Goldsborough A S, Kornberg T B
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12696-700. doi: 10.1073/pnas.91.26.12696.
Changes in levels of transcription can be difficult to gauge in animals with lethal mutations. For example, mutations in a regulatory region of an essential gene can have secondary consequences that complicate attempts to quantify the transcripts produced by the mutant gene. We describe a method that circumvents this problem by revealing the relative amount of transcript produced from each allele in a heterozygote. With this method, recessive lethal mutations can be analyzed in animals that are phenotypically wild type. We used this technique to analyze mutations in the regulatory region of the Drosophila engrailed gene and found that truncations reduce transcription to levels that depend both upon the tissue and upon the location of the chromosomal break. We also found that these mutations affect expression of the linked invected gene, suggesting that engrailed and invected share a complex set of regulatory elements that operate over at least 85 kb. We suggest that this technique will have general utility for the quantitation of allele-specific transcripts, even when amounts of tissue are limiting.
对于具有致死突变的动物而言，转录水平的变化可能难以衡量。例如，必需基因调控区域的突变可能会产生次级效应，使量化突变基因产生的转录本的尝试变得复杂。我们描述了一种方法，通过揭示杂合子中每个等位基因产生的转录本的相对量来规避这个问题。利用这种方法，可以在表型为野生型的动物中分析隐性致死突变。我们使用这项技术分析了果蝇engrailed基因调控区域的突变，发现截短会将转录降低到取决于组织和染色体断裂位置的水平。我们还发现这些突变会影响连锁的invected基因的表达，这表明engrailed和invected共享一组复杂的调控元件，这些元件在至少85 kb的范围内起作用。我们认为，即使组织量有限，这项技术对于等位基因特异性转录本的定量也将具有普遍用途。
