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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD018066-10
Application #
3315045
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1984-06-01
Project End
1996-05-31
Budget Start
1993-06-01
Budget End
1994-05-31
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
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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