The genomic distribution of L1 elements: the role of insertion bias and natural selection

LINE-1 (L1) retrotransposons constitute the most successful family of retroelements in mammals and account for as much as 20% of mammalian DNA. L1 elements can be found in all genomic regions but they are far more abundant in AT-rich, gene-poor, and low-recombining regions of the genome. In addition, the sex chromosomes and some genes seem disproportionately enriched in L1 elements. Insertion bias and selective processes can both account for this biased distribution of L1 elements. L1 elements do not appear to insert randomly in the genome and this insertion bias can at least partially explain the genomic distribution of L1. The contrasted distribution of L1 and Alu elements suggests that postinsertional processes play a major role in shaping L1 distribution. The most likely mechanism is the loss of recently integrated L1 elements that are deleterious (negative selection) either because of disruption of gene function or their ability to mediate ectopic recombination. By comparison, the retention of L1 elements because of some positive effect is limited to a small fraction of the genome. Understanding the respective importance of insertion bias and selection will require a better knowledge of insertion mechanisms and the dynamics of L1 inserts in populations.     

The role of Alu repeat clusters as mediators of recurrent chromosomal aberrations in tumors

There is increasing evidence for the involvement of repetitive DNA sequences as facilitators of some of the recurrent chromosomal rearrangements observed in human tumors. The high densities of repetitive DNA, such as Alu elements, at some chromosomal translocation breakpoint regions has led to the suggestion that these sequences could provide hot spots for homologous recombination, and could mediate the translocation process and elevate the likelihood of other types of chromosomal rearrangements taking place. The Alu core sequence itself has been suggested to promote DNA strand exchange and genomic rearrangement, and it has striking sequence similarity to chi (which has been shown to stimulate recBCD-mediated recombination in Escherichia coli). Alu repeats have been shown to be involved in the generation of many constitutional gene mutations in meiotic cells, attributed to unequal homologous recombination and consequent deletions and/or duplication events. It has recently been demonstrated that similar deletion events can take place in neoplasia because several types of leukemia-associated chromosomal rearrangements frequently have submicroscopic deletions immediately adjacent to the translocation breakpoint regions. Significantly, these types of deletions appear to be more likely to take place when the regions subject to rearrangement contain a high density of Alu repeats. With the completion of the Human Genome Project, it will soon be possible to create more comprehensive maps of the distribution and densities of repetitive sequences, such as Alu, throughout the genome. Such maps will offer unique insights into the relative distribution of cancer translocation breakpoints and the localization of clusters of repetitive DNA.     

Nonrandom distribution of interspersed repeat elements in the BCR and ABL1 genes and its relation to breakpoint cluster regions

The Philadelphia translocation, t(9;22)(q34;q11), is the microscopically visible product of recombination between two genes, ABL1 on chromosome 9 and BCR on chromosome 22, and gives rise to a functional hybrid BCR-ABL1 gene with demonstrated leukemogenic properties. Breakpoints in BCR occur mostly within one of two regions: a 5 kb major breakpoint cluster region (M-Bcr) and a larger 35 kb minor breakpoint cluster region (m-Bcr) towards the 3' end of the first BCR intron. By contrast, breakpoints in ABL1 are reported to occur more widely across a >200 kb region which spans the large first and second introns. The mechanisms that determine preferential breakage sites in BCR, and which cause recombination between BCR and ABL1, are presently unknown. In some cases, Alu repeats have been identified at or near sequenced breakpoint sites in both genes, providing indications, albeit controversial, that they may be relevant. For the present study, we carried out a detailed analysis of genomic BCR and ABL1 sequences to identify, classify, and locate interspersed repeat sequences and to relate their distribution to precisely mapped BCR-ABL1 recombination sites. Our findings confirm that Alu are the most abundant class of repeat in both genes, but that they occupy fewer sites than previously estimated and that they are distributed nonrandomly. r-Scan statistics were applied to provide a measure of repeat distribution and to evaluate extremes in repeat spacing. A significant lack of Alu elements was observed across the major and minor breakpoint cluster regions of BCR and across a 25-kb region showing a high frequency of breakage in ABL1. These findings counter the suggestion that occurrence of Alu at BCR-ABL1 recombination sites is likely by chance because of the high density of Alu in these two genes. Instead, as yet unidentified DNA conformation or nucleotide characteristics peculiar to the preferentially recombining regions, including those Alu elements present within them, more likely influence their fragility.     

Genomic deletions and precise removal of transposable elements mediated by short identical DNA segments in primates

Insertion of transposable elements is a major cause of genomic expansion in eukaryotes. Less is understood, however, about mechanisms underlying contraction of genomes. In this study, we show that retroelements can, in rare cases, be precisely deleted from primate genomes, most likely via recombination between 10- to 20-bp target site duplications (TSDs) flanking the retroelement. The deleted loci are indistinguishable from pre-integration sites, effectively reversing the insertion. Through human-chimpanzee-Rhesus monkey genomic comparisons, we estimate that 0.5%-1% of apparent retroelement "insertions" distinguishing humans and chimpanzees actually represent deletions. Furthermore, we demonstrate that 19% of genomic deletions of 200-500 bp that have occurred since the human-chimpanzee divergence are associated with flanking identical repeats of at least 10 bp. A large number of deletions internal to Alu elements were also found flanked by homologies. These results suggest that illegitimate recombination between short direct repeats has played a significant role in human genome evolution. Moreover, this study lends perspective to the view that insertions of retroelements represent unidirectional genetic events.     

Shuffling of genes within low-copy repeats on 22q11 (LCR22) by Alu-mediated recombination events during evolution

Low-copy repeats, or segmental duplications, are highly dynamic regions in the genome. The low-copy repeats on chromosome 22q11.2 (LCR22) are a complex mosaic of genes and pseudogenes formed by duplication processes; they mediate chromosome rearrangements associated with velo-cardio-facial syndrome/DiGeorge syndrome, der(22) syndrome, and cat-eye syndrome. The ability to trace the substrates and products of recombination events provides a unique opportunity to identify the mechanisms responsible for shaping LCR22s. We examined the genomic sequence of known LCR22 genes and their duplicated derivatives. We found Alu (SINE) elements at the breakpoints in the substrates and at the junctions in the truncated products of recombination for USP18, GGT, and GGTLA, consistent with Alu-mediated unequal crossing-over events. In addition, we were able to trace a likely interchromosomal Alu-mediated fusion between IGSF3 on 1p13.1 and GGT on 22q11.2. Breakpoints occurred inside Alu elements as well as in the 5' or 3' ends of them. A possible stimulus for the 5' or 3' terminal rearrangements may be the high sequence similarities between different Alu elements, combined with a potential recombinogenic role of retrotransposon target-site duplications flanking the Alu element, containing potentially kinkable DNA sites. Such sites may represent focal points for recombination. Thus, genome shuffling by Alu-mediated rearrangements has contributed to genome architecture during primate evolution.     

Mammalian retroelements

The eukaryotic genome has undergone a series of epidemics of amplification of mobile elements that have resulted in most eukaryotic genomes containing much more of this 'junk' DNA than actual coding DNA. The majority of these elements utilize an RNA intermediate and are termed retroelements. Most of these retroelements appear to amplify in evolutionary waves that insert in the genome and then gradually diverge. In humans, almost half of the genome is recognizably derived from retroelements, with the two elements that are currently actively amplifying, L1 and Alu, making up about 25% of the genome and contributing extensively to disease. The mechanisms of this amplification process are beginning to be understood, although there are still more questions than answers. Insertion of new retroelements may directly damage the genome, and the presence of multiple copies of these elements throughout the genome has longer-term influences on recombination events in the genome and more subtle influences on gene expression.     

Alu repeats and human disease.

Alu elements have amplified in primate genomes through a RNA-dependent mechanism, termed retroposition, and have reached a copy number in excess of 500,000 copies per human genome. These elements have been proposed to have a number of functions in the human genome, and have certainly had a major impact on genomic architecture. Alu elements continue to amplify at a rate of about one insertion every 200 new births. We have found 16 examples of diseases caused by the insertion of Alu elements, suggesting that they may contribute to about 0.1% of human genetic disorders by this mechanism. The large number of Alu elements within primate genomes also provides abundant opportunities for unequal homologous recombination events. These events often occur intrachromosomally, resulting in deletion or duplication of exons in a gene, but they also can occur interchromosomally, causing more complex chromosomal abnormalities. We have found 33 cases of germ-line genetic diseases and 16 cases of cancer caused by unequal homologous recombination between Alu repeats. We estimate that this mode of mutagenesis accounts for another 0.3% of human genetic diseases. Between these different mechanisms, Alu elements have not only contributed a great deal to the evolution of the genome but also continue to contribute to a significant portion of human genetic diseases.     

Long-range sequence composition mirrors linkage disequilibrium pattern in a 1.13 Mb region of human chromosome 22.

Association studies, the most powerful tool for the identification of genes underlying complex traits, depend on the observation of linkage disequilibrium (LD) between marker alleles and the trait. The LD pattern of the human genome which determines the regional density of required markers is non-uniform, with regions of long-range LD over several hundred kilobases and regions where LD extends only over a few kilobases. Studying LD in the NF1 gene region we encountered a transition from long-range to short-range LD which coincides with a switch in the isochore pattern. This observation prompted us to investigate the regional variation in the extent of LD more systematically and we selected an isochore transition within the MN1/PITPNB gene region on chromosome 22q12.1. Long-range LD characterizes the GC-poor (40% GC) parts of the sequences. No LD can be observed between closely spaced markers throughout the whole range of the GC-rich (50% GC) parts. In both cases, the NF1 and the MN1/PITPNB gene region, a clear-cut transition of the long-range GC content precisely coincides with a change in the extent of observable LD. The results can be explained by a 72-fold lower recombination frequency in the GC-poor, compared to the GC-rich isochores. Although recombination is not the only factor governing LD, our findings can help to predict levels of LD and marker densities required for association studies on the basis of regional GC content.     

Retrotransposons and their recognition of pol II promoters: a comprehensive survey of the transposable elements from the complete genome sequence of Schizosaccharomyces pombe

The complete DNA sequence of the genome of Schizosaccharomyces pombe provides the opportunity to investigate the entire complement of transposable elements (TEs), their association with specific sequences, their chromosomal distribution, and their evolution. Using homology-based sequence identification, we found that the sequenced strain of S. pombe contained only one family of full-length transposons. This family, Tf2, consisted of 13 full-length copies of a long terminal repeat (LTR) retrotransposon. We found that LTR-LTR recombination of previously existing transposons had resulted in extensive populations of solo LTRs. These included 35 solo LTRs of Tf2, as well as 139 solo LTRs from other Tf families. Phylogenetic analysis of solo Tf LTRs reveals that Tf1 and Tf2 were the most recently active elements within the genome. The solo LTRs also served as footprints for previous insertion events by the Tf retrotransposons. Analysis of 186 genomic insertion events revealed a close association with RNA polymerase II promoters. These insertions clustered in the promoter-proximal regions of genes, upstream of protein coding regions by 100 to 400 nucleotides. The association of Tf insertions with pol II promoters was very similar to the preference previously observed for Tf1 integration. We found that the recently active Tf elements were absent from centromeres and pericentromeric regions of the genome containing tandem tRNA gene clusters. In addition, our analysis revealed that chromosome III has twice the density of insertion events compared to the other two chromosomes. Finally we describe a novel repetitive sequence, wtf, which was also preferentially located on chromosome III, and was often located near solo LTRs of Tf elements.     

Recombination rate and the distribution of transposable elements in the Drosophila melanogaster genome

We analyzed the distribution of 54 families of transposable elements (TEs; transposons, LTR retrotransposons, and non-LTR retrotransposons) in the chromosomes of Drosophila melanogaster, using data from the sequenced genome. The density of LTR and non-LTR retrotransposons (RNA-based elements) was high in regions with low recombination rates, but there was no clear tendency to parallel the recombination rate. However, the density of transposons (DNA-based elements) was significantly negatively correlated with recombination rate. The accumulation of TEs in regions of reduced recombination rate is compatible with selection acting against TEs, as selection is expected to be weaker in regions with lower recombination. The differences in the relationship between recombination rate and TE density that exist between chromosome arms suggest that TE distribution depends on specific characteristics of the chromosomes (chromatin structure, distribution of other sequences), the TEs themselves (transposition mechanism), and the species (reproductive system, effective population size, etc.), that have differing influences on the effect of natural selection acting against the TE insertions.     

cDNA-based gene mapping and GC3 profiling in the soft-shelled turtle suggest a chromosomal size-dependent GC bias shared by sauropsids

Mammalian and avian genomes comprise several classes of chromosomal segments that vary dramatically in GC-content. Especially in chicken, microchromosomes exhibit a higher GC-content and a higher gene density than macrochromosomes. To understand the evolutionary history of the intra-genome GC heterogeneity in amniotes, it is necessary to examine the equivalence of this GC heterogeneity at the nucleotide level between these animals including reptiles, from which birds diverged. We isolated cDNAs for 39 protein-coding genes from the Chinese soft-shelled turtle, Pelodiscus sinensis, and performed chromosome mapping of 31 genes. The GC-content of exonic third positions (GC₃) of P. sinensis genes showed a heterogeneous distribution, and exhibited a significant positive correlation with that of chicken and human orthologs, indicating that the last common ancestor of extant amniotes had already established a GC-compartmentalized genomic structure. Furthermore, chromosome mapping in P. sinensis revealed that microchromosomes tend to contain more GC-rich genes than GC-poor genes, as in chicken. These results illustrate two modes of genome evolution in amniotes: mammals elaborated the genomic configuration in which GC-rich and GC-poor regions coexist in individual chromosomes, whereas sauropsids (reptiles and birds) refined the chromosomal size-dependent GC compartmentalization in which GC-rich genomic fractions tend to be confined to microchromosomes.     

Organization and Evolution of a Gene-Rich Region of the Mouse Genome: A 12.7-Mb Region Deleted in the Del(13)Svea36H Mouse

Del(13)Svea36H (Del36H) is a deletion of ~20% of mouse chromosome 13 showing conserved synteny with human chromosome 6p22.1-6p22.3/6p25. The human region is lost in some deletion syndromes and is the site of several disease loci. Heterozygous Del36H mice show numerous phenotypes and may model aspects of human genetic disease. We describe 12.7 Mb of finished, annotated sequence from Del36H. Del36H has a higher gene density than the draft mouse genome, reflecting high local densities of three gene families (vomeronasal receptors, serpins, and prolactins) which are greatly expanded relative to human. Transposable elements are concentrated near these gene families. We therefore suggest that their neighborhoods are gene factories, regions of frequent recombination in which gene duplication is more frequent. The gene families show different proportions of pseudogenes, likely reflecting different strengths of purifying selection and/or gene conversion. They are also associated with relatively low simple sequence concentrations, which vary across the region with a periodicity of ~5 Mb. Del36H contains numerous evolutionarily conserved regions (ECRs). Many lie in noncoding regions, are detectable in species as distant as Ciona intestinalis, and therefore are candidate regulatory sequences. This analysis will facilitate functional genomic analysis of Del36H and provides insights into mouse genome evolution.     

Periodic explosive expansion of human retroelements associated with the evolution of the hominoid primate

Five retroelement families, L1 and L2 (long interspersed nuclear element, LINE), Alu and MIR (short interspersed nuclear element, SINE), and LTR (long terminal repeat), comprise almost half of the human genome. This genome-wide analysis on the time-scaled expansion of retroelements sheds light on the chronologically synchronous amplification peaks of each retroelement family in variable heights across human chromosomes. Especially, L1s and LTRs in the highest density on sex chromosomes Xq and Y, respectively, disclose peak activities that are obscured in autosomes. The periods of young L1, Alu, LTR, and old L1 peak activities calibrated based on sequence divergence coincide with the divergence of the three major hominoid divergence as well as early eutherian radiation while the amplification peaks of old MIR and L2 account for the marsupial-placental split. Overall, the peaks of autonomous LINE (young and old L1s and L2s) peaks and non-autonomous SINE (Alus and MIRs) have alternated repeatedly for 150 million years. In addition, a single burst of LTR parallels the Cretaceous-Tertiary (K-T) boundary, an exceptional global event. These findings suggest that the periodic explosive expansions of LINEs and SINEs and an exceptional burst of LTR comprise the genome dynamics underlying the macroevolution of the hominoid primate lineage.     

Covariation in frequencies of substitution,deletion, transposition,and recombination during eutherian evolution

Six measures of evolutionary change in the human genome were studied, three derived from the aligned human and mouse genomes in conjunction with the Mouse Genome Sequencing Consortium, consisting of (1) nucleotide substitution per fourfold degenerate site in coding regions, (2) nucleotide substitution per site in relics of transposable elements active only before the human-mouse speciation, and (3) the nonaligning fraction of human DNA that is nonrepetitive or in ancestral repeats; and three derived from human genome data alone, consisting of (4) SNP density, (5) frequency of insertion of transposable elements, and (6) rate of recombination. Features 1 and 2 are measures of nucleotide substitutions at two classes of "neutral" sites, whereas 4 is a measure of recent mutations. Feature 3 is a measure dominated by deletions in mouse, whereas 5 represents insertions in human. It was found that all six vary significantly in megabase-sized regions genome-wide, and many vary together. This indicates that some regions of a genome change slowly by all processes that alter DNA, and others change faster. Regional variation in all processes is correlated with, but not completely accounted for, by GC content in human and the difference between GC content in human and mouse.     

Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements

The reported human genome sequence includes about 400 gaps of unknown sequence that were not found in the bacterial artificial chromosome (BAC) and cosmid libraries used for sequencing of the genome. These missing sequences correspond to approximately 1% of euchromatic regions of the human genome. Gap filling is a laborious process because it relies on analysis of random clones of numerous genomic BAC or cosmid libraries. In this work we demonstrate that closing the gaps can be accelerated by a selective recombinational capture of missing chromosomal segments in yeast. The use of both methodologies allowed us to close the four remaining gaps on the human chromosome 19. Analysis of the gap sequences revealed that they contain several abnormalities that could result in instability of the sequences in microbe hosts, including large blocks of micro- and minisatellites and a high density of Alu repeats. Sequencing of the gap regions, in both BAC and YAC forms, allowed us to generate a complete sequence of four genes, including the neuronal cell signaling gene SCK1/SLI. The SCK1/SLI gene contains a record number of minisatellites, most of which are polymorphic and transmitted through meiosis following a Mendelian inheritance. In conclusion, the use of the alternative recombinational cloning system in yeast may greatly accelerate work on closing the remaining gaps in the human genome (as well as in other complex genomes) to achieve the goal of annotation of all human genes.     

Exceptionally high levels of recombination across the honey bee genome

The first draft of the honey bee genome sequence and improved genetic maps are utilized to analyze a genome displaying 10 times higher levels of recombination (19 cM/Mb) than previously analyzed genomes of higher eukaryotes. The exceptionally high recombination rate is distributed genome-wide, but varies by two orders of magnitude. Analysis of chromosome, sequence, and gene parameters with respect to recombination showed that local recombination rate is associated with distance to the telomere, GC content, and the number of simple repeats as described for low-recombining genomes. Recombination rate does not decrease with chromosome size. On average 5.7 recombination events per chromosome pair per meiosis are found in the honey bee genome. This contrasts with a wide range of taxa that have a uniform recombination frequency of about 1.6 per chromosome pair. The excess of recombination activity does not support a mechanistic role of recombination in stabilizing pairs of homologous chromosome during chromosome pairing. Recombination rate is associated with gene size, suggesting that introns are larger in regions of low recombination and may improve the efficacy of selection in these regions. Very few transposons and no retrotransposons are present in the high-recombining genome. We propose evolutionary explanations for the exceptionally high genome-wide recombination rate.