Isoform-Specific Effects of the DOA Kinase on Autophagic Cell Death of the Larval Salivary Glands in Drosophila melanogaster
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Families of protein kinases have been conserved over millions of years, and they play important roles in many cellular processes such as the cell cycle, differentiation, and signal transduction (Neiman, 1993). Doa, or Darkener of apricot, is a locus found in Drosophila melanogaster that encodes a protein kinase belonging to the LAMMER kinase family. There are seven known protein isoforms of the DOA kinase, which differ by molecular weight and developmental specificity (Kpebe and Rabinow, 2008). While the isoforms contain identical Cterminal catalytic domains, the N-terminal noncatalytic domains are different due to the use of alternative promoters (Kpebe and Rabinow, 2008). Given the number of different structures of DOA isoforms and their respective developmental specificity, it is possible that the isoforms are performing different molecular functions (Kpebe and Rabinow, 2008). Macroautophagy, referred to here as autophagy, is a crucial process that aids in the regulation of cellular homeostasis (Lockshin and Zakeri, 2004). Autophagy is lysosomally-mediated, and is an important process involved in the removal and degradation of bulk cytoplasm and long-lived proteins and organelles (Berry and Baehrecke, 2007). The salivary glands in Drosophila melanogaster offer an ideal model for studying developmental autophagic cell death in vivo, since the bulk of the cytoplasm in the larval salivary glands are removed via autophagic cell death during metamorphosis (Lockshin and Zakeri, 2004). In this project, interfering RNA (RNAi) constructs encoding short hairpin RNAs (shRNAs) were used to silence the expression of specific Doa isoforms through the degradation of homologous mRNA. The constructs each target a different Doa isoform mRNA, because the N-termini are all different from one another, and are being expressed from alternative promoters. This approach could hypothetically reveal different functions of the Doa isoforms. The segregation of markers (Tb on the balancer chromosome TM6 or TSTL) is used to follow genotypes of overexpression/RNAi for Doa. Since RNAi in Drosophila is cell-autonomous, the GAL4-UAS system can be used to target the expression of the RNAi constructs. By crossing female flies with a UAS-GFP responder to males with a Salivary Gland (SG) GAL4 driver, it is possible to express GFP in the salivary glands of the progeny. The salivary glands can be visualized via fluorescence microscopy, and images taken at 6 hour intervals should reveal a construct’s effects on autophagic cell death. Differences in levels of autophagy of the salivary glands between flies with the modifier construct and the wild-type could be indicative of an isoform’s involvement in the promotion or inhibition of cell death.
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