Cell proliferation during organ development is controlled, at least in part, by contact-mediated interactions between cells of the growing tissue. The molecular genetic basis of these cell interactions will be investigated using the imaginal discs of Drosophila as a model system. We will continue our molecular analysis of the fat locus, in which recessive lethal mutations cause the imaginal discs to grow by cell proliferation beyond the normal limits. In collaborative work, we have shown that the fat locus potentially encodes an enormous transmembrane molecule related to calcium- dependent cell adhesion molecules (cadherins) and that a dominant mutation of the locus (Gull) is associated with a transposable element insertion. We have also detected DNA alterations in the fat locus associated with three recessive lethal alleles. The exact nature of these and other mutations will be determined by nucleotide sequencing. Antibodies will be produced against synthetic peptides and against fusion gene products corresponding to parts of the predicted protein, and the antisera will be used in Western blotting and immunofluorescence experiments using confocal microscopy to investigate the developmental expression of the gene product and its subcellular location in both normal and mutant discs. The effects of the mutations on gene expression at the RNA will be determined by Northern blotting and RNAse protection experiments, and by in situ hybridization of exon-specific probes to imaginal disc tissues. A newly developed technique for in vitro culture of imaginal disc cells will be used to examine the relationship between cell adhesion and proliferation. Cultures will be established from imaginal discs from wild-type and overgrowth mutants, and the expression of selected growth-control and cell- adhesion molecules investigated. Hormone treatments and transfection will be used to manipulate the expression of these genes in order to investigate whether they alter cell adhesivity or the expression of other known proliferation control genes, and whether these changes are associated with changes in growth properties. The work will contribute to our understanding of how growth is controlled in animals and in the human body, how specific genes are involved in these control mechanisms, and how genetic mutations lead to cancer and other abnormalities of growth.
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