Alu Subfamilies Research Articles

Characterization of Alu repeats that are associated with trinucleotide and tetranucleotide repeat microsatellites.

The association of subclasses of Alu repetitive elements with various classes of trinucleotide and tetranucleotide microsatellites was characterized as a first step toward advancing our understanding of the evolution of microsatellite repeats. In addition, information regarding the association of specific classes of microsatellites with families of Alu elements was used to facilitate the development of genetic markers. Sequences containing Alu repeats were eliminated because unique primers could not be designed. Various classes of microsatellites are associated with different classes of Alu repeats. Very abundant and poly(A)-rich microsatellite classes (ATA, AATA) are frequently associated with an evolutionarily older subclass of Alu repeats, AluSx, whereas most of GATA and CA microsatellites are associated with a recent Alu subfamily, AluY. Our observations support all three possible mechanisms for the association of Alu repeats to microsatellites. Primers designed using a set of sequences from a particular microsatellite class showed higher homology with more sequences of that class than probes designed for other classes. We developed an efficient method of prescreening GGAA and ATA microsatellite clones for Alu repeats with probes designed in this study. We also showed that Alu probes labeled in a single reaction (multiplex labeling) could be used efficiently for prescreening of GGAA clones. Sequencing of these prescreened GGAA microsatellites revealed only 5% Alu repeats. Prescreening with primers designed for ATA microsatellite class resulted in the reduction of the loss of markers from approximately 50% to 10%. The new Alu probes that were designed have also proved to be useful in Alu-Alu fingerprinting.

Recent polymorphic insertion of an Alu repeat in the baboon lipoprotein lipase ( LPL) gene

We have identified a polymorphic insertion in the lipoprotein lipase ( LPL) gene in a captive baboon colony. Mapping and nucleotide (nt) sequence analysis of the polymorphism showed that it is due to the presence or absence of an Alu repetitive element in intron 7 of the baboon LPL gene. This polymorphic Alu repeat has not been reported in humans, and we did not detect the repeat in a survey of the LPL intron 7 gene region in other non-human primates. Comparison of nt at diagnostic positions in this Alu insertion with different Alu subfamily consensus sequences showed that it most closely resembles the young AluY subfamily. These data suggest that this polymorphic Alu repeat inserted independently in the baboon lineage.

Study of Alu sequences at the hypoxanthine phosphoribosyltransferase ( hprt) encoding region of man

The hypoxanthine phosphoribosyltransferase ( hprt) encoding region of man is considered rich in Alu sequences; with 49 sequences present within 57 kilobases. Subfamily classification of the Alu sequences and identification of flanking direct repeats has been carried out to detect past rearrangements associated with their insertion into the region. Members of the Alu-J and three Alu-S subfamilies are present, along with the existence of free left arm sequences. Using available data, a comparison is made of the Alu subfamilies present at different gene regions. The heterogeneity in the number of each subfamily present at different genes shows that no one particular subfamily attained saturation in the genome. Several adjacent insertions of Alu sequences are seen at the hprt region. Furthermore two novel sequences are described, there is an incident where one Alu sequence has inserted into the middle poly(A) tract of an existing sequence at the hprt region; while another resulted from an Alu/ Alu cross-over event elsewhere in the genome, before insertion into the hprt region. Once inserted, the Alu sequences are rarely subject to loss or rearrangement.

Characterization and population diversity of interspersed repeat sequence variants (IRS-morphs)

Inter-Alu PCR is increasingly useful in human genome mapping studies. One use is the generation of alumorphs, polymorphisms resulting from the presence or absence of inter-Alu PCR products. In this study, we have increased the proportion of the genome that can be analyzed by this technique with the use of long interspersed elements (LINEs). The set of polymorphisms detected by both Alu and LINE primers are referred to as interspersed repetitive sequence variants or IRS-morphs. Since a presence-absence variant may have been the result of a recent Alu or LINE insertion, we analyzed 7 isolated IRS-morphs that were generated, in part, with a primer derived from either a consensus LINE or a young Alu subfamily specific sequence, and observed by Southern blot analysis that these variants resulted from other types of genomic alterations. The use of these primers, however, reduces background from the numerous LINEs and Alu elements in the genome, providing sharp DNA fingerprint profiles. We have demonstrated the potential usefulness of these IRS-morph profiles in human population studies. We compared 12 IRS-morphs from a single amplification reaction from five distinct population groups (Caucasian (northern European descent), Hispanic (Mexican-American), Hindu-Indian, Papua New Guinean, and Greenland Eskimo) and observed that most have variable allelic frequencies among populations. The utilization of additional IRS-morph profiles will perpetuate this technique as a tool for DNA fingerprinting and for the analysis of human populations. Key words : Alu elements, DNA fingerprint, human populations, LINEs, SINEs.

A Naturally Occurring T14A11 Tract Blocks Nucleosome Formation Over the Human Neurofibromatosis Type 1 (NF1)-Alu Element

The nature of chromatin organization over Alu repetitive elements is of interest with respect to the maintenance of their transcriptional silencing as well as their potential to influence local chromatin structure. We previously demonstrated that the pattern of nucleosomal organization over Alu elements in native chromatin is specific and similar to the pattern observed with an in vitro reconstituted Alu template. This pattern, distinguished by a nucleosome centered over the 5 -end of the Alu element, is associated with repression of polymerase III-dependent transcription in vitro (Englander, E. W., Wolffe, A. P., and Howard, B. H. (1993) J. Biol. Chem. 268, 19565-19573; Englander, E. W., and Howard, B. H. (1995) J. Biol. Chem. 270, 10091-10096). In the current study, additional templates representing both evolutionarily old and young Alu subfamilies were found to direct a similar pattern of nucleosome assembly, consistent with the view that nucleosome positioning in vitro is shared by a majority of Alus. We discovered however, that the specific nucleosome positioning pattern was disrupted over one member of a young Alu subfamily, which recently transposed immediately downstream to a T14A11 sequence in the neurofibromatosis type 1 locus (Wallace, M. R., Andersen, L. B., Saulino, A. M., Gregory, P. E., Glover, T. W., and Collins, F. S. (1991) Nature 353, 864-866). Upon removal of this sequence motif, the expected pattern of assembly was restored to the neurofibromatosis type 1-Alu template. This finding indicates that, at least in vitro, certain sequences can override the propensity for positioning nucleosomes that is inherent to Alu elements. The finding also raises the possibility that a similar situation may occur in vivo, with potential implications for understanding mechanisms by which certain Alu elements may evade chromatin-mediated transcriptional silencing.

The age of Alu subfamilies

Using Kimura's distance measure we have calculated the average age of all major Alu subfamilies based on the most recent available data. We conclude that AluJ sequences are some 26 Myr older than previously thought. Furthermore, the origin of the FLA (Free Left Arm) Alu family can be traced back to the very beginning of the mammalian radiation. One new minor subfamily is reported and discussed in the context of sequence diversity in major Alu subfamilies.

The role and amplification of the HS Alu subfamily founder gene.

A recently identified Alu element (Leeflang et al. J. Mol. Evol. 1993, 37:559-565), referred to as the "putative founder of the HS (PV) subfamily," was found to be present at orthologous loci in the human, chimpanzee, gorilla, and gibbon lineages. The evolution of this Alu suggested that it is a source gene in the evolution of Alu family repeats for one of the most recent subfamilies, HS. We have determined that this putative founder of the HS subfamily was not present at the orthologous loci in older primates, including old world and new world monkeys. Thus, this particular Alu locus has only been responsible for the establishment of a very small subfamily of Alu sequences. We have further demonstrated that this putative founder Alu was not responsible for the de novo Alu insertion into the neurofibromatosis-1 gene of an individual causing neurofibromatosis. Our data demonstrate that although the putative founder of the HS subfamily found by Leeflang et al. (1993) probably gave rise to one of the most recent subfamilies of Alu sequences, it has not been very active in retroposition.

Flanking sequences of an Alu source stimulate transcription in vitro by interacting with sequence-specific transcription factors

An Alu source gene, called the EPL Alu, was previously isolated by a phylogenetic strategy. Sequences flanking the EPL Alu family member stimulate its RNA polymerase III (Pol III) template activity in vitro. One cis-acting element maps within a 40-nucleotide region immediately upstream to the EPL Alu. This same region contains an Ap1 site which, when mutated, abolishes the transcriptional stimulation provided by this region. The flanking sequence, as assayed by gel mobility shift, forms sequence-specific complexes with several nuclear factors including Ap1. These results demonstrate that an an ancestral Alu source sequence fortuitously acquired positive transcriptional control elements by insertion into the EPL locus, thereby providing biochemical evidence for a model which explains the selective amplification of Alu subfamilies.

Alu: Structure, Origin, Evolution, Significance, and Function of One-Tenth of Human DNA

Publisher Summary Human repeats have been largely identified and cataloged, accounting for a substantial fraction of the genome. Repetitive sequences are ubiquitously interspersed with single-copy sequences throughout the genome. Interspersed repeats are largely composed of only four distinct families (Alu, LINE 1, MIR, and MaLR), which together comprise 10 to 15% of the entire genome. Alu and LINE 1 subfamilies of different evolutionary ages have different degrees of retrotranspositional activity. This chapter focuses on Alu repeats. Although Alus exemplify many properties of both human and nonhuman repeats, their amplification raises unsolved problems in retroposition. A young Alu subfamily appeared by simple drift of its founder, providing a model for the continual turnover of active repeat-sequence families. Repetitive sequences, ubiquitously distributed throughout the genome, cause various genetic effects. Unequal crossing-over among interspersed repeats duplicates or deletes sequences, thereby, mutating genes. Ubiquitous, homologous repeats, such as Alu, might serve as frequent sites for unequal homologous crossing-over, consequently scrambling their flanking direct repeats.. Unequal Alu–Alu crossing-over duplicates, deletes, or scrambles genetic information. Alu–Alu crossing-over within the low-density lipoprotein receptor gene duplicates exons, thereby, inactivating the gene product, leading to hypercholesterolemia.

Sequence diversity and chromosomal distribution of “young” Alu repeats

Members of the recently inserted human-specific (HS)/predicted variant (PV) subfamily of Alu elements were sequenced. A number of these Alu elements share greater than 98% sequence identity with the subfamily consensus sequence, and they are flanked by perfect 5′ and 3′ direct repeats ranging in size from 6 to 15 nucleotides (nt). Based on the low number of random mutations, the estimated average age of these elements was calculated to be 1.5 million years (Myr). All the young Alu subfamily members were restricted to the human genome, as judged by polymerase chain reaction (PCR) amplification of human and non-human primate DNA samples using the unique flanking sequences specific for each Alu element. The chromosomal locations of several Alu elements belonging to the young subfamilies, designated as HS/PV and Sb2, were determined by PCR amplification of DNA samples from human/rodent somatic cell hybrid panels. A statistical analysis of the chromosomal distribution pattern showed that the recently inserted Alu elements appear to integrate randomly in the human genome.

The consensus sequence of a major Alu subfamily contains a functional retinoic acid response element.

Alu repeats are interspersed repetitive DNA elements specific to primates that are present in 500,000 to 1 million copies. We show here that an Alu sequence encodes functional binding sites for retinoic acid receptors, which are members of the nuclear receptor family of transcription factors. The consensus sequences for the evolutionarily recent Alu subclasses contain three hexamer half sites, related to the consensus AGGTCA, arranged as direct repeats with a spacing of 2 bp, which is consistent with the binding specificities of retinoic acid receptors. An analysis was made of the DNA binding and transactivation potential of these sites from an Alu sequence that has been previously implicated in the regulation of the keratin K18 gene. These Alu double half sites are shown to bind bacterially synthesized retinoic acid receptors as assayed by electrophoretic mobility shift assays. These sites are further shown to function as a retinoic acid response element in transiently transfected CV-1 cells, increasing transcription of a reporter gene by a factor of approximately 35-fold. This transactivation requires cotransfection with vectors expressing retinoic acid receptors, as well as the presence of all-trans-retinoic acid, which is consistent with the known function of retinoic acid receptors as ligand-inducible transcription factors. The random insertion of potentially thousands of Alu repeats containing retinoic acid response elements throughout the primate genome is likely to have altered the expression of numerous genes, thereby contributing to evolutionary potential.

Polymorphic human specific Alu insertions as markers for human identification.

Alu sequences represent the largest family of short interspersed repetitive elements (SINEs) in humans with 500 000 copies per genome. Recently, one Alu subfamily was found to be human specific (HS). We originally described the use of polymorphis HS Alu insertions as a tool in population studies and recently as tools in DNA fingerprinting and forensic analysis. In this report, we will use this simple polymerase chain reaction (PCR) base technique for the detection of HS Alu insertion polymorphisms. We will test the resolving power of this DNA profiling approach in both population genetics and paternity assessment. At the population level, we will describe the genotypic distribution of five polymorphic Alu insertions among 3 populations from the American continent, one of African origin, the other two Amerindians. Insight into their relationships will be provided. At the family level, we will examine one European American family of seven individuals and the same pedigree will also be characterized by way of the two systems currently and widely used to ascertain paternity: PCR-sequence specific oligonucleotide probe hybridization (PCR-SSO) and PCR-restriction fragment length polymorphism (PCR-RFLP) of human leucocyte antigen (HLA) class II molecules, and a standard RFLP protocol used in forensic casework and paternity studies. The importance and strengths of the methods as well as its perspectives for future use in filiation studies will be evaluated.

A recent insertion of an alu element on the Y chromosome is a useful marker for human population studies.

A member of the Alu family of repeated DNA elements has been identified on the long arm of the human Y chromosome, Yq11. This element, referred to as the Y Alu polymorphic (YAP) element, is present at a specific site on the Y chromosome in some humans and is absent in others. Phylogenetic comparisons with other Alu sequences reveal that the YAP element is a member of the polymorphic subfamily-3 (PSF-3), a previously undefined subfamily of Alu elements. The evolutionary relationships of PSF-3 to other Alu subfamilies support the hypothesis that recently inserted elements result from multiple source genes. The frequency of the YAP element is described in 340 individuals from 14 populations, and the data are combined with those from other populations. There is both significant heterogeneity among populations and a clear pattern in the frequencies of the insertion: sub-Saharan Africans have the highest frequencies, followed by northern Africans, Europeans, Oceanians, and Asians. An interesting exception is the relatively high frequency of the YAP element in Japanese. The greatest genetic distance is observed between the African and non-African populations. The YAP is especially useful for studying human population history from the perspective of male lineages.

Alu elements of the primate major histocompatibility complex.

The chromosomal region constituting the major histocompatibility complex (MHC) has undergone complex evolution that is often difficult to decipher. An important aid in the elucidation of the MHC evolution is the presence of Alu elements (repeats) which serve as markers for tracing chromosomal rearrangements. As the first step toward the establishment of sets of evolutionary markers for the MHC, Alu elements present in selected MHC haplotypes of the human species, the gorilla, and the chimpanzee were identified. Restriction fragments of cosmid clones from the libraries of the three species were hybridized with Alu-specific probes, Alu elements were amplified by the polymerase chain reaction, and the amplification products were sequenced. In some cases, sequences of the regions flanking the Alu elements were also obtained. Altogether, 31 new Alu elements were identified, representing six Alu subfamilies. The average density of Alu elements in the MHC is one element per four kilobases (kb) of sequence. Alu elements have apparently been inserted steadily into the MHC over the last 65 million years (my). On average, one Alu element is inserted into the primate MHC every 4 my. Analysis of the human DR3 haplotype supports its origin by duplication from an ancestral haplotype consisting of DRB1 and DRB2 genes. The sharing of an old Alu element by the DRB1 and DRB2 genes, in turn, supports their divergence from a common ancestor more than 55 my ago.

The human immunoglobulin kappa locus: pseudogenes, unique and repetitive sequences.

The human kappa locus contains 25 pseudogenes. After seven of them were described earlier the structures of the remaining 18 are reported now, thus completing the description of all human V kappa genes and pseudogenes. Most of the pseudogenes carry several defects each. Alignments of the pseudogene sequences and comparison with the consensus sequences of the potentially functional V kappa genes indicate that, on PCR amplification of genomic DNA aimed at certain genes of the latter class, also some of the pseudogenes would be coamplified. Unique sequences, which qualify as sequence tagged sites (STS), were defined across the locus. The occurrence of 15 repetitive elements of the LINE1 type in the locus is described. The 15 sequenced Alu elements were assigned to the known Alu subfamilies of different evolutionary age. One of the Alu elements was found only in one of the copies of the kappa locus. It must, therefore, have been inserted after the duplication step which may have taken place about one million years ago. This element belongs to an Alu subfamily known to have been mobile until recently. Some aspects of the evolution of the V kappa pseudogenes and orphons (i.e. V kappa genes located outside the kappa locus) are also discussed.

Phylogenetic isolation of a human Alu founder gene: drift to new subfamily identity [corrected

A severe bottleneck in the size of the PV Alu subfamily in the common ancestor of human and gorilla has been used to isolate an Alu source gene. The human PV Alu subfamily consists of about one thousand members which are absent in gorilla and chimpanzee DNA. Exhaustive library screening shows that there are as few as two PV Alus in the gorilla genome. One is gorilla-specific, i.e., absent in the orthologous loci in both human and chimpanzee, suggesting the independent retrotranspositional activity of the PV subfamily in the gorilla lineage. The second of these two gorilla PV Alus is present in both human and chimpanzee DNAs and is the single PV Alu known to precede the radiation of these three species. The orthologous Alu in gibbon DNA resembles the next older Alu subfamily. Thus, this Alu locus is originally templated by a non-PV source gene and acquired characteristic PV sequence variants by mutational drift in situ, consequently becoming the first member and presumptive founder of this PV subfamily.

Mobility of short interspersed repeats within the chimpanzee lineage

The PV subfamily of Alu repeats in human DNA is largely composed of recently inserted members. Here we document additional members of the PV subfamily that are found in chimpanzee but not in the orthologous loci of human and gorilla, confirming the relatively recent and independent expansion of this Alu subfamily in the chimpanzee lineage. As further evidence for the youth of this Alu subfamily, one PV Alu repeat is specific to Pan troglodytes, whereas others are present in Pan paniscus as well. The A-rich tails of these Alu repeats have different lengths in Pan paniscus and Pan troglodytes. The dimorphisms caused by the presence and absence of PV Alu repeats and the length polymorphisms attributed to their A-rich tails should provide valuable genetic markers for molecular-based studies of chimpanzee relationships. The existence of lineage-specific Alu repeats is a major sequence difference between human and chimpanzee DNAs.

Haemophilia B Due to a De Novo Insertion of a Human-Specific Alu Subfamily Member within the Coding Region of the Factor IX Gene

A de novo insertion of an Alu repeated DNA element was found within exon V of the factor IX gene in a patient with severe haemophilia B. The element interrupts the reading frame of the mature factor IX at glutamic acid 96 resulting in a stop codon within the inserted sequence. The Alu repeat is 322 bp long, and the 5' region is shortened by 38 bp. The insertion created a target site duplication of 15 bp consistent with retroposition, and contains a pure polyadenine tract of at least 78 resides at the 3' end. The nucleotide sequence agrees with a consensus for an Alu subfamily which is evolutionarily the most recently inserted, suggesting that it is an exact copy of a putative source gene. These observations indicate that retroposition of Alu elements is a continual process and a mechanism for generating human genetic defects.

Proposed roles for DNA methylation in Alu transcriptional repression and mutational inactivation.

Methylation at CpG dinucleotides to produce 5 methyl cytosine (5me-C) has been proposed to regulate the transcriptional expression of human Alu repeats. Similarly, methylation has been proposed to indirectly favor the transpositional activity of young Alu repeats by transcriptionally inactivating older Alu's through the very rapid transition of 5me-C to T. Both hypotheses are examined here by RNA polymerase III (Pol III) in vitro transcription of Alu templates using HeLa cell extracts. A limiting factor represses the template activity of methylated Alu repeats. Competition by methylated prokaryotic vector DNA's relieves repression, showing that the factor is not sequence specific. This competitor has no effect on the activity of unmethylated templates showing that the repressor is highly specific toward methylated DNA. While methylation of a single pair of CpG dinucleotides in the A box of the Poll III promoter is sufficient to cause repression, methylation elsewhere within the template also causes repression. The repressor causing these effects on the Pol III directed transcription of Alu repeats is thought to be a previously reported, repressor for Pol II directed templates. Young Alu repeats are transcriptionally more active templates than a representative older Alu subfamily member. Also, younger Alu's form stable transcriptional complexes faster, potentially giving them an additional advantage. The mutation of three CpG's to CpA's within and near the A box drastically decreases both the template activity and rate of stable complex formation by a young Alu member. The sensitivity of Alu template activity to CpG transitions within the A box partially explains the selective transpositional advantage enjoyed by young Alu members.

Alu RNA transcripts in human embryonal carcinoma cells: Model of post-transcriptional selection of master sequences

Alu master sequences colonized the human genome using RNA as amplification intermediate. To understand this phenomenon better we isolated and analyzed Alu RNA from NTera2D1 pluripotential cells. Northern hybridization, primer extension. cDNA cloning and sequencing data are congruent and demonstrate a low level of Alu specific transcription. These bona fide RNA Polymerase III Alu transcripts, although enriched in the cytoplasm, are not dominated by a single master species but rather originate from a variety of loci. However, when compared with the genomic average, or to repeats from RNA Polymerase II co-transcripts, they belong to the youngest group of Alu subfamilies ( p < 0.001) and have a higher content of intact CpG-dinucleotides. This suggests that Alu transcription is influenced both by mutations and the genomic context, and points to a possible role of DNA methylation in silencing the bulk of genomic repeats. Because of the heterogeneity of Alu transcripts a post-transcriptional selection mechanism recruiting Alu master sequences for retroposition is required. We propose that Alu RNA masters could have evolved as selfish satellites to a more complex retroposition system equipped with a reverse transcriptase activity and that their structure was conserved through “phenotypic” selection at the RNA level.