Andrew Collick

Characterization of a highly unstable mouse minisatellite locus: Evidence for somatic mutation during early development ☆

A highly unstable mouse minisatellite locus, Ms6-hm, has been identified in mouse DNA fingerprints produced by cross-hybridization with human minisatellite probe 33.6. A 7-kb allele of Ms6-hm was cloned from a C57BL/6J mouse and collapsed to a 400-bp plasmid insert on propagation in Escherichia coli due to loss of the majority of minisatellite repeat units. Sequence analysis revealed that Ms6-hm has evolved by amplification within a member of the MT (mouse transcript) family of interspersed repetitive elements. Linkage analysis localized Ms6-hm near the brown coat color gene (b) on chromosome 4. Multiallelism and heterozygosity at this locus within inbred strains result from a high germline mutation rate to new-length alleles (2.5% per gamete). Mice mosaic for cells carrying a nonparental allele in somatic tissue, and in some cases also in the germline, provide evidence for additional, somatic, mutation events at Ms6-hm. In two mosaic mice the fraction of cells containing the nonparental allele has been shown to be indistinguishable in different adult tissues. These somatic mutation events at Ms6-hm must therefore occur very early in development, preceding the allocation of somatic lineages, and the same pool of primitive ectoderm cells must contribute equally to all somatic tissues. Under low-stringency hybridization conditions the collapsed subclone of Ms6-hm cross-hybridizes to other unstable loci in the mouse genome to generate a novel and highly individual specific mouse DNA fingerprint.

Instability of long inverted repeats within mouse transgenes.

Various sequences in the mammalian genomes are unstable. One class of sequence arrangement is long inverted repeats, which are known to be unstable in bacteria and yeast. While in mammals some evidence suggests that short inverted repeats (<10 bp long) may show instability, nothing is known about the stability of long inverted repeats. Here we describe two unrelated multicopy transgenes in the mouse (loci 109 and OX1–5), each of which contains a long inverted repeat that shows substantial mitotic instability. This instability also occurs in the germline so that mutant transgenes appear within pedigrees at a high frequency. The mutation processes acting at these two inverted repeats are complex and can involve insertion or deletion, and can result in stabilization of the transgene. At transgene 109 mutational events range from very small rearrangements at the centre of the inverted repeat to complete transgene deletion. In addition we show that the rates of mutation at the inverted repeat of transgene OX1–5 can vary between the male and female germlines and between inbred strains of mice, suggesting the possibility of a genetic analysis to identify loci that modulate inverted repeat instability.

Minisatellite variant repeat mapping: Application to DNA typing and mutation analysis

Most DNA typing systems assay allele length variation at tandemly repeated loci such as minisatellites and microsatellites. Allele length measurements are approximate, which impedes the use of such loci in forensic analysis and in studies of allelic variability at hypervariable loci. We now review progress in the development of alternative DNA typing systems based on allelic variation in the interspersion patterns of variant repeat units along minisatellite alleles. Minisatellite variant repeat mapping by PCR (MVR-PCR) not only provides a powerful new digital approach to DNA typing, but also for the first time allows investigation of the true level of allelic variability at minisatellite loci and of the mutational mechanisms that generate ultravariability.

Variable germline and embryonic instability of the human minisatellite MS32 (D1S8) in transgenic mice.

Tandem repeat loci such as minisatellites and trinucleotide repeats frequently show instability. We have investigated mutation at human minisatellite MS32 (locus D1S8) transferred to transgenic mice. Three lines of hemizygous transgenic mice were studied. A single‐copy line (110D) was seen to be relatively stable, whilst two multicopy lines showed structural instability of the transgene in pedigrees (lines 109 and 110A). For both these lines, mutant structures were detected as a result of mutation events having occurred in the germline or early embryo. Structural changes seen included gain or loss of minisatellite repeat units (110A and 109), alteration of DNA flanking the minisatellite repeat array (109 only) or deletion of the entire transgene (109 only). This work demonstrates that tandem repeat transgenes can show instability and thus provide additional systems for the analysis of repetitive DNA structural change in mice.