9806046 Neugebauer This project pioneers a cytological assay that simultaneously localizes particular genes by in situ hybridization and alternative splicing factors by immunocytochemistry. The Principle Investigator has completed a preliminary study, using high resolution fluorescent light microscopy to co-localize splicing factors with active sites of gene transcription by RNA polymerase II (Neugebauer and Roth, 1997. Genes Dev 11:1148-1159). These results and others argue strongly that pre-mRNA is spliced while it is being synthesized at the gene. In addition, she found that a particular alternative splicing factor, SRp20, was located at a subset of the sites where general splicing factors were located, suggesting the gene-specific action of individual splicing factors. Thus, the interphase nucleus of any cell can be studied cytologically to determine which genes are being expressed and which regulators are associated with those active genes. The long term goal is to establish a number of experimental systems to examine regulated gene expression. During the one-year period covered by the POWRE award, Dr. Neugebauer plans to use the high resolution light microscopy system Deltavision, which has been under development at Fred Hutchinson Cancer Research Center by Applied Precision, Inc. This technology is only available at a very few labs and institutions world-wide. Therefore, the POWRE award is being used to support research/educational enhancement. This research project addresses how the cell nucleus carries out its functions. In the cytoplasm, the cell achieves a high degree of order and efficiency by compartmentalizing its functions among the organelles. This work uses precursor messenger RNA (pre-mRNA) maturation as an example with which to probe nuclear structure and function. Sophisticated labeling techniques using antisera and nucleic acids will be developed to determine whether mRNA maturation occurs in specific subdomains of the nucleus and to study its regulation.
