*Unitat de Biologia Evolutiva, Facultat de Ciències de la Salut i de la Vida, Universitat Pompeu Fabra, Barcelona, Catalonia, Spain; and
Department of Forensic Medicine, University of Helsinki, Helsinki, Finland
Alu elements are a family of short interspersed repeats that have mobilized throughout primate genomes by retrotransposition over the past 65 Myr of primate evolution (for a review, see Deininger and Batzer 1993
). In the human genome, Alu elements exist in copy numbers of approximately 500,000 per haploid genome, representing approximately 5% of the genome, and they may be classified into groups of related subfamily members that share common diagnostic substitutions (Batzer et al. 1996b
). The major subfamily branches (J, S, and Y) seem to have appeared at different evolutionary times, with J being older than S, and S being older than Y. Not only have the Alu elements contributed to the evolution of the primate genomes, but they also contribute up to 0.4% of human genetic disease according to Deininger and Batzer (1999)
. Two main mechanisms may produce human diseases: direct insertions of Alu elements within genes (0.1% of human genetic disease), and unequal homologous recombination events between Alu repeats (0.3% of human genetic disease).
Some of the human Alu elements have retroposed so recently that their insertion at a specific location within the human genome remains polymorphic. These polymorphic insertions have been used as genetic markers in human evolution studies due to their particular properties: they are rapid and easy to type, are apparently selectively neutral, and have known ancestral states. The insertion of an Alu element into the human genome is almost certainly a unique event, making any pair of Alu insertion alleles identical by descent and free of homoplasy. The use of these polymorphisms in a worldwide survey of human populations has confirmed the African origin of modern humans (Batzer et al. 1994
; Batzer et al. 1996a
; Stoneking et al. 1997
). One of these polymorphic Alu insertions is the PV92 Alu site that is located in chromosome 16, and it has been proved to be human-specific (Batzer et al. 1994
). The PV92 Alu insertion element belongs to the youngest subfamily of Alu sequences, the Alu Y subfamily, and, within that, to the Ya5 subfamily, which is defined by five diagnostic changes relative to the Y consensus (Batzer et al. 1996b
). The PV92 Alu insertion is most frequent in Amerindians (Novick et al. 1998
) and East Asians (Stoneking et al. 1997
), while it has lower frequencies elsewhere.
In order to understand the evolutionary context of the PV92 Alu insertion in Western Mediterranean populations, a total of 676 autochthonous individuals coming from the Iberian Peninsula (Andalusians, Basques, and Catalans) and North Africa (Morocco, Western Sahara, Algeria, and Tunisia) were typed for the human-specific PV92 Alu insertion polymorphism. The sample comprised unrelated healthy blood donors, and informed consent was obtained from all individuals participating in the study. Genomic DNA was extracted from whole blood using a phenol-chloroform extraction method after digestion with proteinase K. The PCR amplification of the PV92 Alu insertion locus was performed in 30 cycles of 95°C for 1 min, 54°C for 1 min, and 72°C for 1 min, with a final elongation step of 72°C for 7 min. The primers used were PV92A (5'-AACTGGGAAAATTTGAAGAGAAAGT-3') and PV92B (5'-TGAGTTCTCAACTCCTGTGTGTTAG-3'). In order to visualize the results, the PCR product was run in a 2% agarose gel.
The chromosomes without the PV92 Alu insertion yielded an amplified fragment of 129 bp using the present set of primers (PV92A and PV92B), whereas the chromosomes with the insertion yielded an amplified fragment of 443 bp. In the present sample set of 1,352 chromosomes, 970 chromosomes did not bear the insertion, 382 bore the insertion, and the three remaining chromosomes bore a larger-than-expected insertion,
789 bp in length (fig. 1A
). These three chromosomes belonged to three different heterozygous individuals (two Basques and one northern Moroccan) whose other chromosome did not present the insertion.
|
The complete sequence of the individuals selected is shown in figure 1B.
The chromosomes with the larger insertion presented an Alu sequence inserted within the standard PV92 Alu element, which produces an amplified fragment around 346 bp larger than the standard fragment. This Alu element belongs to the Alu Y subfamily, in particular to the Alu Yb8 subfamily defined by eight diagnostic changes relative to the Y consensus (Batzer et al. 1996
b).
Three positions of the sequenced region were found to be polymorphic in the individuals analyzed (fig. 2A ): one position upstream of the Alu insertions (position 53 upstream of the Alu Ya5 insertion site) and two positions within the Alu Ya5 element (positions 100 and 255 downstream of the site of the Alu insertion). The sequence of one of the chromosomes with an absence of the Alu elements presented a T in position -53, while the rest of the chromosomes presented an A. One of the two individuals carrying only the Alu Ya5 element was homozygous for a G in position +100, whereas the other Alu Ya5 homozygous individual presented a G in one chromosome and a C in the other. On the other hand, all of the chromosomes with the Alu Yb8 presented a C in this position. Finally, the chromosomes with only the Alu Ya5 element presented a C in position +255, whereas chromosomes with the Alu Yb8 element presented a G. The sequences of the three chromosomes with the novel Yb8 Alu insertion were identical. The novel Yb8 element was likely to be inserted in an original sequence carrying an A at position -53 and a C at position +100, but the present data do not allow us to elucidate whether the novel element was inserted in an original sequence with a C or a G at position +255 (fig. 2B ). Two possible scenarios would yield the same result: the appearance of the C/G polymorphism in position +255 followed by the insertion of the Alu element or, alternatively, the insertion of the novel Alu element followed by a nucleotide change in position +255. As none of the intermediate sequences have been found in the present data, no conclusions about this sequence of events can be derived.
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The Ya5 and Yb8 Alu subfamilies appear to be actively undergoing concurrent amplification and mobilization within the human genome, as reported in a detailed study of these two subfamilies (Batzer et al. 1995
) as well as evidenced by the de novo Alu insertions that have resulted in diseases (Deininger and Batzer 1999
). The present Yb8 Alu insertion is one of the four youngest repeats reported to date (after three Yb8 Alu insertions already reported in Deininger and Batzer [1999]
), reinforcing the argument that these two Alu subfamilies are undergoing concurrent retroposition in the human genome.
The finding of an Alu element inserted within another polymorphic and human-specific Alu insertion suggests that the insertion of the novel Alu element represents a very recent event. The present polymorphism shows a precise case of migration through the Straits of Gibraltar, an important barrier to human migration (Bosch et al. 1999
). This fact, plus its limited geographical distribution and its absence in a broad set of populations already typed in the literature, makes the novel Alu insertion described in the present study a clear case of private or population-specific polymorphism. A population-specific polymorphism is defined as a genetic variant found in a particular group that might be used as a genetic marker for the genetic affiliation of one individual (Calafell et al. 1999
). Moreover, the description of the Alu element insertions provides a first level of genetic analysis, but there is a second, more precise level of analysis that integrates nucleotidic variations within the Alu elements and even, as in the present case, new insertional events.
Acknowledgements
We thank Kirsti Höök and Marjo Leppälä for technical assistance. This research was supported by Dirección General de Investigación Científica y Técnica in Spain (PB98-1064) and Direcció General de Recerca, Generalitat de Catalunya (1998SGR00009). D.C. was financially supported by the Finnish Ministry of Education and by Centre for International Mobility (CIMO) Scholarships for foreign postgraduates and young researchers. S.P. is the recipient of a predoctoral fellowship (2000FI00696) by the Comissionat per a Universitats i Recerca, Catalan Autonomous Government.
Footnotes
Jeffrey C. Long, Reviewing Editor
1 Keywords: Alu,
PV92
insertion event
2 Address for correspondence and reprints: Jaume Bertranpetit, Unitat de Biologia Evolutiva, Facultat de Ciències de la Salut i de la Vida, Universitat Pompeu Fabra, Doctor Aiguader 80, 08003 Barcelona, Catalonia, Spain. E-mail: jaume.bertranpetit{at}cexs.upf.es
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