Neurofilament Deficiency in Quaff Caused by Nonsense Mutation in Neurofilament-L Gene

The existence of a neurofilament-deficient mutant of Japanese quail was recently documented (Yamasaki, H., C. Itakura, and M. Mizutani. 1991. Acta Neuropathol. 82:427--434), but the genetic events leading to the neurofilament deficiency have yet to be determined. Our molecular biological analyses revealed that the expression of neurofilament-L (NF-L) gene was specifically repressed in neurons of this mutant. To search for mutation(s) responsible for the shutdown of this gene expression, we cloned and sequenced the NF-L genes in the wild-type and mutant quails. It is eventually found that the NF-L gene in the mutant includes a nonsense mutation at the deduced amino acid residue 114, indicating that the mutant is incapable of producing even a trace amount of polymerizationcompetent NF-L protein at any situation. The identification of this nonsense mutation provides us with a solid basis on which molecular mechanisms underlying the alteration in the neuronal cytoskeletal architecture in the mutant should be interpreted. N EUROFILAMENTS (NFs), ~ a member of the intermediate filament (IF) protein family, are major constituents of neuronal cytoskeleton and primarily composed of three different protein subunits referred to as NF-L, NF-M, and NF-H in vertebrates (Shaw, 1991). Although NFs have been actively studied for many years, we can describe the function of NFs only in vague terms. For example, a function of NFs is believed to control axonal calibers as mechanical space filling molecules (Hoffman et al., 1987). However, it is still unclear whether this is the exclusive function of NFs in vivo. Although several lines of evidence have suggested that NFs are not vital for a certain class of neurons, there is no convincing evidence as yet for dispensability of NFs in the nervous system of vertebrates (Palay and Chan-Palay, 1974; Phillips et al., 1983; Lasek et al., 1983, 1985). In this regard, the recent discovery of a NFdeficient mutant of Japanese quail (Coturnix coturnixjaponica), named "quiver" (Quv), strongly arrested our attention because this mutant may shed light on the uncovered function of NFs in vivo (Mizutani et al., 1992; Yamasaki et al., 1991, 1992). Previous studies have characterized the lesions in Quv to some extent: Quv shows no big differences in fertility, hatchability, and posthatching mortality (Mizutani et al., 1992); the lesions in Quv are inherited as a single autosomal recessive trait after Mendelian laws (Mizutani et al., 1992); the axons of central and peripheral nervous system of Quv are deficient of 10-nm filaments (i.e., NFs) but with welldeveloped myelin sheaths, and the size distribution of the axonal calibers significantly shifted to small size classes I. Abbreviations used in this paper: IF, intermediate filament; NF, neurofilament; Quv, quiver. (Yamasaki et al., 1991); NF-L subunit protein cannot be detected in neuronal tissues at all, whereas a nonor hypophosphorylated form of NF-M exists only in a very small population of the neurons (Yamasaki et al., 1992); and the conduction velocity of the NF-deficient neurons is coordinately reduced with the decrease in the axonal calibers (Sakaguchi, T., M. Okada, T. Kitamura, and K. Kawasaki, manuscript submitted for publication). The survival of Quv, without any severe symptoms except for mild generalized quivering, strongly suggests that NFs, unlike microfilaments and microtubules, are dispensable for matured quails. Furthermore, the fact that NF deficiency results in only a certain decrease in the axonal calibers provides us with clear evidence of NFs being not an exclusive but major determinant of the axonal calibers. Nevertheless, a very basic question has yet to be answered: What kind of genetic event(s) resulted in the NF deficiency in Quv? Without answering this question, it is almost impossible for us to discriminate primary results of the genetic defect(s) from secondary ones. Hence, we tried to identify the genetic defect(s) responsible for the NF deficiency in Quv at the gene structural level, and found that a nonsense mutation was introduced in the Quv NF-L gene. Here we discuss the sequence of events leading to the total loss of NFs on the molecular basis of the identified mutation in NF-L gene. Mater ia l s and M e t h o d s