Short tandem-repeat polymorphism/alu haplotype variation at the PLAT locus: implications for modern human origins

Two dinucleotide short tandem-repeat polymorphisms (STRPs) and a polymorphic Alu element spanning a 22-kb region of the PLAT locus on chromosome 8p12-q11.2 were typed in 1,287-1,420 individuals originating from 30 geographically diverse human populations, as well as in 29 great apes. These data were analyzed as haplotypes consisting of each of the dinucleotide repeats and the flanking Alu insertion/deletion polymorphism. The global pattern of STRP/Alu haplotype variation and linkage disequilibrium (LD) is informative for the reconstruction of human evolutionary history. Sub-Saharan African populations have high levels of haplotype diversity within and between populations, relative to non-Africans, and have highly divergent patterns of LD. Non-African populations have both a subset of the haplotype diversity present in Africa and a distinct pattern of LD. The pattern of haplotype variation and LD observed at the PLAT locus suggests a recent common ancestry of non-African populations, from a small population originating in eastern Africa. These data indicate that, throughout much of modern human history, sub-Saharan Africa has maintained both a large effective population size and a high level of population substructure. Additionally, Papua New Guinean and Micronesian populations have rare haplotypes observed otherwise only in African populations, suggesting ancient gene flow from Africa into Papua New Guinea, as well as gene flow between Melanesian and Micronesian populations.     

Impact of copy number of distinct SV40PolyA segments on expression of a GFP reporter gene

The presence of Alu repeats downregulates the expression of the green fluorescent protein(GFP) gene.We found that SV40PolyA(PolyA,240 bp),in either orientation,eliminated the inhibition of GFP gene expression induced by Alu repeats when it was placed between the GFP gene and the Alu repeats.In this study,4 different segments(each 60 bp) were amplified from antisense PolyA(PolyAas) by PCR,and inserted upstream of Alu14 in pAlu14 plasmid(14 Alu repeats inserted downstream of the GFP gene in vector pEGFP-C1 in...     

A high frequency of length polymorphisms in repeated sequences adjacent to Alu sequences

We describe a new class of DNA length polymorphism that is due to a variation in the number of tandem repeats associated with Alu sequences (Alu sequence-related polymorphisms). The polymerase chain reaction was used to selectively amplify a (TTA)n repeat identified in the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from genomic DNA of 41 human subjects, and the size of the amplified products was determined by gel electrophoresis. Seven alleles were found that differed in size by integrals of three nucleotides. The allele frequencies ranged from 1.5% to 52%, and the overall heterozygosity index was 62%. The polymorphic TTA repeat was located adjacent to a repetitive sequence of the Alu family. A homology search of human genomic DNA sequences for the trinucleotide TTA (at least five members in length) revealed tandem repeats in six other genes. Three of the six (TTA)n repeats were located adjacent to Alu sequences, and two of the three (in the genes for beta-tubulin and interleukin-1 alpha) were found to be polymorphic in length. Tandemly repetitive sequences found in association with Alu sequences may be frequent sites of length polymorphism that can be used as genetic markers for gene mapping or linkage analysis.     

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.     

Association of some potential hormone response elements in human genes with the Alu family repeats.

Short interspersed repeats of the Alu family located in promoters of some human genes contain high-affinity binding sites for thyroid hormone receptor, retinoic acid receptor and estrogen receptor. The standard binding sites for the receptors represent variants of duplicated AGGTCA motif with different spacing and orientation (direct, DR, or inverted, IR), and Alu sequences were found to have functional DR-4, DR-2 or variant IR-3/IR-17 elements. In this study we analyzed distribution and abundance of the elements in a set of human genomic sequences from GenBank and their association with Alu repeats. Our results indicate that a major fraction of potentially active DR-4, DR-2 and variant IR-3/IR-17 elements in the genes is located within Alu repeats. Alu-associated DR-2 elements are conserved in primate evolution. However, very few Alu have potential DR-3 glucocorticoid-response elements. Gel-shift experiments with the probe (AUB) corresponding to the consensus Alu sequence just upstream of the RNA polymerase III promoter B-box and containing duplicated AGGTCA motif indicate that the probe interacts in a sequence-specific manner with human nuclear proteins which bind to standard IR-0, DR-1, DR-4 or DR-5 elements. The AUB sequence was also able to promote thyroid hormone-dependent trans-activation of a reporter gene. The results support the view that Alu retroposons played an important role in evolution of regulation of the primate gene expression by nuclear hormone receptors.     

Junctions between genes in the haptoglobin gene cluster of primates.

To investigate the nature of the recombination that generated the haptoglobin three-gene cluster in Old World primates, we sequenced the region between the second gene (HPR) and the third gene (HPP) in chimpanzees (15 kb), as well as the region 3' to the cluster in humans (14 kb). Comparison to the previously sequenced human haptoglobin (HP) and HPR genes showed that the junction point between HP and HPR in humans (junction 1) was not identical to the junction point between the HPR and HPP genes of the chimpanzee (junction 2). An Alu sequence was found at each junction, but both Alu sequences lacked short direct repeats of the flanking genomic DNA. The lack of direct repeats implies that both junction Alu sequences are the products of recombination between different Alu elements. In addition, other insertion and deletion events are clustered in the regions near the junction Alu sequences. The observation that Alu sequences define the junctions between genes in the haptoglobin gene cluster emphasizes the importance of Alu sequences in the evolution of multigene families.     

Identification and characterization of two critical sequences in SV40PolyA that activate the green fluorescent protein reporter gene

Alu repeats or Line-1-ORF2 (ORF2) inhibit expression of the green fluorescent protein (GFP) gene when inserted downstream of this gene in the vector pEGFP-C1. In this work, we studied cis-acting elements that eliminated the repression of GFP gene expression induced by Alu and ORF2 and sequence characteristics of these elements. We found that sense and antisense PolyA of simian virus 40 (SV40PolyA, 240 bp) eliminated the repression of GFP gene expression when inserted between the GFP gene and the Alu (283 bp) repeats or ORF2 (3825 bp) in pAlu14 (14 tandem Alu repeats were inserted downstream of the GFP gene in the vector pEGFP-C1) or pORF2. Antisense SV40PolyA (PolyAas) induced stronger gene expression than its sense orientation (PolyA). Of four 60-bp segments of PolyAas (1F1R, 2F2R, 3F3R and 4F4R) inserted independently into pAlu14, only two (2F2R and 3F3R) eliminated the inhibition of GFP gene expression induced by Alu repeats. Deletion analysis revealed that a 17 nucleotide AT repeat (17ntAT; 5'-AAAAAAATGCTTTATTT-3') in 2F2R and the fragment 3F38d9 (5'-ATAAACAAGTTAACAACA ACAATTGCATT-3') in 3F3R were critical sequences for activating the GFP gene. Sequence and structural analyses showed that 17ntAT and 3F38d9 included imperfect palindromes and may form a variety of unstable stem-loops. We suggest that the presence of imperfect palindromes and unstable stem-loops in DNA enhancer elements plays an important role in GFP gene activation.     

Enrichment for histone H3 lysine 9 methylation at Alu repeats in human cells

The aim of this study was to identify in human cells common targets of histone H3 lysine 9 (H3-Lys9) methylation, a modification that is generally associated with gene silencing. After chromatin immunoprecipitation using an H3-Lys9 methylated antibody, we cloned the recovered DNA and sequenced 47 independent clones. Of these, 38 clones (81%) contained repetitive elements, either short interspersed transposable element (SINE or Alu elements), long terminal repeat (LTR), long interspersed transposable element (LINE), or satellite region (ALR/Alpha) DNA, and three additional clones were near Alu elements. Further characterization of these repetitive elements revealed that 32 clones (68%) were Alu repeats, corresponding to both old Alu (23 clones) and young Alu (9 clones) subfamilies. Association of H3-Lys9 methylation was confirmed by chromatin immunoprecipitation-PCR using conserved Alu primers. In addition, we randomly selected 5 Alu repeats from the recovered clones and confirmed association with H3-Lys9 by PCR using primer sets flanking the Alu elements. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly decreased the level of H3-Lys9 methylation in the Alu elements, suggesting that H3-Lys9 methylation may be related to the suppression of Alu elements through DNA methylation. Thus H3-Lys9 methylation is enriched at human repetitive elements, particularly Alu elements, and may play a role in the suppression of recombination by these elements.     

Sequence composition similarities with the 7SL RNA are highly predictive of functional genomic features

Transposable elements derived from the 7SL RNA gene, such as Alu elements in primates, have had remarkable success in several mammalian lineages. The results presented here show a broad spectrum of functions for genomic segments that display sequence composition similarities with the 7SL RNA gene. Using thoroughly documented loci, we report that DNaseI-hypersensitive sites can be singled out in large genomic sequences by an assessment of sequence composition similarities with the 7SL RNA gene. We apply a root word frequency approach to illustrate a distinctive relationship between the sequence of the 7SL RNA gene and several classes of functional genomic features that are not presumed to be of transposable origin. Transposable elements that show noticeable similarities with the 7SL sequence include Alu sequences, as expected, but also long terminal repeats and the 5′-untranslated regions of long interspersed repetitive elements. In sequences masked for repeated elements, we find, when using the 7SL RNA gene as query sequence, distinctive similarities with promoters, exons and distal gene regulatory regions. The latter being the most notoriously difficult to detect, this approach may be useful for finding genomic segments that have regulatory functions and that may have escaped detection by existing methods.     

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.     

An unusual Alu repeat sequence within the CAD gene

Summary There are several hundred thousand members of the Alu repeat family in the human genome. Those Alu elements sequenced to date appear to fit into subfamilies. A novel Alu has been found in an intron of the human CAD gene: it appears to be due to rearrangement between Alu repeats belonging to two different subfamilies. Further sequence data from this intron suggest that the Alu element may have rearranged prior to its entry into the CAD gene. Such findings indicate that, in addition to single nucleotide substitutions and deletions, DNA rearrangments may be a factor in generating the diversity of Alu repeats found in primate genomes.     

AluElements: Repetitive DNA as Facilitators of Chromosomal Rearrangement

Alu repeats are the most common type of repetitive DNA sequences dispersed throughout the human genome. Technical advances in the field of cytogenetics and molecular biology have facilitated the analysis of epithelial tumors and hematologic malignancies which has led to the observation of Alu elements in and near sites often involved in chromosomal rearrangements. Repair mechanisms of double strand breaks (DSB) such as homol-ogous recombination (HR) may rely on the sequence homology of Alu repeats, potentially leading to chromosomal rearrange-ments. Databases have confirmed the strong association between Alu repeats, specifically the 26 bp consensus sequence and chro-mosomal regions involved in deletions and translocations. Although the Alu repetitive sequence is a potential "hotspot" during homologous recombination, there are other cellular mech-anisms that may play a more prominent role in the initiation of chromosomal rearrangements.     

Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.     

Using Alu J Elements as Molecular Clocks to Trace the Evolutionary Relationships Between Duplicated HLA Class I Genomic Segments

The class I region of the major histocompatibility complex contains two subgenomic blocks (250–350 kb each), known as the alpha and beta blocks. These blocks contain members of multicopy gene families including HLA class I, HERV-16 (previously called P5 sequences), and PERB11 (MIC). We have previously shown that each block consists of imperfect duplicated segments (duplicons) containing linked members of different gene families, retroelements and transposons that have coevolved as part of two separate evolutionary events. Another region provisionally designated here as the kappa block is located between the alpha and the beta blocks and contains HLA-E, -30, and -92, HERV-16 (P5.3), and PERB11.3 (MICC) within about 250 kb of sequence. Using Alu elements to trace the evolutionary relationships between different class I duplicons, we have found that (a) the kappa block contains paralogous (duplicated) Alu J sequences and other retroelement patterns more in common with the beta than the alpha block; (b) the retroelement pattern associated with the HLA-E duplicon is different from all other HLA class I duplicons, indicating a more complex evolution; (c) the HLA-92 duplicon, although substantially shorter, is closely related in sequence to the HLA-B and -C duplicons; (d) two of the six paralogous Alu J elements within the HLA-B and -C duplicons are associated with the HLA-X duplicon, confirming their evolutionary relationships within the beta block; and (e) the paralogous Alu J elements within the alpha block are distinctly different from those identified within the beta and kappa blocks. The sequence conservation and location of duplicated (paralogous) Alu J elements in the MHC class I region show that the beta and kappa blocks have evolved separately from the alpha block beginning at a time before or during the evolution of Alu J elements in primates. Received: 22 September 1999 / Accepted: 24 January 2000     

Invasion of the human albumin-alpha-fetoprotein gene family by Alu, Kpn, and two novel repetitive DNA elements

The human albumin-alpha-fetoprotein genomic domain contains 13 repetitive DNA elements randomly distributed throughout the symmetrical structures of these genes. These repeated sequences are located at different sites within the two genes. The human albumin gene contains five Alu elements within four of its 14 intervening sequences. Two of these repeats are located in intron 2, and the remaining three are located in introns 7, 8, and 11. The human alpha-fetoprotein gene contains three of these Alu elements, one in intron 4 and the remaining two in the 3'-untranslated region. In addition, the human alpha-fetoprotein gene contains a Kpn repeat and two classes of novel repeats that are absent from the human albumin gene. Six of the Alu elements within the two genes are bound by short direct repeats that harbor five base substitutions in 120 possible positions (60 bp times 2 termini). The absence of Alu repeats from analogous positions in rodents indicates that these repeats invaded the albumin-alpha-fetoprotein domain less than 85 Myr ago (the time of mammalian radiation). Furthermore, considering the conservation of terminal repeats flanking the Alu sequences of the albumin-alpha-fetoprotein domain (0.042 changes per site), we submit that the average time of Alu insertion into this gene family could have been as recently as 15-30 Myr ago.     

Recombinational resolution in primate cells of two homologous human DNA segments with a gradient of sequence divergence.

Human alpha-thalassemia-2 genotype -alpha 4.2 is the result of meiotic recombination between two 1.3 kb long, homologous DNA segments, X(alpha 2) and X(alpha 1), located in the adult alpha globin locus. The two segments can also undergo intramolecular recombination on extrachromosomal vectors transfected into mitotically dividing primate cells (COS 7). The existence of a gradient of sequence divergence between X(alpha 2) and X(alpha 1) makes them an interesting system to study the relationship between efficiencies of homologous DNA recombination and the extent of dispersed and localized base mismatches. By partial restriction mapping and DNA sequencing of plasmids recombined in COS 7 cells and rescued from bacteria HB 101, we have determined the distribution of recombinational resolution sites along the two X blocks. Most, if not all, of the homologous recombination events between the two X blocks appear to be single crossing-over without efficient gene correction or repair of base mismatches. The distribution of the sites of recombinational resolution is inversely correlated with that of the gradient of sequence divergence, with only approximately 7% of the X recombinants resolved within the 3' third of the X blocks where two diverged Alu family repeats reside. The Alu sequence within which one of the X recombinants resolved is homologous to a previously characterized alpha thalassemia deletion point.