Fibronectin and the fibronectin receptor promote cell-cell and cell-matrix interactions which are essential for spatial organization and differentiation of cells during embryonic development. Thus, the genes encoding these proteins are likely to be developmentally regulated. The objective of the proposed research is to increase our understanding of the molecular mechanisms that control fibronectin levels during limb development, and to assess the role of the fibronectin receptor in this process. A combination of molecular biological and histological methods will be used to determine: A) stage- or region-specific changes in fibronectin or fibronectin receptor mRNA levels in the chicken limb; B) the level of gene expression at which regulation occurs; and, C) whether specific DNA sequences are involved in regulatory control. To accomplish these goals, the specific aims are: 1) To analyse fibronectin and fibronectin receptor mRNA levels in limb buds from different stage chick embryos using chicken fibronectin and fibronectin receptor cDNA clones as probes for A) in situ hybridization; and, B) slot blot and """"""""Northern"""""""" blot analysis. 2) To analyse the types and levels of alternatively spliced fibronectin mRNAs in the limb bud of different stage chick embryos by using appropriate portions of chicken fibronectin cDNA as probes for A) in situ hybridizaton; and, B) S1 nuclease analysis. 3) To determine rates of transcription initiation for the fibronectin gene and the fibronectin receptor gene by nuclear runoff mRNA synthesis using nuclei isolated from different developmental stage chick limb buds. 4) To construct hybrid genes such that DNA sequences from potential regulatory regions of the fibronectin gene or the fibronectin receptor gene control the expression of a different gene, encoding an assayable """"""""reporter"""""""" molecule. Hybrid genes will be inserted into a src-, Rous Sarcoma Virus vector, and viral stocks carrying these constructions prepared. Mesenchymal cell cultures derived from different stage limb buds will be infected and studied for expression of fibronectin or fibronectin receptor and the """"""""reporter"""""""" molecule. DNA sequences involved in regulating fibronectin or fibronectin receptor mRNAs levels during limb development will be identified since they will cause similar changes in the level of the """"""""reporter"""""""" molecule as a function of developmental stage. The health relatedness of this project derives from its potential contribution to our understanding of the basic molecular mechanisms regulating gene expression in normal development.
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