Using numts

Generating a data set of paralogous Numt sequences

Such a data set is useful for assessing patterns of molecular evolution (for example, Bensasson et al, 2001 ) or to generate a resource of known potential nuclear pseudogene contaminants for the study of mtDNA (for example, Vartanian and Wain-Hobson, 2002).

Approaches: PCR, clone and sequence mtDNA-like PCR products (Bensasson et al, 2001; Vartanian and Wain-Hobson, 2002).An alternative approach is to screen large-fragment genomic libraries by PCR (Yuan et al, 1999) .

Tips for the study of molecular evolution: Choice of a fast evolving mitochondrial region for PCR should maximise the differences between mtDNA and its nuclear copies. Prior to PCR, decrease the proportion of amplified mtDNA by enriching for nuclear DNA (Zhang and Hewitt, 1996) , by using tissue that is rich in nuclear DNA (e.g. sperm heads, Zischler et al, 1995) , or by digesting total genomic DNA with a restriction enzyme that will cut mtDNA at a single unconserved site: if any Numts differ from the mtDNA at this site, they will predominate in the subsequent PCR product (Yuan et al, 1999) . The pre-PCR digestion approach can also be used to focus on Numts of a particular evolutionary age. PCR products can be cloned and sequenced directly, screened with restriction enzymes, or screened by CDCE (Li-Sucholeiki et al, 1999) or SSCP (Sunnucks et al, 2000) .

Testing whether pseudogenes arose through independent transfers

If a pairwise comparison of Numts reveals significant codon position bias in the differences between them, this implies that they are descended from different functional (mitochondrial) ancestors and are therefore the result of independent transfers to the nucleus (Bensasson et al, 2000; Mundy et al, 2000) .

Distinguishing between substitutions arising in nucleus or mitochondrion

For orthologous Numt sequences this distinction is straightforward (Arctander, 1995) . For paralogous sequences, substitutions on Numt branches can be identified by parsimony analysis or maximum likelihood, and non-coding Numt changes can be identified as “unique” changes in an alignment of Numts and mtDNA (Sunnucks and Hales, 1996; Bensasson et al, 2001) .

Dating transfers

Where divergence dates are known for mitochondrial lineages involved in the analysis, the date of transfer can be estimated using the method of Li et al. (1981), as in Refs. (Lopez et al, 1994) and (DeWoody et al, 1999) .

Identifying and dating nuclear DNA duplications

If two numts have homology that extends into their flanking DNA they probably arose by DNA duplication in the nucleus (Bensasson et al, 2003) . If mtDNA sequences with homology to the numts are also known, a phylogenetic approach can be used to date the DNA duplications observed (Bensasson et al, 2003) .

References

Arctander P (1995). Comparison of a mitochondrial gene and a corresponding nuclear pseudogene. Proc. R. Soc. Lond. B 262: 13-19.

Bensasson D, Feldman MW, Petrov DA (2003). Rates of DNA duplication and mtDNA insertion in the human genome. Journal of Molecular Evolution 57: 343-354.

Bensasson D, Petrov DA, Zhang D-X, Hartl DL, Hewitt GM (2001). Genomic gigantism: DNA loss is slow in mountain grasshoppers. Molecular Biology and Evolution 18: 246-253.

Bensasson D, Zhang D-X, Hewitt GM (2000). Frequent assimilation of mitochondrial DNA by grasshopper nuclear genomes. Mol.Biol.Evol. 17: 406-415.

DeWoody JA, Chesser RK, Baker RJ (1999). A Translocated Mitochondrial Cytochrome b Pseudogene in Voles (Rodentia: Microtus). Journal of Molecular Evolution 48: 380-382.

Li W-H, Gojobori T, Nei M (1981). Pseudogenes as a paradigm of neutral evolution. Nature 292: 237-239.

Li-Sucholeiki X-C, Khrapko K, Andre PC, Marcelino LA, Karger BL, Thilly WG (1999). Applications of constant denaturant capillary electrophoresis / high fidelity polymerase chain reaction to human genetic analysis. Electrophoresis 20: 1224-1232.

Lopez JV, Yuhki N, Masuda R, Modi W, O'Brien SJO (1994). Numt, a Recent Transfer and Tandem Amplification of Mitochondrial DNA to the Nuclear Genome of the Domestic Cat. Journal of Molecular Evolution 39: 174-190.

Mundy NI, Pissinatti A, Woodruff DS (2000). Multiple Nuclear Insertions of Mitochondrial Cytochrome b Sequences in Callitrichine Primates. Molecular Biology and Evolution 17: 1075-1080.

Sunnucks P, Hales DF (1996). Numerous Transposed Sequences of Mitochondrial Cytochrome Oxidase I-II in Aphids of the Genus Sitobion (Hemiptera: Aphididae). Molecular Biology and Evolution 13: 510-524.

Sunnucks P, Wilson ACC, Beheregaray LB, Zenger K, French J, Taylor C (2000). SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. Molecular Ecology 9: 1699-1710.

Vartanian J-P, Wain-Hobson S (2002). Analysis of a library of macaque nuclear mitochondrial sequences confirms macaque origin of divergent sequences from old oral polio vaccine samples. PNAS 99: 7566-7569.

Yuan JD, Shi JX, Meng GX, An LG, Hu GX (1999). Nuclear pseudogenes of mitochondrial DNA as a variable part of the human genome. Cell Research 9: 281-290.

Zhang D-X, Hewitt GM (1996). Highly conserved nuclear copies of the mitochondrial control region in the desert locust Schistocerca gregaria: some implications for population studies. Molecular Ecology 5: 295-300.

Zischler H, Geisart H, von Haeseler A, Paabo S (1995). A nuclear 'fossil' of the mitochondrial D-loop and the origin of modern humans. Nature 378: 489-492.