| Abstrakt: |
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. |
