A technique for genome-wide identification of differences in the interspersed repeats integrations between closely related genomes.

Anton Buzdin^a*, Ilgar Mamedov^a, Konstantin Khodosevich^a, Tatyana Vinogradovaa, Yuri Lebedev^a, Gerhard Hunsmann^b, Eugene Sverdlov^a

^aShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St, Moscow 117871, Russia
^bGerman Primate Centre, Department for Virology and Immunology, Kellnerweg 4, D-37077 Goettingen, Germany

ABSTRACT
A method of Targeted Genomic Difference Analysis (TGDA) was developed allowing genome-wide detection of differences in integration sites of interspersed repeats between related genomes. The method includes two principal steps: (i) a whole genome selective amplification of the flanks adjacent to target interspersed repetitive elements in both genomic DNAs under comparison and (ii) subtractive hybridization of the selected amplicons. Differences between human and chimpanzee’s genomes in the integration sites of HERV-K(HML-2) human endogenous retroviruses and related solitary long terminal repeats (LTRs) were analyzed. Of 55 randomly chosen sequenced clones from a library enriched with human specific integration (HSI) sites, 33 (60%) represented HSIs, 14 (25%) were found also in the chimpanzee’s genome and 8 (15%) could not be unambiguously assigned. The number of the HSI sites in the library was estimated to be 67 that corresponds to 2-3% of all HERV-K(HML-2) LTRs integrations in the human genome. Together with the HSIs described by other authors, the number of the characterized LTR HSIs is now 39. All the revealed HSI LTRs belong to two closely related evolutionary young groups, thus suggesting parallel activities of two master genes in the hominid lineage. No deletion/insertion polymorphism was detected for any of the LTR HSIs by genomic PCR of DNA samples from 25 unrelated Caucasoid individuals. The HSI LTRs were assigned to the human genome sequenced loci and the neighboring genes were identified. Many of the LTRs were found to be located close to genes or within gene introns. The technique developed can be applied to any type of moderate repeats in mammalian genomes, including young subfamilies of Alu and L1 repeats.