Minisatellite instability at the Adh locus reveals somatic polymorphism in amphioxus

Abstract

Amphioxus (subphylum Cephalochordata) is the closest living relative to vertebrates and widely used for phylogenetic analyses of vertebrate gene evolution. Amphioxus genes are highly polymorphic, but the origin and nature of this variability is unknown. We have analyzed the alcohol dehydrogenase locus (Adh3) in two amphioxus species (Branchiostoma lanceolatum and Branchiostoma floridae) and found that genetic variation is related to repetitive DNA sequences, mainly minisatellites. Small pool-PCR assays indicated that allelic variants are generated by minisatellite instability. We conclude that the generation of new forms was not preferentially linked to germline processes but rather to somatic events leading to mosaic adult animals. Furthermore, most Adh minisatellites belong to a novel class, which we have named mirages. Their distinctive feature is that the repeat subunit spans the exon-intron boundaries and generates potential duplications of the splice sites. However, splicing may not be compromised as no aberrant mRNA variants were detected.

Figure 1

Southern analysis of BlAdh3 (accession nos AF156698, AF156700 and AF156705) and BfAdh3 (AF266713–AF266719). Genomic DNA from single individuals was digested with PstI and probed with BlAdh3 fragments containing exon 3 (A), 4–6 (B) and 8–9 (C), and exons 2–3 (D), 5 (E) and 8 (F) of BfAdh3. The expected band size from the characterized Adh3 genes (under each panel) and the DNA size markers (at the left of each panel) are depicted. Genomic structure of Adh3 (central part): exons (boxes); EcoRI (E) PstI (P, in bold) and XbaI (X) restriction sites, mirages (arrows), other minisatellites (circles) and probes (horizontal lines) are shown. Notice that more than two bands are shown in lane 11 (B), and lane 7 (D–F).

Figure 2

(A) Schematic representation of a GT-mirage overlapping the donor splice site at the boundary of exon 6–intron 6 of BlAdh3. Arrow-boxes indicate mirage subunits and the boxed sequence corresponds to the exon. The exon–intron boundary is numbered 0 and each following mirage subunit is consecutively numbered 1–10 (5′–3′). The splice donor site is indicated by an oblique arrow. (B) Sequence alignment of the intron-6 GT-mirage array of two BlAdh3 alleles. The segment corresponding to the coding region (capital letters in the dark gray box) and intron sequences (lower-case letters) are shown. The splice donor site (bold lower case) and its reiteration are depicted on light gray background. Nucleotide substitutions in the repeated sequences are shown, identities are represented by dots and deletions by dashes.

Figure 3

RT–PCR assays to detect aberrant splicing variants of mirages containing BlAdh3 introns (accession nos AF156698–AF156708) with oligonucleotides located at the flanking exons of intron 2 (lane 1, oligonucleotide pairs L1S–1R), intron 6 (lane 2, L6S–L14A), intron 7 (lane 3, F7S–CR2), intron 8 (lane 4, L8S–L10A) and the segment encompassing intron 6–exon 7–intron 7–exon 8–intron 8 (lane 5, F7S–L10A). Splicing of intron 1 and 9 (Table 1) were also assayed (data not shown). In all samples, no aberrant forms were detected and a single band with the expected size of the properly spliced product was observed. MWM, φX174 DNA HinfI.

Figure 4

Allelic length variants of BlAdh3 in four single animals (A–D) by SP-PCR. Examples of abnormal length mutants (*) corresponding to somatic (left) and gonadal-enriched DNA fractions (right), of intron 1 (top) and intron 6 (bottom) Adh3. Animals showed one progenitor molecule (B and C for intron 1; D for intron 6) or two progenitor molecules (A for intron 1 and C for intron 6). PCR products were electrophoretically resolved and abnormal-length mutants (*, one-subunit; **, two-subunit variation) were detected by blotting and hybridization.