Davies J A, Addison C F, Delaney S J, Sunkel C, Glover D M
J Mol Biol. 1986 May 5;189(1):13-24. doi: 10.1016/0022-2836(86)90377-3.
We have linked the protein coding region of the prokaryotic gene for chloramphenicol acetyl transferase (CAT) to the promoter region of the Drosophila genes for larval serum protein 1 (LSP1). These regions consist of 1.65 X 10(3) and 2.25 X 10(3) base long DNA segments upstream from the LSP1 alpha and beta genes, respectively. The hybrid genes have been inserted into a P-element transformation vector and the constructs introduced into cultured Drosophila Kc cells by calcium phosphate-mediated transfection, or into the germ-lines of flies by P-element-mediated transformation. CAT expression occurs in approximately 1% of the cultured cells following transfection and the accumulation of protein is maximal three to four days after transfection; and it is dependent upon the presence of the LSP1 sequences. We have also obtained several lines of transformed flies that carry the LSP1-CAT genes in their germ-line. The onset of synthesis of functional chloramphenicol acetyl transferase in these organisms occurs in the late third larval instar, rising to a maximum at puparium formation. We continue to detect CAT until the first few hours of adulthood. Assays of CAT activity in the homogenates of dissected tissues indicated that the genes are only expressed in the fat body, as are the endogenous LSP1 genes. We have confirmed this tissue-specific localization of CAT in indirect immunofluorescence using an anti-CAT monoclonal antibody. Northern blots indicate that CAT transcripts are found only in the fat body but their abundance is an order of magnitude lower than endogenous LSP1 transcripts. Primer extension experiments show that transcription of the hybrid genes is initiated at the same nucleotide as the endogenous LSP1 genes. Taken together these data indicate that the 1.65 X 10(3) and 2.25 X 10(3) base segments of DNA upstream from the LSP1 alpha and LSP1 beta genes contain cis-acting regulatory elements necessary for correct tissue and temporal specificity of LSP1 gene expression.
我们已将氯霉素乙酰转移酶（CAT）的原核基因的蛋白质编码区与果蝇幼虫血清蛋白1（LSP1）基因的启动子区域相连。这些区域分别由LSP1α和β基因上游1.65×10³和2.25×10³个碱基长的DNA片段组成。杂种基因已被插入到一个P因子转化载体中，并通过磷酸钙介导的转染将构建体导入培养的果蝇Kc细胞，或通过P因子介导的转化导入果蝇的种系中。转染后，约1%的培养细胞中出现CAT表达，蛋白质积累在转染后三到四天达到最大值；并且它依赖于LSP1序列的存在。我们还获得了几条在种系中携带LSP1 - CAT基因的转化果蝇品系。在这些生物体中，功能性氯霉素乙酰转移酶的合成起始于第三龄幼虫后期，在化蛹时达到最大值。我们在成年后的最初几个小时内仍能检测到CAT。对解剖组织匀浆进行的CAT活性测定表明，这些基因仅在脂肪体中表达，内源性LSP1基因也是如此。我们使用抗CAT单克隆抗体通过间接免疫荧光证实了CAT的这种组织特异性定位。Northern印迹表明，仅在脂肪体中发现CAT转录本，但其丰度比内源性LSP1转录本低一个数量级。引物延伸实验表明，杂种基因的转录起始于与内源性LSP1基因相同的核苷酸。综合这些数据表明，LSP1α和LSP1β基因上游1.65×10³和2.25×10³个碱基的DNA片段包含LSP1基因正确的组织和时间特异性表达所必需的顺式作用调节元件。
