IN SITU HYBIDIZATION VISUALIZED USING BIOTINATED PROBES

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date12/31/895/31/96

ASJC

  • Genetics
  • Medicine(all)
  • Embryology

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