Phylogenetic roots of Alu-mediated rearrangements leading to cancer.

There are over a million Alu repetitive elements dispersed throughout the human genome, and a high level of Alu-sequence similarity ensures a strong propensity for unequal crossover events, some of which have lead to deleterious oncogenic rearrangements. Furthermore, Alu insertions introduce consensus 3' splice sites, which potentially facilitate alternative splicing. Not surprisingly, Alu-mediated defective splicing has also been associated with cancer. To investigate a possible correlation between the expansion of Alu repeats associated with primate divergence and predisposition to cancer, 4 Alu-mediated rearrangements--known to be the basis of cancer--were selected for phylogenetic analysis of the necessary genotype. In these 4 cases, it was determined that the different phylogenetic age of the oncogenic recombination-prone genotype reflected the evolutionary history of Alu repeats spreading to new genomic sites. Our data implies that the evolutionary expansion of Alu repeats to new genomic locations establishes new predispositions to cancer in various primate species.     

Various aspects of evolution of Alu repeats in mammals

A complex study on various evolutionary peculiarities of the mammalia dispersed Alu repeats (Alu repeats of primates and B1 of rodents) has been carried out by phylogenetic analysis. A phylogenetic tree, containing the 7SL RNA genes and the Alu repeats of primates and rodents has been constructed. It has been shown that the branch of the phyletic line leading to the Alu repeats of primates and B1 of rodents from the 7SL RNA genes occurred after the divergence of the 7SL RNA genes of amphibia and mammalia, but before the divergence of the 7SL RNA genes of primates and rodents (250.10 years ago). A statistically reliable slowing down in the evolutionary rates of one of two monomers for the human Alu repeats has been proved. It may be caused by the functional load of the corresponding monomer in connection with the presence of a definit regulatory site in it.     

A young Alu subfamily amplified independently in human and African great apes lineages.

A variety of Alu subfamilies amplified in primate genomes at different evolutionary time periods. Alu Sb2 belongs to a group of young subfamilies with a characteristic two-nucleotide deletion at positions 65/66. It consists of repeats having a 7-nucleotide duplication of a sequence segment involving positions 246 through 252. The presence of Sb2 inserts was examined in five genomic loci in 120 human DNA samples as well as in DNAs of higher primates. The lack of the insertional polymorphism seen at four human loci and the absence of orthologous inserts in apes indicated that the examined repeats retroposed early in the human lineage, but following the divergence of great apes. On the other hand, similar analysis of the fifth locus (butyrylcholinesterase gene) suggested contemporary retropositional activity of this subfamily. By a semi-quantitative PCR, using a primer pair specific for Sb2 repeats, we estimated their copy number at about 1500 per human haploid genome; the corresponding numbers in chimpanzee and gorilla were two orders of magnitude lower, while in orangutan and gibbon the presence of Sb2 Alu was hardly detectable. Sequence analysis of PCR-amplified Sb2 repeats from human and African great apes is consistent with the model in which the founding of Sb2 subfamily variants occurred independently in chimpanzee, gorilla and human lineages.     

Evolution of Alu repeats: dynamics of distribution in genome

A mathematical model of evolutionary dynamics of Alu repeats' number in the human genome has been worked out. The model permitted us to observe the dynamics of propagation of Alu repeats within the genome and to evaluate such important parameters of the process mentioned as the rates of transposition (insertion of new copies into the genome) and excision of repeats. The peculiarities of the control of Alu repeats' number in the genome have been discussed, based on the data obtained.     

Alu repeats in the human genome

Highly repetitive DNA sequences account for more than 50% of the human genome. The L1 and Alu families harbor the most common mammalian long (LINEs) and short (SINEs) interspersed elements. Alu elements are each a dimer of similar, but not identical, fragments of total size about 300 bp, and originate from the 7SL RNA gene. Each element contains a bipartite promoter for RNA polymerase III, a poly(A) tract located between the monomers, a 3'-terminal poly(A) tract, and numerous CpG islands, and is flanked by short direct repeats. Alu repeats comprise more than 10% of the human genome and are capable of retroposition. Possibly, these elements played an important part in genome evolution. Insertion of an Alu element into a functionally important genome region or other Alu-dependent alterations of gene functions cause various hereditary disorders and are probably associated with carcinogenesis. In total, 14 Alu families differing in diagnostic mutations are known. Some of these, which are present in the human genome, are polymorphic and relatively recently inserted into new loci. Alu copies transposed during ethnic divergence of the human population are useful markers for evolutionary genetic studies.     

Evolution of a Hypervariable Region of the Low Density Lipoprotein Receptor (LDLR) Gene in Humans and other Hominoids

Alu repeats in primates have been shown to evolve at a neutral mutation rate, as anticipated for non-coding autosomal loci. However, we have identified Alu elements within the 3' untranslated region (UTR) of the low density lipoprotein receptor (LDLR) gene that exhibited highly accelerated rates of evolution. In humans, a 100- and 25-fold increase in average divergence, for an upstream Alu (Alu U) and a downstream Alu (Alu D) respectively, was estimated based on sequence analysis among eight individuals of diverse ethnic backgrounds. None of these individuals demonstrated identical sequences within a 950 base region consisting of these two Alu elements. The hypervariability of this genetic region in the nuclear genome yields a potentially powerful tool for human population studies, forensics and paternity. Additionally, the mutation rate of Alu U among non-human hominoids was also accelerated, although to a lesser extent of roughly 3-fold that of other Alu elements. Sequence analysis of various Hominoidea species demonstrated its utility as a phylogenetic tool. The mechanism for the hypervariability in mutation rates is unclear, but may be accelerated as a result of Alu-mediated gene conversion in the human lineage.     

Distribution of Alu repeats along the human genome: formation of clusters and features of insertion regions

The contextual analysis of nucleotide sequences of 22 Alu repeats arrangement regions in the human genome has been carried out and some of their peculiarities have been revealed. In particular, the occurrence of marked and statistical non-random homology between the repeats and the regions of their integration has been shown. A mechanism of choosing the Alu repeats insertion regions in the genome has been suggested taking into account these peculiarities. Using a sample of the 80 human Alu repeats sequences peculiarities of these repeats location within the genome has been investigated. A tendency to the formation of Alu repeats clusters in various regions of the genome was revealed. A range of possible mechanisms on such Alu clusters emergence is considered. On the basis of the data obtained an "attraction" mechanism, according to which integration of Alu repeats into the definite region of the genome increases the insertion probability of other Alu repeats into the same region, are proposed.     

Origin and phylogenetic distribution of Alu DNA repeats: irreversible events in the evolution of primates.

Over the past 60 million years, or so, approximately one million copies of Alu DNA repeats have accumulated in the genome of primates, in what appears to be an ongoing process. We determined the phylogenetic distribution of specific Alu (and other) DNA repeats in the genome of several primates: human, chimpanzee, gorilla, orangutan, baboon, rhesus, and macaque. At the population level studied, the majority of the repeats was found to be fixed in the primate species. Our data suggest that new Alu elements arise in unique, irreversible events, in a mechanism that seems to preclude precise excision and loss. The same insertions did not arise independently in two species. Once inserted and genetically fixed, the DNA elements are retained in all descendant lineages. The irreversible expansion of Alu s introduces a vector of time into the evolutionary process, and provides realistic (rather than statistical) answers to questions on phylogenies. In contrast to point mutations, the present distribution of individual Alu s is congruent with just one phylogeny. We submit that only irreversible and taxonomically relevant events are at the molecular basis of evolution. Most point mutations do not belong to this category.     

Growth and decline of introns

To gauge the processes that might direct the length of introns, I studied the balance of indels (insertions or deletions, determined using Alu and LINE1 retroposon repeats) and the density of these repeats in the introns of the human genome. The indel balance is biased in favour of deletions and correlated with the divergence of repeats. At fixed repeat divergence, the indel bias correlated with the intron size: the shorter the intron, the more deletions were favoured over insertions. This correlation with the intron size was stronger than with the gene-wide or isochore-wide parameters. The density of repeats (the number of repeats in a unit of intron length) correlated positively with the intron size. Thus, quite different mechanisms, the indel bias and the integration and/or persistence of retroposons, act in the same direction in regards to intron size, which suggests selection for the size of individual introns.     

The Biased Distribution of Alus in Human Isochores Might Be Driven by Recombination

Alu retrotransposons do not show a homogeneous distribution over the human genome but have a higher density in GC-rich (H) than in AT-rich (L) isochores. However, since they preferentially insert into the L isochores, the question arises: What is the evolutionary mechanism that shifts the Alu density maximum from L to H isochores? To disclose the role played by each of the potential mechanisms involved in such biased distribution, we carried out a genome-wide analysis of the density of the Alus as a function of their evolutionary age, isochore membership, and intron vs. intergene location. Since Alus depend on the retrotransposase encoded by the LINE1 elements, we also studied the distribution of LINE1 to provide a complete evolutionary scenario. We consecutively check, and discard, the contributions of the Alu/LINE1 competition for retrotransposase, compositional matching pressure, and Alu overrepresentation in introns. In analyzing the role played by unequal recombination, we scan the genome for Alu trimers, a direct product of Alu–Alu recombination. Through computer simulations, we show that such trimers are much more frequent than expected, the observed/expected ratio being higher in L than in H isochores. This result, together with the known higher selective disadvantage of recombination products in H isochores, points to Alu–Alu recombination as the main agent provoking the density shift of Alus toward the GC-rich parts of the genome. Two independent pieces of evidence—the lower evolutionary divergence shown by recently inserted Alu subfamilies and the higher frequency of old stand-alone Alus in L isochores—support such a conclusion. Other evolutionary factors, such as population bottlenecks during primate speciation, may have accelerated the fast accumulation of Alus in GC-rich isochores.     

Evolution of an Alu DNA element of type Sx in the lineage of primates and the origin of an associated tetranucleotide microsatellite.

A 394-bp DNA fragment, which in human is on chromosome 6 near the MOG (myelin oligodendrocyte glycoprotein) gene and encompasses an Alu element and an associated tetranucleotide microsatellite, was sequenced from a large range of primate species to follow its evolutionary divergence and to understand the origin of the microsatellite. This Alu element is found at the same orthologous position in all primates sequenced, but the tetranucleotide repeat is present only in Catarrhini between the 3'-oligo(dA) of the Alu element and the 3' flanking direct repeat. Little intraspecific variation was found. Sequence identity values for this orthologous primate Alu averaged 90% (82-99%) with transitions comprising between 70% and 100% of the observed nucleotide substitutions. Although the insertion of the Alu element predates the separation of these species, the original sequence of the site of integration can still be identified. This identification of the direct repeats suggests an active role of the oligo(dA) of the Alu element in the origin of the tetranucleotide repeats. The microsatellite probably appeared after the insertion of the Alu element, early in the lineage leading to the common ancestor of the hominoids and the Old World monkeys.     

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     

Recently integrated human Alu repeats: finding needles in the haystack

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Non-traditional Alu evolution and primate genomic diversity

Alu elements belonging to the previously identified "young" subfamilies are thought to have inserted in the human genome after the divergence of humans from non-human primates and therefore should not be present in non-human primate genomes. Polymerase chain reaction (PCR) based screening of over 500 Alu insertion loci resulted in the recovery of a few "young" Alu elements that also resided at orthologous positions in non-human primate genomes. Sequence analysis demonstrated these "young" Alu insertions represented gene conversion events of pre-existing ancient Alu elements or independent parallel insertions of older Alu elements in the same genomic region. The level of gene conversion between Alu elements suggests that it may have a significant influence on the single nucleotide diversity within the genome. All the instances of multiple independent Alu insertions within the same small genomic regions were recovered from the owl monkey genome, indicating a higher Alu amplification rate in owl monkeys relative to many other primates. This study suggests that the majority of Alu insertions in primate genomes are the products of unique evolutionary events.     

Increased gene coverage and Alu frequency in large linkage disequilibrium blocks of the human genome

The human genome has linkage disequilibrium (LD) blocks, within which single-nucleotide polymorphisms show strong association with each other. We examined data from the International HapMap Project to define LD blocks and to detect DNA sequence features inside of them. We used permutation tests to determine the empirical significance of the association of LD blocks with genes and Alu repeats. Very large LD blocks (>200 kb) have significantly higher gene coverage and Alu frequency than the outcome obtained from permutation-based simulation, whereas there was no significant positive correlation between gene density and block size. We also observed a reduced frequency of Alu repeats at the gaps between large LD blocks, indicating that their enrichment in large LD blocks does not introduce recombination hotspots that would cause these gaps.     

Origin and instability of GAA repeats: insights from Alu elements

Expansion of GAA repeats in the intron of the frataxin gene is involved in the autosomal recessive Friedreich's ataxia (FRDA). The GAA repeats arise from a stretch of adenine residues of an Alu element. These repeats have a size ranging from 7- 38 in the normal population, and expand to thousands in the affected individuals. The mechanism of origin of GAA repeats, their polymorphism and stability are not well understood. In this study, we have carried out an extensive analysis of GAA repeats at several loci in the humans. This analysis indicates the association of a majority of GAA repeats with the 3' end of an "A" stretch present in the Alu repeats. Further, the prevalence of GAA repeats correlates with the evolutionary age of Alu subfamilies as well as with their relative frequency in the genome. Our study on GAA repeat polymorphism at some loci in the normal population reveals that the length of the GAA repeats is determined by the relative length of the flanking A stretch. Based on these observations, a possible mechanism for origin of GAA repeats and modulatory effects of flanking sequences on repeat instability mediated by DNA triplex is proposed.