Mismer D, Rubin G M
Department of Biochemistry, University of California, Berkeley 94720.
Genetics. 1989 Jan;121(1):77-87. doi: 10.1093/genetics/121.1.77.
We have analyzed the cis-acting regulatory sequences of the Rh1 (ninaE) gene in Drosophila melanogaster by P-element-mediated germline transformation of indicator genes transcribed from mutant ninaE promoter sequences. We have previously shown that a 200-bp region extending from -120 to +67 relative to the transcription start site is sufficient to obtain eye-specific expression from the ninaE promoter. In the present study, 22 different 4-13-bp sequences in the -120/+67 promoter region were altered by oligonucleotide-directed mutagenesis. Several of these sequences were found to be required for proper promoter function; two of these are conserved in the promoter of the homologous gene isolated from the related species Drosophila virilis. Alteration of a conserved 9-bp sequence results in aberrant, low level expression in the body. Alteration of a separate 11-bp sequence, found in the promoter regions of several photoreceptor-specific genes of Drosophila, results in an approximately 15-fold reduction in promoter efficiency but without apparent alteration of tissue-specificity. A protein factor capable of interacting with this 11-bp sequence has been detected by DNaseI footprinting in embryonic nuclear extracts. Finally, we have further characterized two separable enhancer sequences previously shown to be required for normal levels of expression from this promoter.
我们通过P因子介导的从突变型ninaE启动子序列转录的指示基因的种系转化，分析了黑腹果蝇Rh1（ninaE）基因的顺式作用调控序列。我们先前已经表明，相对于转录起始位点从-120延伸至+67的200bp区域足以从ninaE启动子获得眼特异性表达。在本研究中，通过寡核苷酸定向诱变改变了-120/+67启动子区域中的22个不同的4-13bp序列。发现这些序列中的几个是正常启动子功能所必需的；其中两个在从相关物种果蝇中分离的同源基因的启动子中是保守的。一个保守的9bp序列的改变导致身体中异常的低水平表达。在果蝇的几个光感受器特异性基因的启动子区域中发现的一个单独的11bp序列的改变，导致启动子效率降低约15倍，但组织特异性没有明显改变。通过胚胎核提取物中的DNaseI足迹法检测到一种能够与该11bp序列相互作用的蛋白质因子。最后，我们进一步表征了先前显示为该启动子正常表达水平所必需的两个可分离的增强子序列。
