次。为鉴定Rgt1调控的转录本，我们将野生型菌株BWP17与rgt1突变菌株CM18的白色念珠菌细胞，置于含5%甘油的合成培养基中于37℃培养。收集细胞后提取总RNA，对其进行标记并与微阵列杂交。每项实验均包含野生型与突变菌株来源的总RNA样本的成对竞争性杂交，并设置了反向染料翻转重复实验。由于每个菌株均设置了两次生物学重复，因此共使用了4张DNA微阵列玻片。杂交结束后立即使用ScanArray express HT扫描仪（珀金埃尔默，Perkin-Elmer）对玻片进行扫描。设置扫描仪的光电倍增管（PMT）参数，以获得最优信号强度与最低背景噪音。针对每张玻片额外进行一次低PMT参数扫描，将PMT值调整至仅1%以下的探针点信号饱和，以此表征高PMT设置下出现信号饱和的探针点。