The long-term objective of this project is to obtain an understanding of how an elaborate developmental process is accomplished by the integrated expression of a large number of genes. Failure to integrate the expression of various structural genes is the basis for a variety of human developmental defects. The model system chosen for this study is the production of the chorion (eggshell) by the monolayer of follicle cells surrounding the maturing oocyte of the silkmoth, Bombyx mori. In these cells the synthesis of over 100 chorion proteins follows a precise temporal pattern corresponding to a program of specific mRNA production. One useful property of this system is that all chorion genes are tightly clustered on a small segment of one chromosome. The size of this region is estimated to be 700 kilobases. Recovery of the entire locus on recombinant clones (500 kb of which is already accomplished) along with detailed characterizations of the genes encountered, will provide a rare opportunity to trace the evolution of a complex developmental process, as well as provide the groundwork for studies designed to understand how the locus is coordinately regulated. Our study of gene regulation will involve two major approaches. First, utilizing the synchronous populations of cells that can be dissected from the silkmoth ovary, we will examine the structural changes of the chorion genes within the nucleus prior to and during their expression. These structural changes will be monitored by determining the accessibility of the genes to DNase I digestions. Such experiments address the important question of how genes are marked in the nucleus for subsequent expression. Second, we will determine whether the chorion locus is divided into looped chromosomal domains. Evidence for such loops will be obtained by if segments of the chorion locus are found to be associated with the nuclear matrix, an elaborate protein network within the nucleus. The use of mutations containing breakage points within the chorion locus will be extremely helpful in determining the functional role of such putative attachment sites.
