Retroprocessed pseudogenes, or retropseudogenes, are reverse transcripts of mature mRNAs retrotransposed to new locales within the genome [1]. Recently, these loci have received increasing attention [2]. Goncalves et al. [3] have shown that retropseudogenes are quite common in mammalian genomes; 23,000 to 33,000 are estimated to reside in the human genome. Studies of both point mutations [4] and indels (insertions/deletions) [5] in retropseudogenes have shown them to be excellent sources of background genetic information in a wide range of species. Thus, one of the emerging utilities of retropseudogenes is their role in providing markers for phylogenetic studies between species or between populations within species [6,7,8,9,10].

Among the retropseudogenes studied to date, the high-mobility-group (HMG) pseudogene HMG1L3 is a member of a rare class in which all or part of the encoded protein is still expressed [11]. Seeler et al. [12] reported that the nuclear protein SP100 displays a number of alternative splice variants. One of these, called SP100-HMG, is an 879 amino acid protein whose carboxy-terminal 170 residues bear a close similarity to the family of HMG proteins. Rogalla et al. [13] identified five retropseudogenes for which the antecedent gene is HMG1. Subsequently, Rogalla et al. [14] demonstrated that the carboxy-terminal extension of SP100-HMG is encoded by part of one of these HMG-1 retropseudogenes. Denoted HMG1L3, this retrotranscribed copy was inserted at the 3' end of the SP100 gene and has become incorporated into the 3' end of the SP100 locus as an exon, resulting in the addition of a DNA-binding function to the SP100 protein.

Rogalla et al. [14] performed a number of PCR amplifications using primer sequences from the 3' end of the SP100 locus. Different PCR primer combinations produced ampli-cons variously containing: the penultimate exon encoding a 14 amino acid joining region between SP100 and HMG1L3; the last SP100 intron; and the entire HMG1L3 pseudogene. Genomic DNA from human, chimpanzee, gorilla, gibbon and rhesus macaque was used in their study. Results suggest that the retro-transposition of HMG1L3 into the SP100 locus occurred at least 35 million years ago. In addition, a PCR amplicon produced from the rhesus macaque revealed the presence of an Alu sequence between the penultimate SP100 exon and the HMG1L3 insertion site that is not present in hominoid genomes. Here, I have used an expanded panel of New World and Old World monkey species to refine dating of both the HMG1L3 retrotransposition and the Alu insertion events.

Major features of the 3' end of the SP100 locus are shown in Figure 1. In addition to the spatial relationship among these features, the locations of PCR primers used in this study are indicated. Rogalla et al. [14] primers PICauf1 and a1PICdo amplify a 614 base pair (bp) amplicon in genomic DNA from human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and gibbon (Hylobates lar) and a 900 bp amplicon in the rhesus macaque (Macaca mulatta). Here, this same primer pair is used against genomic DNA from H. sapiens and M. mulatta as well as additional Old World monkey species including the baboons Papio anubis and Papio hamadryas, the vervet monkey Cercopithecus aethiops, and the Asian macaque Macaca assamensis. In addition, genomic DNAs from three New World monkey species: spider monkey (Ateles paniscus), tamarin (Leontopithecus saguinus) and marmoset (Callithrix jacchus) are examined. Results of these PCR amplifications are shown in Figure 2; H. sapiens yields the expected 614 bp amplicon and all five Old World monkey species display the 900 bp amplicon. This indicates that the Alu sequence previously found in the rhesus macaque is present in a wide range of Old World monkey genomes. On the other hand, none of the three New World monkey species produced an amplicon with these primers, suggesting that neither the HMG1L3 retropseudogene nor the Alu sequence is present in New World monkey genomes.

In support of the above suggestion, a third PCR primer, SP100-HMG3, was chosen from SP100 genomic sequence upstream of the 5' HMG1L3 insertion site. Amplification with this primer and a1PICdo yields a 292 bp amplicon in human and Old World monkey samples but no product in the New World monkey samples (Figure 2). Together, these results demonstrate that New World monkey species do not have HMG1L3, but that it is probably present throughout the Old World monkeys as well as ape and human (Hominoidea) genomes. Clearly, the reverse transcription and retrotransposition of HMG-1 that resulted in the creation of HMG1L3 occurred after divergence of Old World primate species (Catarrhini) from New World primates (Platyrrhini), but prior to the divergence from the Catarrhini of the lineage leading to apes and humans. Estimates of the origin and subsequent phylogenetic radiation of the Anthropoidea offered by Kay et al. [15], places these events in late Eocene to middle Oligocene, or between 30 and 40 million years ago.

Results illustrated in Figure 2 also show that the 300 bp Alu sequence found in the region between the penultimate exon of SP100 and the HMG1L3 insertion site in the genome of Macaca mulatta is present in the genomes of other Old World monkey species from Asia, the Indian subcontinent and Africa. Previous results [14] clearly show that the Alu is not present in any hominoid genome. Again, relying on the anthropoid phylogeny of Kay et al. [15], insertion of the Alu would have to have occurred after the divergence of the hominoids, or not more than 25 million years ago. An alternative view is that the Alu sequence insertion in SP100 occurred prior to the divergence of the hominoids, perhaps even at the same time as the HMG1L3 insertion, but that it was lost in the line leading to Hominoidea after divergence. However, the latter possibility is unlikely, for the following reasons: individual Alu sequences arise via unique insertion events; they are inserted in a sequence-independent manner into breaks in genomic DNA; and those breaks are subsequently repaired with the Alu embedded at the break point [16]. Once inserted, Alu sequences remain stable features of the host genome [17]. Although Alu sequences have been lost from host genomes, their excision is never as clean as their insertion. Either only part of the Alu sequence is lost or a loss of flanking genomic DNA occurs along with loss of the Alu sequence [18,19].

To determine which of the two scenarios is applicable to the SP100-HMG Alu, PICauf1/a1PICdo amplicons from human, baboon, vervet monkey and three macaque genomes were cloned and sequenced (GenBank Accession numbers AF377332, AF377333, AF377334, AF377335, AF377336 and AF378670). Consensus amplicon sequences from the five Old World monkey species and from three unrelated humans are presented in Figure 3. Comparison of the Old World monkey consensus sequence with the human consensus sequence shows that loss of the Alu among the Hominoidea subsequent to divergence from the Catarrhini would have required a perfect reversal of the insertion. In fact, the only sequence deletion is seen among the Old World monkey amplicons. This 22 base deletion (position 140-162) is near the position 186 Alu insertion site. If the sequence of this deletion is the same or nearly the same as that retained in the human consensus, it can form a hairpin with flanking poly(T)s and may, thus, have been lost during the repair process that occurred as part of the Alu insertion event.

An alignment of the Alu sequences from the five Old World Monkey species is presented in Figure 4. Two features of these sequences suggest a late, that is, post-divergence, origin of the insertion. First, all five sequences are consistent with a ClassIV Alu based on the classification of Britten et al. [20] and, more specifically, with the AluY group from the nomenclature of Batzer et al. [21], both of which are regarded as late origin 'master' Alu sequences. Second, disregarding both diagnostic sites and CpG dimers, there are few sequence variations among the five species. With the exception of four mutations found only in one or another of the five species, the variations that are in evidence fall into two types. One type, composed of fourteen single base changes and one deletion, is shared among all five species and the other type, composed of ten single base changes and one deletion, is common to either the African species P. anubis and C. aethiops or the Asian macaque species but not both. The shared variants could be a feature of the ancestral Alu, but those that are segregated clearly arose after insertion and after the divergence of the macaques from the rest of the catarrhines some 8 to 10 million years ago [22,23].

On the basis of these results, the most parsimonious scenario involves insertion of the Alu into the 3' region of the catarrhine SP100 gene and loss of the 22 base upstream sequence after hominid-catarrhine divergence between 20 and 25 million years ago. The most recent point at which these events might have occurred is 10 million years ago, the time at which the Cercopithecidae, represented by C. aethiops, and the Papionidae, represented by baboons and macaques, diverged [22,23]. This gives a window of 10-15 million years for the Alu insertion. Should members of the Colobinae, such as Colobus, Presbytis or Nasalis, have the Alu, the upper limit would be pushed back to 16-18 million years ago and restrict the insertion window to only 5-10 million years [24]. Taking an estimate of 5 × 10^-9 nucleotide substitutions per site per year for pseudogenes [25], mutations in the Alu sequences shown here suggest a date on the order of 19 million years ago for the insertion event. This is consistent with both the molecular and paleontologic data.