We propose research to identify and characterize proteins controlling cell death and the rolling genes controlling these proteins. The most experimentally useful paradigm for cell death is insect metamorphosis, in which large masses of homogeneous tissues die synchronously and at scheduled times. Our previous studies have employed the intersegmental muscles and labial gland of Manduca sexta, a 10 g insect which provides substantial amounts of each tissue. During cell death in these tissues, synthesis of most proteins drops drastically while simultaneously synthesis of a highly reproducible small set of proteins begins, increases dramatically, or continues unabated, suggesting that these latter proteins are intimately involved in the destruction of the cell. Our earlier observation that protein synthesis is required for cell death has been widely corroborated. We are currently isolating libraries of the genes which are up-regulated during cell death, with the goal of identifying those genes responsible for determining the lifespan of the cell. The proposed research will extend these efforts, and compare the genes of the hornworm to those in Drosophila, in order to utilize and extend the high connectivity of Drosophila I genetics and biology. We will correlate morphological changes with appearance of specific messages and proteins, and determine which proteins are common to Manduca muscle and labial gland. We will also sequence the up-regulated proteins in order to generate antibodies and impute functions if possible. The antibodies will be used to locate the proteins in the dying cells. Second, we will isolate and characterize common cDNAs activated during different stages of involution and likewise use these to characterize the activity of the genes. Finally, we will search for homologous genes in Drosophila. Identification of these genes in Drosophila will permit us to locate them on the Drosophila chromosome and by selection of appropriate mutants or P-element transformation control the activity of the genes in order to determine the extent to which they control cell death. Identification of Manduca in Drosophila will select for conserved genes. The knowledge gained is expected to be applicable to mammalian systems. Knowledge of how cells define their own vulnerability should be valuable for therapeutic considerations, as similar genes are expected to be found in embryonic differentiation, malignant growth and invasion, and aging. It is also likely that a better understanding of normal cell turnover will come from the study of programmed cell death.
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