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 Previous Article
Journal of Neuroscience, Vol 7, 2590-2599, Copyright © 1987 by Society for Neuroscience
Complete amino acid sequence and in vitro expression of rat NF-M, the middle molecular weight neurofilament protein
EW Napolitano, SS Chin, DR Colman and RK Liem
A lambda gtII 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 development
from postnatal day 0 (PO) to adulthood. Using pNF-M3C as a probe, a second
cDNA clone was isolated from a lambda gtII 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 radiolabeled 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.
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