IN SITU HYBRIDIZATION VISUALIZED USING BIOTINATED PROBES

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.