Hope I A
Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Development. 1991 Oct;113(2):399-408. doi: 10.1242/dev.113.2.399.
A screen of gene expression patterns has been developed for the nematode Caenorhabditis elegans. Promoter-reporter gene fusions were constructed in vitro by ligating C. elegans genomic DNA fragments upstream of a lacZ gene. Patterns of beta-galactosidase expression were examined by histochemical staining of C. elegans lines transformed with the constructs. beta-galactosidase expression depended on translational fusion, so constructs were assayed in large pools to expedite detection of the low proportion that were active. Expression in a variety of cell types and temporal patterns was observed with different construct pools. The most striking expression patterns were obtained when the beta-galactosidase activity was localized to subcellular structures by the C. elegans portion of the fusion protein. The active constructs of three selected pools were identified subsequently by an efficient combinatorial procedure. The genomic locations of the DNA fragments from the active constructs were determined and appear to define previously uncharacterized genetic loci.
已开发出一种用于线虫秀丽隐杆线虫的基因表达模式筛选方法。通过将秀丽隐杆线虫基因组DNA片段连接到lacZ基因上游,在体外构建启动子-报告基因融合体。通过对用构建体转化的秀丽隐杆线虫品系进行组织化学染色,检测β-半乳糖苷酶的表达模式。β-半乳糖苷酶的表达依赖于翻译融合,因此对构建体在大量样本中进行检测,以加快对低比例活性构建体的检测。用不同的构建体样本观察到了在多种细胞类型中的表达以及不同的时间模式。当β-半乳糖苷酶活性通过融合蛋白的秀丽隐杆线虫部分定位于亚细胞结构时,获得了最显著的表达模式。随后通过一种有效的组合方法鉴定了三个选定样本中的活性构建体。确定了来自活性构建体的DNA片段的基因组位置,这些位置似乎定义了以前未表征的基因座。