Picciani Renata G, Diaz Anthony, Lee Richard K, Bhattacharya Sanjoy K
Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
Invest Ophthalmol Vis Sci. 2009 Jul;50(7):3106-11. doi: 10.1167/iovs.08-3106. Epub 2008 Dec 20.
To determine the existence of a relatively higher abundance of potential TFs in glaucomatous trabecular meshwork (TM) that may bind putative promoter regions and affect cochlin protein expression in glaucomatous compared to normal TM.
Combinatorial bioinformatics and biochemical analyses, using human glaucomatous and normal donor tissue (n = 4 each). Biochemical analysis included electrophoretic mobility shift assays (EMSAs), filter binding assays (FBAs), coupled in vitro transcription-translation (TNT) assays and promoter mutation analysis.
Combinatorial bioinformatics and biochemical analyses revealed the existence of a higher abundance of TFs in glaucomatous than in normal TM nuclear extracts. The evidence of a relatively high abundance of TFs, leading to increased expression of cochlin predicted by bioinformatic and biochemical analyses (EMSA and FBA), was further supported by TNT and promoter mutation TNT assays.
These results support the finding that the observed increased cochlin expression in glaucomatous TM is due to relative elevated abundance of TFs. The results also demonstrate the utility of combinatorial bioinformatic and biochemical analyses for genes with uncharacterized promoter regions.
确定与正常小梁网（TM）相比，青光眼性小梁网中可能结合假定启动子区域并影响耳蜗素蛋白表达的潜在转录因子（TFs）是否相对丰度更高。
采用组合生物信息学和生化分析方法，使用人类青光眼供体组织和正常供体组织（各n = 4）。生化分析包括电泳迁移率变动分析（EMSA）、滤膜结合分析（FBA）、体外转录-翻译偶联（TNT）分析和启动子突变分析。
组合生物信息学和生化分析显示，青光眼性TM核提取物中TFs的丰度高于正常TM。生物信息学和生化分析（EMSA和FBA）预测TFs相对丰度较高会导致耳蜗素表达增加，TNT分析和启动子突变TNT分析进一步支持了这一证据。
这些结果支持以下发现，即观察到的青光眼性TM中耳蜗素表达增加是由于TFs相对丰度升高所致。结果还证明了组合生物信息学和生化分析对于启动子区域未明确的基因的实用性。
