IN SITU HYBRIDIZATION VISUALIZED USING BIOTINATED PROBES

Project: Research project

Project Details

Description

This proposal investigates the spatial organization of mRNAs and their
relationship to cellular structure and function. The sorting of proteins
to their proper destinations within the cell may be assisted by the sorting
of their cognate mRNA so that synthesis of proteins can occur at their site
of function. Our goals in this proposal are twofold: to investigate the
association of mRNAs which are functionally related and to improve in situ
hybridization methodology so that low concentration mRNA molecules are
detectable at the high resolution of light and electron microscopy. For
the first of these goals, the compartmentalization of mRNA will be
investigated in the lamella of motile chicken embryo fibroblasts and
myoblasts. Actin mRNA has been shown to be localized in this structure,
and the localization of the mRNAs for actin-binding proteins such as
tropomyosin, alpha-actinin or myosin I will be investigated to determine
their relationship to the spatial distribution of actin mRNA. Likewise the
developing myofibril presents a good system to investigate actin-binding
proteins which provide for the assembly of the sarcomere. The myosins as
well as troponins and desmin, tropomyosin and alpha-actinin will be
investigated by digital imaging and electron microscopy. Within the cell,
a high resolution study will reveal how mRNAs associate with cellular
structures and whether physiologically related mRNAs are attached to these
same structures. For the second goal, in order to investigate mRNA
distribution to detect mRNAs of low concentration, high sensitivity methods
will be developed using digital imaging microscopy, silver enhanced
colloidal gold, or reverse transcriptase used in situ with oligonucleotide
primers. Eventually we wish to use fluorochromeconjugated probes
microinjected into cells to follow the movement of nucleic acids in vivo.
The goals of this proposal would provide needed information linking the
expression of specific genes to spatial compartments within the cell and
hence to the control of cell structure, function and differentiation.
StatusFinished
Effective start/end date6/1/925/31/93

Funding

  • Eunice Kennedy Shriver National Institute of Child Health and Human Development

ASJC

  • Molecular Biology
  • Biophysics
  • Structural Biology
  • Cell Biology
  • Histology

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