Molecular instability in theCOII–tRNA^Lysintergenic region of the human mitochondrial genome: multiple origins of the 9–bp deletion and heteroplasmy for expanded repeats

Abstract

We have identified two individuals from Glasgow in Scotland who have a deletion of one of two copies of the intergenic 9-bp sequence motif CCCCCTCTA, located between the cytochrome oxidase II (COII) and lysine tRNA (tRNA(Lys)) genes of the human mitochondrial genome. Although this polymorphism is common in Africa and Asia, it has not been reported in Northern Europe. Analysis of the mitochondrial DNA control region sequences of these two individuals suggests that they belong to a lineage that originated independently of the previously characterized African and Asian 9-bp deleted lineages. Among the Scottish population we have also identified a maternal lineage of three generations exhibiting heteroplasmy for two, three and four copies of the CCCCCTCTA motif. Polymerase chain reaction amplification across the COII-tRNA(Lys) intergenic region of these individuals gives different ratios of the three product lengths that are dependent on the concentration of the DNA-binding dye crystal violet. To investigate whether changes in repeat number were generated de novo, we constructed clones containing known numbers of the CCCCCTCTA motif. In the presence of high concentrations of crystal violet we obtained two, three and four copies of this motif when the amplification template contained only four copies. Various DNA-binding drugs are known to stabilize bulged structures in DNA and contribute to the process of slipped-strand mispairing during DNA replication. These results suggest that the COII-tRNA(Lys) intergenic region is unstable owing to slipped-strand mispairing. Although sequences containing four copies of the CCCCCTCTA motif are less stable in vitro, we observed an increase in the proportion of mitochondrial genomes with four repeats between-a mother and a daughter in the heteroplasmic lineage. From this we conclude that drift in the germ-line lineage is a main factor in the maintenance or loss of heteroplasmy.

Evolutionary history of the mtDNA 9-bp deletion in Chinese populations and its relevance to the peopling of east and southeast Asia

“…The deletion motif occurs at varying frequencies in Asian, Oceanic, and Amerindian populations (Ballinger et al 1992;Harihara et al 1992;Passarino et al 1993;Sykes et al 1995;Betty et al 1996;Melton et al 1995Melton et al , 1998Lum et al 1998;Merriwether et al 1999;Watkins et al 1999;Shurr et al 1990;Ward et al 1991;Shields et al 1992;Kolman et al 1995). Additional reports have demonstrated the presence of the 9-bp deletion in African (Soodyall et al 1996;Watkins et al 1999) and European (Torroni et al 1995;Thomas et al 1998) populations. The coalescent times of the 9-bp deletion haplotypes are estimated to be 50,000-70,000 years before present (YBP; Redd et al 1995;Soodyall et al 1996; Thomas et al 1998).…”

“…Additional reports have demonstrated the presence of the 9-bp deletion in African (Soodyall et al 1996;Watkins et al 1999) and European (Torroni et al 1995;Thomas et al 1998) populations. The coalescent times of the 9-bp deletion haplotypes are estimated to be 50,000-70,000 years before present (YBP; Redd et al 1995;Soodyall et al 1996; Thomas et al 1998). Phylogenetic analyses of these mtDNA haplotypes also suggest multiple independent origins of the 9-bp deletion in Africa (Soodyall et al 1996;Watkins et al 1999), Europe (Torroni et al 1995;Thomas et al 1998), and Asia (Ballinger et al 1992;Redd et al 1995;Watkins et al 1999).…”

Molecular and morphological evidence on the phylogeny of the Elephantidae

“…First-round ampli¢cations were carried out as described p reviously (Thomas et al 1998), except that each reaction contained 0.08 mg ml 71 bovine serum albumin (BSA) (Boehringer, Lewes, Sussex, UK) and 1.3 units of Taq DNA p olymerase premixed with TaqStart antibody (Clontech, Palo Alto, CA, USA) in a molar ratio of 1:14. PCR cycling parameters: pre-incubation at 95 8C for 1min, followed by 55 cycles of denaturation at 93 8C for 1min, annealing at 55 8C for 1min and extension at 72 8C for 1min.…”

“…The Loxodonta individuals were all savannah elephants from southern and eastern Africa. Each DNA sample was prepared for sequencing as described in Thomas et al (1997), and DNA sequencing was carried out according to Thomas et al (1998).…”

Molecular genetic evidence for the human settlement of the Pacific: analysis of mitochondrial DNA, Y chromosome and HLA markers

“…The DNA sequences were aligned manually. For the analysis of the intergenic 9-bp deletion, PCR ampli¢cation was carried out as described previously (Hagelberg & Clegg 1993;Thomas et al 1998).…”