Isolation of the chicken middle-molecular weight neurofilament (NF-M) gene and characterization of its promoter

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Abstract
We have isolated and sequenced genomic DNA clones covering the coding region of the chicken mid-size neurofilament (NF-M) gene and > 1 kb of its 5'' upstream region. The NF-M gene contains two introns which both are located within the highly conversed C-terminal region of the rod domain. The 5'' end of the corresponding mRNA was assigned to a G residue 40 nucleotides upstream of the translation start site and in appropriate distance from a potential TATA box. To functionally analyze the NF-M promoter, constructs carrying 112, 222, and 1026 nucleotides of the 5'' upstream region in front of a luciferase reporter gene were tested for their capability to direct luciferase expression after transient transfection into various cell lines. Significant luciferase activity was recorded both in rat phaeochromocytoma (PC12) cells and murine fibroblasts. In PC12 cells, in which neurite outgrowth is induced by nerve growth factor (NGF), expression was stimulated up to 13-fold within 3 days of NGF treatment. This closely resembles expression of the endogenous NF-M gene in response to this hormone.