Musarrat Javed, Hashsham Syed A
Department of Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh, India.
Teratog Carcinog Mutagen. 2003;Suppl 1:283-94. doi: 10.1002/tcm.10054.
DNA microarray is a powerful tool for parallel detection of multiple target genes in biological systems. In this study, a low-density DNA microarray has been custom designed by using Pseudomonas stutzeri strain KC ORFs that are implicated in carbon tetrachloride degradation. PCR amplified strain KC probes of varying lengths were obtained using ORF-specific primers. Purified short probes (80-120 bp) and full-length amplicons were directly immobilized on gamma-aminosilane coated and superaldehyde trade mark glass substrates without any chemical modification. The full-length amplicons exhibited a much higher signal compared to the shorter probes upon hybridization with the Cy5/Cy3-labeled unfragmented cDNA targets. The meager signal with the shorter probes limits the advantage of using the multiple probes of the same genes for enhancing the specificity of hybridization with environmental samples. Nevertheless, expression analysis of strain KC genome, under controlled laboratory conditions, revealed the constitutive expression of at least 11 putative ORFs of the pdt operon. Comparatively weaker hybridization signals with the cDNA from mutant cells suggested a low abundance of mRNA transcripts in the KC 1896 mutant. Similar expression levels of the pdt ORFs I, J, K, M, N, O, P, and fur gene both under iron-limiting conditions and in presence of iron (20 micro M Fe(3+)) suggested metal ion-independent regulation of the pdt operon. The tailor-made array with strain KC gene-specific probes served as a model for demonstrating the utility of cDNA microarray technology in monitoring the expression of environmentally important genes in bacteria.
DNA微阵列是一种用于并行检测生物系统中多个靶基因的强大工具。在本研究中，利用与四氯化碳降解相关的施氏假单胞菌菌株KC的开放阅读框（ORF）定制设计了一种低密度DNA微阵列。使用ORF特异性引物获得了不同长度的PCR扩增菌株KC探针。纯化后的短探针（80 - 120 bp）和全长扩增子直接固定在γ-氨基硅烷包被的和商标为superaldehyde的玻璃基板上，无需任何化学修饰。与用Cy5/Cy3标记的未片段化cDNA靶标杂交时，全长扩增子比短探针表现出高得多的信号。短探针产生的微弱信号限制了使用同一基因的多个探针来提高与环境样品杂交特异性的优势。然而，在受控实验室条件下对菌株KC基因组的表达分析表明，pdt操纵子的至少11个推定ORF呈组成型表达。与突变体细胞的cDNA杂交信号相对较弱，表明KC 1896突变体中mRNA转录本丰度较低。在铁限制条件下和存在铁（20 μM Fe(3+)）时，pdt ORF I、J、K、M、N、O、P和fur基因的表达水平相似，表明pdt操纵子的调控不依赖金属离子。带有菌株KC基因特异性探针的定制阵列作为一个模型，证明了cDNA微阵列技术在监测细菌中对环境重要基因表达方面的实用性。
