Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 16/10 Miklukho-Maklaya Street, 117997 Moscow, Russia
Correspondence
Ilgar Z. Mamedov
Imam{at}humgen.siobc.ras.ru
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ABSTRACT |
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Supplementary data associated with this article are available at http://humgen.siobc.ras.ru/supplement/suppl.html.
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MAIN TEXT |
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In the host genome, almost all HERVs and solo LTRs are flanked by short direct repeats (SDRs) or target site duplications (TSDs) at the sites of insertion. TSDs represent duplicated genomic sequences introduced through the mechanism of retroviral integration. SDRs of HERV-related elements of 46 nt long are identical at the moment of integration but can significantly diverge with time.
In this report we describe unusually long TSDs flanking some of the retroviral LTRs in the human genome and discuss their origin.
The LTR insertions with unusually long TSDs were first identified in a library of human-specific LTR HERV-K integrations, which was obtained using a new method described by us recently (Mamedov et al., 2002). Human specificity of individual LTRs was confirmed by comparison of PCR amplification products for human and great ape DNA samples. This stage was performed with primers targeted at unique sequences flanking the LTR integration sites at the 5' and 3' ends. The amplification product of a locus with an LTR insert is generally about 960 bp (the LTR length) longer than that derived from an orthologous locus lacking the LTR. However, this was not the case for two human-specific LTRs. For the fragments amplified from the LTR AC006035 insert located on human chromosome 7 and an orthologous site in chimpanzee lacking the LTR, the difference in length was approximately 250 bp higher than expected (i.e.
1210 bp instead of 960 bp; see Fig. 1
for details). Here a standard PCR protocol with the Gibco PCR Reagents System and primers 5'-AACCACGTGAATACACTTTCTCA-3' (forward) and 5'-GTCCAGTTAGACCCCTCAACTAG-3' (reverse) was used. The samples were amplified for 28 cycles at 94 °C for 20 s, 65 °C for 20 s and 72 °C for 40 s. A similar peculiar deviation was also observed for the LTR AC009132 located on chromosome 16. In this case the amplification profile was 28 cycles of 94 °C for 20 s, 53 °C for 20 s and 72 °C for 40 s, and primers 5'-ACGAGATTGGGTAGTTAAAATCC-3' (forward) and 5'-TACACTGTAACATGAATGTACCA-3' (reverse) were employed.
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Both the LTRs were specific to the human genome and belonged to the youngest subfamily amplified late in primate evolution (Table 1). Their pre-integration states were characterized by shorter PCR fragments amplified from orthologous loci in chimpanzee genomic DNA. The absence of the LTR insertions in orthologous genomic loci of chimpanzee and gorilla suggested that the LTRs were integrated after the split of the chimpanzee and human lineages, which occurred 56 million years ago. A low divergence of TSD sequences in human DNA implies recent duplication events. To evaluate the duplication time, PCR products obtained from chimpanzee and gorilla genomic DNAs from the locus orthologous to the LTR AC009132 were cloned using a PCR products T-easy cloning kit (Promega) and sequenced using an Applied Biosystems 373 automatic DNA sequencer. An analysis of the obtained sequences and sequenced fragments of the chimpanzee genome revealed that the orthologous loci of the two closely related primate genomes lacked the duplication (see supplementary data), thus confirming that this duplication did occur after the divergence of the human and chimpanzee lineages.
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To examine whether the duplication arose due to the LTR insertion, PCR assays of various primate genomic DNAs were performed using primers 5'-TCATAGATAGAAACAAGGTCCTCCT-3' and 5'-CCCCAGTGGCTCGTACTAGAG-3' targeted at the LTR AC108063 flanks. The amplification was performed for 30 cycles at 94 °C for 20 s, 63 °C for 20 s and 72 °C for 40 s. The PCR fragments corresponding to the LTR AC108063 insertion site with 96 bp long TSDs were detected in DNA samples from human, chimpanzee and gorilla. With several gibbon DNA samples presumably lacking the LTR, the corresponding PCR fragments were shorter. A PCR product derived from the gibbon genome of a Hylobates lar individual was cloned and sequenced (accession no. AY536064), confirming the absence of duplication in the pre-integration site.
The LTR Z95704 belongs to an old subfamily and is integrated into an L1 retrotransposon. Its direct repeats are highly (8 %) diverged. The sequences of the sites corresponding to the human LTRs AC108063 and Z95704, taken from the recently published chimpanzee genome (available at http://genome.ucsc.edu), revealed similar duplications flanking the LTRs.
So far only a few examples of long TSDs flanking other transposable elements in the sites of their integration are known, among them a 214 bp long TSD produced due to the integration of a human L1 (Feng et al., 1996), 952 bp long repeats surrounding an IS476 element in a recombinant plasmid (Chen et al., 1999
) and 82 bp long repeats flanking the intracisternal A particle (IAP) in the mouse genome (Tanaka & Ishihara, 1995
). The long interspersed elements (LINE) retrotransposition mechanism differs from that of retroviruses and includes nicking of the target DNA. Feng et al. (1996)
suggested that the formation of such long TSDs was due to peculiarities of helicase activity at the site of integration. In the case of the bacterial insertion sequence (IS) element, the duplication was suggested to be due to plasmid recombination at the site of insertion (Chen et al., 1999
). Similar to retroviruses, the integrated form of an IAP element has gag, pol and env genes between two LTRs and uses the same mechanism of retrotransposition. In this context, the closest example is an 82 bp long TSD flanking the de novo integration of an IAP element into the IL-3 gene of myeloid leukaemia cells, generated by whole-body irradiation of mice. Tanaka & Ishihara (1995)
suggested that such a long target site duplication was somehow associated with the impact of radiation, which might cause rearrangements or induce an unusual mechanism of retrotransposition.
It has been hypothesized (Morrish et al., 2002) that retrotransposons sometimes take part in the reparation of DNA breaks caused by various reasons. A similar reparation process coupled with HERV-K retrotransposition might form the LTRs described in this work. However, real forces and participants responsible for the integration-associated duplication remain unclear. Hopefully, they will be identified when other mammalian genomes are sequenced and more examples of such rare events are available. The study of the mechanism of the long TSD formation will give us a deeper insight into retroviral transposition and interactions of endogenous retroviruses with the host genome.
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ACKNOWLEDGEMENTS |
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Received 14 October 2003;
accepted 22 February 2004.
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