Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing
Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing
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DOI：
10.1093/embo-reports/kvd058
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发表时间：
2000-09-01
期刊：
影响因子：
7.7
通讯作者：
Wiemann, S
中科院分区：
文献类型：
--
作者：
Simpson, JC;Wellenreuther, R;Wiemann, S
作为比生物信息分析更全面的 cDNA 功能表征的第一步，生物信息分析只能对约一半的已测序 cDNA 进行功能预测，我们开发并测试了一种策略，允许其系统且快速的亚细胞定位。我们使用一种新颖的克隆技术快速生成 cDNA 的 Nand C 端绿色荧光蛋白融合物，以检查活细胞中 > 100 个表达的融合蛋白的细胞内定位。整个分析适合自动化，这对于扩大吞吐量非常重要。对于超过 80% 的新蛋白质，可以确定其在已知结构或细胞器中的清晰细胞内定位。对于生物信息学分析能够预测可能身份的 cDNA，定位能够在 75% 的情况下支持这些预测。对于那些无法预测同源性的 cDNA，定位数据代表了第一个信息。
As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate Nand C-terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of >100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For >80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.