SEQUENCE AND EXPRESSION PATTERNS OF 2 FORMS OF THE MIDDLE MOLECULAR-WEIGHT NEUROFILAMENT PROTEIN (NF-M) OF XENOPUS-LAEVIS

The middle molecular weight neurofilament protein (NF-M) is relevant t o our understanding of vertebrate neurofilaments in growing axons, bot h because it exists in all vertebrates and because it undergoes charac teristic changes in its phosphorylation state during axonal developmen t. indeed, all vertebrate neurofilament proteins are believed to have originated by gene duplication from an ancestral, NF-M-like protein. T he role of NF-M in axonal development has been studied extensively in the frog, Xenopus laevis, through the use of monoclonal antibodies. To acquire a better understanding of the relationship of X. laevis NF-M to that of other vertebrates and to obtain additional reagents to stud y and perturb neurofilaments in developing axons, we isolated cDNA clo nes from the nervous system. These clones encoded two forms of NF-M, w hich exhibited 93% amino acid identity overall and 94%, 96% and 90% id entity over their head, rod, and tail domains, respectively. Synonymou s nucleotide substitution rates between the two forms tied their origi n to an ancestral duplication of the Xenopus genome, which occurred ap proximately 30 million years ago. Non-synonymous substitution rates in dicated that the tail domain is evolving more rapidly than the rod dom ain. Both forms shared structural features in common with other verteb rate NF-Ms but had only a single example of the KSP phosphorylation mo tif that is repeated multiple times in the NF-Ms of bird, goldfish and mammal. In post-metamorphic frogs, the NF-M(1) transcript was consist ently expressed at higher levels than that of NF-M(2), although their anatomical patterns of expression were qualitatively similar. During d evelopment, however, only NF-M(2) was detectable in retinal ganglion c ells through stage 42. We speculate that the differences observed betw een these two forms may represent early stages of protein diversificat ion akin to what occurred after the gene duplications that gave rise t o other vertebrate neurofilament proteins.