Stroeher V L, Kennedy B P, Millen K J, Schroeder D F, Hawkins M G, Goszczynski B, McGhee J D
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Dev Biol. 1994 Jun;163(2):367-80. doi: 10.1006/dbio.1994.1155.
We describe an experimental system in which to investigate DNA-protein interactions in the early Caenorhabditis elegans embryo. A homogeneous population of developmentally blocked mid-proliferation stage embryos can be produced by exposure to the deoxynucleotide analog fluorodeoxyuridine. These blocked embryos remain viable for days and express a number of biochemical markers of early differentiation, for example, gut granules, the gut esterase ges-1, and two regulatory genes, mab-5 and hlh-1. Using the techniques of gel mobility shift and DNase I footprinting, we show that nuclear extracts prepared from these embryos contain factors that bind to the 5'-promoter sequences of the C. elegans gut-specific ges-1 gene. In particular, we examine a putative gut "activator" region, which was previously identified by deletion-transformation analysis and which contains two copies of a consensus GATA-factor binding sequence. Factors that bind to double-stranded oligonucleotides containing the ges-1 GATA sequences are present predominantly in nuclear extracts of embryos but are found neither in cytoplasmic nor in nuclear extracts of unfertilized oocytes. Two proteins, of 43 and 60 kDa, can be uv-crosslinked to double-stranded oligonucleotides containing the ges-1 GATA sequences. The sizes of these proteins correspond to the sizes expected for the elt-1 protein and for the skn-1 protein, two regulatory factors present in early C. elegans embryos and possible candidates for ges-1 control. However, we show that homozygous deficiency embryos (mDf7/mDf7 embryos and eDf19/eDf19 embryos, both of which lack the elt-1 gene, and nDf41/nDf41 embryos, which have no skn-1 gene), still express the ges-1 esterase. We conclude that neither the elt-1 gene nor the skn-1 gene is necessary zygotically for ges-1 expression. We suggest that neither the elt-1 protein nor the skn-1 protein interacts directly with the ges-1 gene and that the observed binding proteins must correspond to products of other genes. More generally, the present experimental system should allow the biochemical study of any gene expressed during early C. elegans embryogenesis.
我们描述了一个用于研究秀丽隐杆线虫早期胚胎中DNA-蛋白质相互作用的实验系统。通过暴露于脱氧核苷酸类似物氟脱氧尿苷，可以产生处于发育阻滞的增殖中期胚胎的同质群体。这些阻滞的胚胎可以存活数天，并表达一些早期分化的生化标志物，例如肠道颗粒、肠道酯酶ges-1以及两个调控基因mab-5和hlh-1。使用凝胶迁移率变动分析和DNase I足迹分析技术，我们表明从这些胚胎制备的核提取物中含有与秀丽隐杆线虫肠道特异性ges-1基因的5'-启动子序列结合的因子。特别是，我们研究了一个假定的肠道“激活剂”区域，该区域先前通过缺失-转化分析确定，并且包含两个共有GATA因子结合序列的拷贝。与含有ges-1 GATA序列的双链寡核苷酸结合的因子主要存在于胚胎的核提取物中，但在未受精卵母细胞的细胞质或核提取物中均未发现。两种蛋白质，分子量分别为43 kDa和60 kDa，可以通过紫外线交联到含有ges-1 GATA序列的双链寡核苷酸上。这些蛋白质的大小与秀丽隐杆线虫早期胚胎中存在的两种调控因子elt-1蛋白和skn-1蛋白预期的大小相对应，它们可能是ges-1调控的候选因子。然而，我们表明纯合缺失胚胎（mDf7/mDf7胚胎和eDf19/eDf19胚胎，两者均缺乏elt-1基因，以及nDf41/nDf41胚胎，其没有skn-1基因）仍然表达ges-1酯酶。我们得出结论，elt-1基因和skn-1基因在合子阶段对于ges-1表达都不是必需的。我们认为elt-1蛋白和skn-1蛋白都不直接与ges-1基因相互作用，并且观察到的结合蛋白必定对应于其他基因的产物。更一般地说，本实验系统应该允许对秀丽隐杆线虫早期胚胎发育过程中表达的任何基因进行生化研究。
