COMPLETE AMINO-ACID-SEQUENCE AND INVITRO EXPRESSION OF RAT NF-M, THE MIDDLE MOLECULAR-WEIGHT NEUROFILAMENT PROTEIN

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  • 1 August 1987
    • journal article
    • research article
    • Vol. 7  (8) , 2590-2599
Abstract
A .lambda.gtll expression library was prepared from rat brain and screened with a polyclonal antiserum, which recognizes both NF-H and NF-M. An NF-M cDNA clone (pNF-M3C = 1.6 kb) was isolated and characterized. The fusion protein of NF-M3C, when used as an affinity matrix for the anti-neurofilament serum, isolated a subpopulation of antibodies specific for NF-M. Northern analysis demonstrates a single band of approximately 3000 nt and a constant message level for NF-M during postnatal developmental from postnatal day 0 (PO) to adulthood. Using pNF-M3C as a probe, a second cDNA was isolated from a .lambda.gtll rat brain expression library (pNF-M2D = 2.7 kb). The 2 clones were sequenced and pNF-M2D was found to encode the entire rat NF-M protein. The calculated molecular weight is 95,600, which is only 65% of the molecular weight determined by SDS-PAGE. The amino acid sequence of rat NF-M shows the conserved rod segment present in all intermediate filament proteins. The molecule also contains an unusual C-terminal extension with stretches of glutamic acid, which could contribute to the anomalous migration of this protein on SDS-PAGE and the fact that NF-M does not readily assemble into filaments. The pNF-M2D clone was transcribed and translated in vitro utilizing a rabbit reticulocyte lysate system. The resulting radiolabeld translation products were unexpectedly shown to comigrate with purified rat NF-M on 1- and 2-dimensional gels, even though the translated protein is not phosphorylated.