ABSTRACT
Drosophila PTEN (dPTEN) plays indispensable roles in the development of Drosophila melanogaster by controlling cell size and number. Although three potential spliced forms of dPTEN have been isolated, functional distinction among these forms remains elusive. In this study, we demonstrate that all spliced forms of dPTEN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)); however, PI(3,4,5)P(3)-dependent activation of Drosophila Akt is suppressed specifically by one of three spliced forms, dPTEN3. Further, dPTEN3 dramatically changes its expression during the Drosophila development, while the other forms are expressed throughout the development. Our results suggest that dPTEN3 is the predominant spliced form that participates in PI(3,4,5)P(3)-mediated signaling pathways.
Subject(s)
Alternative Splicing , Drosophila Proteins/chemistry , Phosphoinositide-3 Kinase Inhibitors , Phosphoric Monoester Hydrolases/chemistry , Signal Transduction , Animals , Cell Line , DNA, Complementary/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster , Glutathione Transferase/metabolism , Microscopy, Confocal , Mutation , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Plasmids/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , TransfectionABSTRACT
We have devised a novel method for automated microsatellite analysis using "universal" fluorescent labeling. This system is based on polymerase chain reactions driven by sequence-specific primers and a reporter primer labeled with a fluorescent dye at its 5' end. The forward sequence-specific primer is designed with a tag region bearing no homology to any human genomic sequence. Complementary tag sequences act as templates for the 6-carboxyfluorescein-labeled reporter primer, and those products can be analyzed with an autosequencer. The results we achieved with this assay system were consistent with the results of conventional assays using radioisotope-labeled primers, and diagnosis required less time. Furthermore, the fluorescent-labeled reporter primer is "universal" in that it can be used with different sequence-specific primers designed to carry the appropriate tag sequence at their 5'-ends. Our observations suggest that the "universal" fluorescent labeling method is an efficient tool for analyzing sequence variations in human DNA.
