Nerve growth factor (NGF) is a 118 amino acid polypeptide that is derived from a larger precursor and plays an important role in the development and survival of peripheral and central neurons. To study the physiology and regulation of NGF biosynthesis the NGF gene has been isolated from a gene library. The structure and functional organization of the NGF gene will be determined by restriction enzyme analysis, DNA blotting, and nucleotide sequence analysis. The primary focus of these studies will be the as yet unknown promoter and 5' flanking regions. The structural information and cloned gene will be used for subsequent studies of NGF physiology and gene expression. To study the developmental tissue and cell-specific regulation of NGF gene expression, we will use in situ hybridization to determine the specific cellular sites of NGF mRNA production, and RNA blot hybridization to examine the specific mRNAs that are expressed. These data will be correlated with immunocytochemical detection of NGF and NGF precursors, and localization of NGF receptors by historadioreceptor assay. The hybridization probes for tissue sections and RNA blots are available in the laboratory, were shown to be sufficiently sensitive to detect NGF mRNA in brain, and consist of cloned mouse NGF gene sequences, a cloned NGF cDNA for preparation of cRNA probes, and region (exon-specific) oligonucleotides. Several antisera suitable for immunocytochemical detection of NGF are available. One antiserum has been used to detect NGF in mouse submaxillary gland. Additional antisera to 2.5S mouse NGF and to peptides corresponding to several regions of the NGF precursor will be raised and used for detection of NGF precursors and peptides that may be generated from proNGF. The proposed studies provide a coherent and focussed analysis of NGF gene expression during development and are relevant because they will (i) extend knowledge of NGF gene structure and regulation of its expression, (ii) delineate the specific cellular sites of NGF biosynthesis during development, (iii) determine the specific mRNAs and encoded peptides derived from the NGF gene at these cellular sites and (iv) help elucidate the role of NGF in development and as a potential factor in degenerative diseases of the nervous system.
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