Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL͞Wld S . Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld S . The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld S brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld S mice. However, it is undetectable on Northern blots of Wld S brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld S mutant for studies of ubiquitin and retinoid metabolism.
SUMMARYFour monoclonal antibodies to glycoprotein D (gD) of herpes simplex virus (HSV) types 1 and 2 neutralized virus in the presence of complement but exhibited diverse activities in its absence. Amino acid substitutions that conferred resistance to neutralization by each antibody were identified by deriving the nucleotide sequence of the gD gene from resistant mutants. Each antibody selected a substitution from different parts of the molecule and mutants resistant to a single antibody always arose from the same mutation. One of the antibodies reacted with a synthetic oligopeptide corresponding to the region of the molecule in which amino acid substitution conferred resistance, but the remaining three antibodies failed to react with predicted oligopeptide targets. These antibodies may therefore react with 'discontinuous' epitopes, a view supported by the observation that two of these three antibodies competed with each other in binding assays despite the fact that substitutions conferring resistance to neutralization arose nearly 100 residues apart in the primary sequence. The four antibodies had very different biological properties. One antibody neutralized infectivity but did not inhibit cell fusion, one antibody inhibited cell fusion but did not neutralize, while a third antibody had both activities. One antibody had neither activity but enhanced the infectivity of HSV-2 in a type-specific manner. The ability of antibodies to inhibit cell fusion by syncytial virus strains correlated with an ability to prevent plaque enlargement by a non-syncytial virus strain, implying a role for gD in the intercellular spread of virus that is independent of the syncytial phenotype. We found no correlation between neutralizing activity and anti-fusion activity suggesting that, while gD is involved in cell fusion, it has at least one other function which is required for infectivity.
Wallerian degeneration is the degeneration of the distal stump of an injured axon. It normally occurs over a time course of around 24 hr but it is delayed in the slow Wallerian degeneration mutant mouse (C57BL͞Wld s ) for up to 3 weeks. The gene, which protects from rapid Wallerian degeneration, Wld, previously has been mapped to distal chromosome 4. This paper reports the fine genetic mapping of the Wld locus, the generation of a 1.4-Mb bacterial artificial chromosome and P1 artificial chromosome contig, and the identification of an 85-kb tandem triplication mapping within the candidate region. The mutation is unique to C57BL͞Wld s among 36 strains tested and therefore is a strong candidate for the mutation that leads to delayed Wallerian degeneration. There are very few reports of tandem triplications in a vertebrate and no evidence for a mutation mechanism so this unusual mutation was characterized in more detail. Sequence analysis of the boundaries of the repeat unit revealed a minisatellite array at the distal boundary and a matching 8-bp sequence at the proximal boundary. This finding suggests that recombination between short homologous sequences (''illegitimate'' or ''nonhomologous'' recombination) was involved in the rearrangement. In addition, a duplication allele was identified in two Wld s mice, indicating some instability in the repeat copy number and suggesting that the triplication arose from a duplication by unequal crossing over.
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