An 85-kb tandem triplication in the slow Wallerian degeneration (Wlds) mouse

Abstract

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 (C57BL/Wlds) 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 C57BL/Wlds 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 Wlds mice, indicating some instability in the repeat copy number and suggesting that the triplication arose from a duplication by unequal crossing over.

Figure 1

Physical and genetic map of the Wld region. Wld maps between Nppa and D4Mit127 in distal mouse chromosome 4. Five recombinations lying within the span of a single P1 (236-D23) define the proximal end of the Wld region, whereas a single recombination defines the distal end. A BAC and P1 contig estimated at 1.4 Mb was generated by using as a starting point three nonrecombinant markers, D4Mit49, D4Mit225, and D4Mit310. BACs (Research Genetics) are labeled “B” on their left side on this diagram and the other clones are P1s (Resource Centre Primary Database, RZPD). The coordinates of each clone from the respective libraries are shown. Five P1 and BAC end clones are shown to lie within the repeated region. L23/R and 75/R are the closest flanking end clones that are not repeated, and the location of 5P1 (used in Fig. 2a) is also shown.

Figure 2

Identification by genomic Southern blotting of a tandemly repeated region on Wld^s chromosome 4. (a) Three probes (56/L, 320/L, and 269/L) lying within 30 kb of one another show an amplified signal when hybridized to a Southern blot of Wld genomic DNA. Markers on either side (L23/R and 5P1) show an approximately equal signal in C57BL/Wld^s and C57BL/6J when hybridized to the same filter. (b) Probes located centrally within the repeat unit (320/L is shown here) consistently show an increase in signal intensity but no difference in restriction fragment size with a range of frequent-cutter restriction enzymes. (c) Probe 59 (i) and (iii) located within the repeat unit immediately adjacent to the proximal boundary, hybridizes to a fragment of identical size in C57BL/Wld^s and in C57BL/6J, but also to a Wld-specific fragment of altered size and with an increased intensity. A probe located 0.8–1.0 kb from the distal end of the repeat unit (ii and iv) detects the same Wld-specific fragment (indicated by arrows) as probe 59, together with a different fragment that is common to both C57BL/6J and C57BL/Wld^s. (d) Tandem repeat model to account for the above observations. Probe 1 represents probes used in a and b. It lies centrally in the repeated region and detects a restriction fragment of increased dosage but unaltered size in C57BL/Wld^s. Probe 2 represents probe 59 in c. It detects one restriction fragment that is present as a single copy in both C57BL/6J and C57BL/Wld^s. However, when the target sequence of this probe is repeated in C57BL/Wld^s, a shifted band appears because of the formation of a junction fragment. This band may be shifted up or down depending on the location of the restriction site shown on the left. Probe 3 is located at the other end of the repeat unit and detects the same shifted fragment as probe 2, but a different fragment that is common to both C57BL/6J and C57BL/Wld^s.

Figure 3

The genomic rearrangement is unique to C57BL/Wld^s. (a) A BamHI genomic Southern blot was hybridized with a probe located close to the proximal boundary, giving a C57BL/Wld^s-specific shifted band. (b) A BlpI Southern blot was hybridized with 269/L, located centrally within the repeat unit, and with 5P1 as a single-copy control. Closely related C57 strains are shown in a and a representative group of other strains in b. Additional strains tested and not shown were 129/J, AKR/J, BALB/cJ, BDP/J, BUB/BnJ, CAST/Ei, C3H, DBA, HRS/J, I/LnJ, LT/Sv-A<y>/A, MA/MyJ, and NZW/LacJ. In both assays, the Wld^s allele was unique.

Figure 4

Identification of 170- and 85-kb insertions in C57BL/Wld^s. Pulsed-field gel analysis of C57BL/Wld^s and C57BL/6J genomic DNA digested with NotI or SmaI. After electrophoresis the gel was Southern-blotted and the DNA was hybridized with the probe 271/L, located centrally within the repeat unit (Fig. 1). WldA is representative of five of seven DNAs analyzed, whereas Wlds C and D were unique.

Figure 5

Sequencing of the boundaries of the repeat unit. (a) Sequence of the proximal boundary of the repeat unit showing the normal sequence (upper line) and the inverse PCR product (lower line), representing the inner junctions formed in Wld^s. The two sequences can be aligned exactly, except for 19 bp (angled) that are unique to the inverse PCR product. These 19 bp are derived from the distal end of the repeat unit, having been juxtaposed in C57BL/Wld^s by the formation of the junction (Fig. 2d). PstI sites shown left and right are the sites of circularization during inverse PCR. The 8-bp sequence shared between the proximal and distal boundaries is boxed. Sequences of PCR primers, represented by horizontal arrows, are 54 = 5′-CGTTGGCTCTAAGGACAGCAC-3′ and 59 = 5′-CGCCTCTTTGATCCCTACAGA-3′. (b) Sequence of the distal boundary of the repeat unit showing the approximate site of the boundary and the 8-bp motif common to the distal and proximal boundaries (boxed). The inverse PCR product diverges at a slightly different point from the normal distal and proximal sequences. This, and the possibility that one or two bases may be coincidentally shared between sequences, makes it difficult to define the exact site of each boundary.