High-density microarray-mediated gene expression profiling of Escherichia coli
High-density microarray-mediated gene expression profiling of Escherichia coli
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DOI：
10.1128/jb.183.2.545-556.2001
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发表时间：
2001-01-01
影响因子：
3.2
通讯作者：
LaRossa, RA
中科院分区：
文献类型：
--
作者：
Wei, Y;Lee, JM;LaRossa, RA
一个几乎完整的4,290个大肠杆菌开放阅读框的集合被扩增并高密度排列在玻片上。为开发该试剂，已建立了从E、Coli细胞中提取RNA、伴随荧光染料掺入的cDNA生产、DNA-DNA杂交和杂交定量的条件。短暂的异丙基-β-D-硫代半乳糖苷(IPTG)处理使LacZ、Lacy和LacA转录本的含量增加了约30倍；相反，大多数其他转录本的滴度保持不变。对指数生长和过渡生长阶段的大肠杆菌培养物之间不同的RNA表达模式进行了分类，以及与在最少和丰富的培养基中培养的差异进行了分类。通过使用基因组DNA衍生的荧光标记探针的杂交作为校正因子来估计每个转录本的相对丰度。这份清单提供了每一种mRNA的稳态水平的定量视图，并列举了用该方法检测到的基因的表达，并将结果与目前对大肠杆菌生理学的理解进行了比较。
A nearly complete collection of 4,290 Escherichia coli open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from E, coli cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established. A brief isopropyl-beta -D-thiogalaetopyranoside (IPTG) treatment elevated lacZ, lacY, and lacA transcript content about 30-fold; in contrast, most other transcript titers remained unchanged. Distinct RNA expression patterns between E. coli cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media. The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor. This inventory provided a quantitative view of the steady-state level of each mRNA species, Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of E. coli physiology.