Couderc J L, Sobrier M L, Giraud G, Becker J L, Dastugue B
J Mol Biol. 1983 Mar 5;164(3):419-30. doi: 10.1016/0022-2836(83)90059-1.
The steroid hormone ecdysterone induced characteristic and specific changes of morphology, enzymatic activities and protein synthesis in a Kc 0% Drosophila melanogaster cell line. To study the ecdysterone action at a molecular level, a Drosophila genomic library was screened by differential hybridization to poly(A)+ RNA from control and ecdysterone-treated cells. Two recombinant phages were selected for hybridizing very intensively with poly(A)+ RNA of ecdysterone-treated cells and very weakly with poly(A)+ RNA of untreated ones. These two clones (lambda Dm 1632 and lambda Dm A5A1) mapped at the 5 C locus on polytene chromosomes; they overlap for a 9000 base-pair sequence that contains an abundantly transcribed region in ecdysterone-treated cells of about 2000 base-pairs. This region permits the selection of mRNA that gives, after translation in vitro, two polypeptides identified as cytoplasmic actin II and III. We demonstrated that these two recombinant phages, hybridizing preferentially with poly(A)+ RNA of ecdysterone-treated cells, contain the 5 C actin gene. Poly(A)+ RNA prepared from various times of treatment of cells were electrophoresed on agarose gels, transferred to nitrocellulose paper and then hybridized with the cloned actin probe. Results of these experiments indicate that there is a sharp increase in the level of RNA coding for actin after ecdysterone treatment of the cell, and that there are two forms of actin-specific RNA in the D. melanogaster cells. Using genomic blots with specific probes derived from lambda Dm 1632, we show that there are six actin genes per haploid Drosophila cell genome contained on six EcoRI fragments, as in Drosophila embryos, indicating that there is no rearrangement of these sequences in cultured cells. Our results suggest that the expression of actin genes in D. melanogaster Kc 0% cells is modulated by ecdysterone.
甾体激素蜕皮甾酮在Kc 0%黑腹果蝇细胞系中诱导了形态、酶活性和蛋白质合成的特征性及特异性变化。为了在分子水平上研究蜕皮甾酮的作用，通过与对照细胞和经蜕皮甾酮处理的细胞的聚腺苷酸加尾RNA（poly(A)+ RNA）进行差异杂交，筛选了一个果蝇基因组文库。选择了两个重组噬菌体，它们与经蜕皮甾酮处理的细胞的poly(A)+ RNA强烈杂交，而与未处理细胞的poly(A)+ RNA杂交很弱。这两个克隆（λDm 1632和λDm A5A1）定位于多线染色体的5C位点；它们在一个9000个碱基对的序列上重叠，该序列在经蜕皮甾酮处理的细胞中包含一个约2000个碱基对的高转录区域。该区域允许选择在体外翻译后产生两种被鉴定为细胞质肌动蛋白II和III的多肽的mRNA。我们证明，这两个优先与经蜕皮甾酮处理的细胞的poly(A)+ RNA杂交的重组噬菌体包含5C肌动蛋白基因。从细胞不同处理时间制备的poly(A)+ RNA在琼脂糖凝胶上进行电泳，转移到硝酸纤维素纸上，然后与克隆的肌动蛋白探针杂交。这些实验结果表明，在细胞经蜕皮甾酮处理后，编码肌动蛋白的RNA水平急剧增加，并且在黑腹果蝇细胞中有两种肌动蛋白特异性RNA形式。使用来自λDm 1632的特异性探针进行基因组印迹分析，我们表明每个单倍体果蝇细胞基因组在六个EcoRI片段上包含六个肌动蛋白基因，如同在果蝇胚胎中一样，这表明在培养细胞中这些序列没有重排。我们的结果表明，黑腹果蝇Kc 0%细胞中肌动蛋白基因的表达受蜕皮甾酮调节。
