The objective of this proposal is to continue to develop and apply in situ hybridization methodology for an understanding of the organization and expression of nucleic acid sequences within the cell. In situ hybridization, particularly in conjunction with non- isotopic detection, is a powerful tool for describing the molecular biolgoy of a single cell. As such advances in this technology have important ramifications spanning both basic and clinical sciences. Having built a firm methodological foundation during the last grant period, we are in a strong position for applying in situ hybridization in new ways to obtain biological information of a fundamental and far-reaching nature. The unusually gentle and sensitive hybridization methodology we have developed has allowed us to investigate the intracellular distribution of specific mRNAs, leading to the discovery that mRNAs for different cytoskeletal proteins exhibit specific and distinct patterns of localization. In the work proposed here we will extend this analysis of mRNA localization to include a variety of cell types as well as other mRNAs for cytoskeletal-associated and non- cytoskeletal proteins. Moreover, we will employ several approaches including light and electron microscopy to analyze the mechanism of mRNA association with the cytoskeleton. Furthermore, recent developments in our lab make it possible to detect with high resolution and efficiency, a single copy sequence within interphase nuclei or on chromosomes. This methodology in conjunction with image processing now makes it possible to study the organization of genes in their functional state within interphase chromatin, using the ordered array of nuclei within the skeletal myofibre as a model system. Hence, we can investigate the molecular cytology of gene expression as a continuum from the production of transcripts within the nucleus to localization and translation of mRNA in the cytoplasm. Finally, the particular hybridization and non-isotopic detection methodology we continue to advance has significant impact on several fields as diverse as clinical diagnostics, human gene mapping (prenatal diagnosis), and virology.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Molecular Cytology Study Section (CTY)
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University of Massachusetts Medical School Worcester
Schools of Medicine
United States
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Politz, J C; Singer, R H (1999) In situ reverse transcription for detection of hybridization between oligonucleotides and their intracellular targets. Methods 18:281-5
Jacobson, M R; Cao, L G; Taneja, K et al. (1997) Nuclear domains of the RNA subunit of RNase P. J Cell Sci 110 ( Pt 7):829-37
Singer, R H (1996) Triplet repeats and human disease. Mol Med Today 2:65-9
Taneja, K L; McCurrach, M; Schalling, M et al. (1995) Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J Cell Biol 128:995-1002
Politz, J C; Taneja, K L; Singer, R H (1995) Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells. Nucleic Acids Res 23:4946-53
Latham Jr, V M; Kislauskis, E H; Singer, R H et al. (1994) Beta-actin mRNA localization is regulated by signal transduction mechanisms. J Cell Biol 126:1211-9
Bassell, G J; Powers, C M; Taneja, K L et al. (1994) Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts. J Cell Biol 126:863-76
Moores Jr, R R; Carter, B S; Meschia, G et al. (1994) Placental and fetal serine fluxes at midgestation in the fetal lamb. Am J Physiol 267:E150-5
Kislauskis, E H; Zhu, X; Singer, R H (1994) Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype. J Cell Biol 127:441-51
Bassell, G J; Taneja, K L; Kislauskis, E H et al. (1994) Actin filaments and the spatial positioning of mRNAS. Adv Exp Med Biol 358:183-9

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