Origin of the Alu family: a family of Alu-like monomers gave birth to the left and the right arms of the Alu elements.

The Alu dimeric elements are a common feature of the primate genomes, where they constitute a family of related sequences (1). The identification of a free left Alu monomer (FLAM) family plus a free right Alu monomer (FRAM) family suggests that the dimeric structure results from the fusion of a FLAM sequence with a FRAM sequence (2). Here, we describe a very old Alu-like monomeric family, referred to as FAM for fossil Alu monomer. This family arose from a 7SL RNA sequence and gave birth to the FLAM and FRAM families. From the results obtained, the evolution of the Alu family can be subdivided into two phases. The first phase, which involves only monomeric elements, is characterized by deep remodelling of the progenitor sequences and ends with the appearance of the first Alu dimeric element through the fusion of a FLAM and a FRAM element. The second phase, still in progress, starts with the first Alu dimeric element. This phase is characterized by the stabilization of the progenitor sequences.     

A master sequence related to a free left Alu monomer (FLAM) at the origin of the B1 family in rodent genomes.

The question of the origin of the B1 family of rodents is addressed. The modern B1 elements are similar to the left Alu monomer, but with a 9 bp deletion and a 29 bp duplication. Search of databases for B1 elements that do not exhibit those modern features revealed sequence fragments that are very similar to the free left Alu monomers (FLAMs) described in the primate genomes. In addition, the analysis reveals elements that have 10 bp or 7 bp deletion in place of the 9 bp deletion but without the 29 bp tandem duplication. The elements described define families of proto B1 elements (referred as PB1, PB1D10 and PB1D7) that appeared before the first modern B1 element. A phylogenetic reconstruction suggest that the origin of Alu and B1 families took place before the divergence between the primate and the rodent lineages and that each family has followed different evolutionary routes since this radiation.     

Monophyletic Origin of Alu Elements in Primates

To get insight into the early evolution of the primate Alu elements, we characterized sequences of these repeats from the Malagasy prosimians, lemurs (Lemuridae) and sifakas (Indriidae), as well as from galagos (Lorisidae). These sequences were compared with the oldest Alu species known from the human genome: dimeric Alu J and S and free Alu monomers. Our analysis indicates that about 60 Myr ago, before the prosimian divergence, free left and right monomers formed an Alu heterodimer connected by a 19-nucleotide-long A-rich linker. The resulting elements successfully propagated in diverging primate lineages until about ∼20 Myr ago, conserving similar sequence features and essentially the same Alu RNA secondary structure. We suggest that until that time the same ``retropositional niche', molecular machinery making possible the proliferation by retroposition, constrained the evolution of Alu elements in extant primate species. These constraints became subsequently relaxed. In the Malagasy prosimians the dimeric Alu continued to amplify after acquiring a 34- to 36-nucleotide extension of their linker segment, whereas in the galago genome the ``retropositional niche' was occupied by novel short elements. Received: 1 December 1997 / Accepted: 30 January 1998     

Free left arms as precursor molecules in the evolution of Alu sequences

Summary The dimeric Alu molecule of human and other primates is composed of a left and a right arm that are very similar but show characteristic differences. If the Alu sequence has arisen through the fusion of monomeric precursor molecules, the traces of such precursor genes are expected still to be present in contemporary primate DNA. We report finding seven independent human DNA sequences that qualify s descendants of a left-arm precursor gene. Some characteristics in primary and secondary structures of these sequences are described.     

Emergence of master sequences in families of retroposons derived from 7sl RNA

The past few years have brought new insight into the evolution of families of retroposons. These are composed of a very small number of master sequences able to duplicate, and a large majority of copies that are inactive for retroposition. During the course of time, successive replacements of master sequences have produced waves of amplification that are recognizable as subfamilies. In the Alu and the B1 families, one can distinguish two evolutionary periods. The first involves only monomeric elements that are now extinguished (fossil elements) and is characterized by deep remodeling of the sequences. This period ends, in primates, with the fusion of a free left and a free right Alu monomer, producing the first modern Alu dimeric element; in rodents it ends with a tandem duplication of 29 bp to create the first modern B1 element. The second period is characterized by a great stability of the master sequences. The observed turn-over of master sequences is still an enigma. However, analysis of the contemporary master sequences and of the oldest master sequences provide some clues. Here, we review the very first stages of the appearance of the Alu and the B1 families in mammalian genomes.     

Structure and Origin of a Novel Dimeric Retroposon B1-dID

Here we describe a new short retroposon family of rodents. Like the primate Alu element consisting of two similar monomers, it is dimeric, but the left and right monomers are different and descend from B1 and ID short retroposons, respectively. Such elements (B1-dID) were found in the genomes of Gliridae, Sciuridae, Castoridae, Caviidae, and Hystricidae. Nucleotide sequences of this retroposon can be assigned to several structural variants. Phylogenetic analysis of B1-dID and related sequences suggests a possible scenario of B1-dID evolution in the context of rodent evolution. Received: 30 August 1999 / Accepted: 20 March 2000     

A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.     

Evolution of secondary structure in the family of 7SL-like RNAs

Primate and rodent genomes are populated with hundreds of thousands copies of Alu and B1 elements dispersed by retroposition, i.e., by genomic reintegration of their reverse transcribed RNAs. These, as well as primate BC200 and rodent 4.5S RNAs, are ancestrally related to the terminal portions of 7SL RNA sequence. The secondary structure of 7SL RNA (an integral component of the signal recognition particle) is conserved from prokaryotes to distant eukaryotic species. Yet only in primates and rodents did this molecule give rise to retroposing Alu and B1 RNAs and to apparently functional BC200 and 4.5S RNAs. To understand this transition and the underlying molecular events, we examined, by comparative analysis, the evolution of RNA structure in this family of molecules derived from 7SL RNA.RNA sequences of different simian (mostly human) and prosimian Alu subfamilies as well as rodent B1 repeats were derived from their genomic consensus sequences taken from the literature and our unpublished results (prosimian and New World Monkey). RNA secondary structures were determined by enzymatic studies (new data on 4.5S RNA are presented) and/or energy minimization analyses followed by phylogenetic comparison. Although, with the exception of 4.5S RNA, all 7SL-derived RNA species maintain the cruciform structure of their progenitor, the details of 7SL RNA folding domains are modified to a different extent in various RNA groups. Novel motifs found in retropositionally active RNAs are conserved among Alu and B1 subfamilies in different genomes. In RNAs that do not proliferate by retroposition these motifs are modified further. This indicates structural adaptation of 7SL-like RNA molecules to novel functions, presumably mediated by specific interactions with proteins; these functions were either useful for the host or served the selfish propagation of RNA templates within the host genome.Abbreviations FAM fossil Alu element - FLAM free left Alu monomer - FRAM free right Alu monomer - L-Alu left Alu subunit - R-Alu right Alu subunit Correspondence to: D. LabudaDedicated to Dr. Robert Cedergren on the occasion of his 25th anniversary at the University of Montreal     

Integration site preferences of the Alu family and similar repetitive DNA sequences.

Numerous flanking nucleotide sequences from two primate interspersed repetitive DNA families have been aligned to determine the integration site preferences of each repetitive family. This analysis indicates that both the human Alu and galago Monomer families were preferentially inserted into short d(A+T)-rich regions. Moreover, both primate repeat families demonstrated an orientation specific integration with respect to dA-rich sequences within the flanking direct repeats. These observations suggest that a common mechanism exists for the insertion of many repetitive DNA families into new genomic sites. A modified mechanism for site-specific integration of primate repetitive DNA sequences is provided which requires insertion into dA-rich sequences in the genome. This model is consistent with the observed relationship between galago Type II subfamilies suggesting that they have arisen not by mere mutation but by independent integration events.     

Comprehensive analysis of two Alu Yd subfamilies

Alu elements have inserted in the human genome throughout primate evolution. A small number of Alu insertions have occurred after the divergence of humans from nonhuman primates and therefore should not be present in nonhuman primate genomes. Most of these recently integrated Alu elements are contained with a series of discrete Alu subfamilies that are related to each other based upon diagnostic nucleotide substitutions. We have extracted members of the Alu Yd subfamily that are derivatives of the Alu Y subfamily that share a common 12-bp deletion that defines the Yd lineage from the draft sequence of the human genome. Analysis of the Yd Alu elements resulted in the recovery of two new Alu subfamilies, Yd3 and Yd6, which contain a total of 295 members (198 Yd3 and 97 Yd6). DNA sequence analysis of each of the Alu Yd subfamilies yielded age estimates of 8.02 and 1.20 million years old for the Alu Yd3 and Yd6 subfamilies, respectively. Two hundred Alu Yd3 and Yd6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and associated levels of human genomic diversity. The Alu Yd3 subfamily appears to have started amplifying relatively early in primate evolution and continued propagating albeit at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Only two of the elements are polymorphic in the human genome and absent from the genomes of nonhuman primates. By contrast all of the members of the Alu Yd6 subfamily are restricted to the human genome, with 12% of the elements representing insertion polymorphisms in human populations. A single Alu Yd6 locus contained an independent parallel forward insertion of a paralogous Alu Sq sequence in the owl monkey. These Alu subfamilies are a source of genomic fossil relics for the study of primate phylogenetics and human population genetics.     

Birth of a gene: locus of neuronal BC200 snmRNA in three prosimians and human BC200 pseudogenes as archives of change in the Anthropoidea lineage

The gene encoding brain-specific dendritic BC200 small non-messenger RNA is limited to the primate order and arose from a monomeric Alu element. It is present and neuronally expressed in all Anthropoidea examined. By comparing the human sequence of about 13.2 kb with each of the prosimian (lemur 14.6 kb, galago 12 kb, and tarsier 13.8 kb) orthologous loci, we could establish that the BC200 RNA gene is absent from the prosimian lineages. In Strepsirhini (lemurs and lorises), a dimeric AluJ-like element integrated very close to the BC200 insertion point, while the corresponding tarsier region is devoid of any repetitive element. Consequently, insertion of the Alu monomer that gave rise to the BC200 RNA gene must have occurred after the anthropoid lineage diverged from the prosimian lineage(s). Shared insertions of other repetitive elements favor proximity of simians and tarsiers in support of their grouping into Haplorhini and the omomyid hypothesis. On the other hand, the nucleotide sequences in the segment that is available for comparison in all four species reveal less exchanges between Strepsirhini (lemur and galago) and human than between tarsier and human. Our data imply that the early activity of dimeric Alu sequences must have been concurrent with the activity of monomeric Alu elements that persisted longer than is usually thought. As BC200 RNA gave rise to more than 200 pseudogenes, we used their consensus sequence variations as a molecular archive recording the BC200 RNA sequence changes in the anthropoid lineage leading to Homo sapiens and timed these alterations over the past 35-55 million years.     

Short Retroposons of the B2 Superfamily: Evolution and Application for the Study of Rodent Phylogeny

Short retroposons can be used as natural phylogenetic markers. By means of hybridization and PCR analysis, we demonstrate that B2 retroposon copies are present only in the three rodent families: Muridae, Cricetidae, and Spalacidae. This observation highlights the close phylogenetic relation between these families. Two novel B2-related retroposon families, named DIP and MEN elements, are described. DIP elements are found only in the genomes of jerboas (family Dipodidae) and birch mice (family Zapodidae), demonstrating the close relationship between these rodents. MEN element copies were isolated from the squirrel, Menetes berdmorei, but were not detected in three other species from the family Sciuridae. The MEN element has an unusual dimeric structure: the left and right monomers are B2- and B1-related sequences, respectively. Comparison of the B2, DIP, MEN, and 4.5S₁ RNA elements revealed an 80-bp core sequence located at the beginning of the B2 superfamily retroposons. This observation suggests that these retroposon families descended from a common progenitor. A likely candidate for this direct progenitor could be the ID retroposon. Received: 20 December 1996 / Accepted: 17 June 1997