The vitellogenin gene family from the nematode, Caenorhabditis elegans is being studied. The genes are expressed abundantly in the intestine of the adult hermaphrodite worm, but are not transcribed in the male, in any of the four larval stages, or inother hermaphrodite tissues. In C. elegans five closely-related genes and one distant member of the gene family encode two distinct classess of vitellogenins. The main objective of this research is to understand the nature of the developmental signals which control transcription of these genes and the detailed mechanisms by which the regulation is effected. By sequencing the promoter regions of the six vitellogenin genes plus those from a related nematode species, two highly conserved heptameric sequences which are likely to be involve in the regulation have been identified. Development of a nematode transformation system to test these hypotheses has just recently been accomplished. A vitellogenin gene promoter has been fused to the structural gene for E. coli beta-glucuronidase and several transgenic worm strains containing the fusion gene integrated in their genomes have been obtained. These lines will be tested for regulated expression. The regions of the promoter required for particular aspects of the regulation will be identified using deletions, linker-scanning mutagenesis, and synthesis of putative regulatory elements. The regulatory roles of the different heptameric element will also be tested in competition experiments: Transformants containing long tandem arrays of the elements will be tested for inappropriate expression. Sequences bound by proteins in crude extracts will be identified by footprinting assays. The proteins that bind to the heptamers will be purified and the genes that encode them will be cloned. Regulatory mutants will be selected and their effects of the binding proteins measured. Mutants that express the vitellogenins in males or that fail to express them in hermaphrodites will be analyzed. Sequence analysis has revealed the presence of potential stem- loop structures at the 5' ends of the vitellogenin mRNAs. Evolutionary evidence strongly suggests that such stems exist. Direct evidence for their existence will be obtained by use of single-and double-strand specific nuclease digestion. Possible functions for the stems will be investigated, and the importance of the stems in performance of the proposed functions studied in vivo using transgenic worms containing the stem-containing region fused to a reporter gene.
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