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.     

Homologous recombination-mediated irreversible genome damage underlies telomere-induced senescence

Loss of telomeric DNA leads to telomere uncapping, which triggers a persistent, p53-centric DNA damage response that sustains a stable senescence-associated proliferation arrest. Here, we show that in normal cells telomere uncapping triggers a focal telomeric DNA damage response accompanied by a transient cell cycle arrest. Subsequent cell division with dysfunctional telomeres resulted in sporadic telomeric sister chromatid fusions that gave rise to next-mitosis genome instability, including non-telomeric DNA lesions responsible for a stable, p53-mediated, senescence-associated proliferation arrest. Unexpectedly, the blocking of Rad51/RPA-mediated homologous recombination, but not non-homologous end joining (NHEJ), prevented senescence despite multiple dysfunctional telomeres. When cells approached natural replicative senescence, interphase senescent cells displayed genome instability, whereas near-senescent cells that underwent mitosis despite the presence of uncapped telomeres did not. This suggests that these near-senescent cells had not yet acquired irreversible telomeric fusions. We propose a new model for telomere-initiated senescence where tolerance of telomere uncapping eventually results in irreversible non-telomeric DNA lesions leading to stable senescence. Paradoxically, our work reveals that senescence-associated tumor suppression from telomere shortening requires irreversible genome instability at the single-cell level, which suggests that interventions to repair telomeres in the pre-senescent state could prevent senescence and genome instability.     

Alu insertion profiling: array-based methods to detect Alu insertions in the human genome

The analysis of the genetic variability associated to Alu sequences was hampered by the absence of genome-wide methodologies able to efficiently detect new polymorphisms/mutations among these repetitive elements. Here we describe two Alu insertion profiling (AIP) methods based on the hybridization of Alu-flanking genomic fragments on tiling microarrays. Protocols are designed to preferentially detect active Alu subfamilies. We tested AIP methods by analyzing chromosomes 1 and 6 in two genomic samples. In genomic regions covered by array-features, with a sensitivity of 2% (AIP1) -4% (AIP2) and 5% (AIP1) -8% (AIP2) for the old J and S Alu lineages respectively, we obtained a sensitivity of 67% (AIP1) -90% (AIP2) for the young Ya subfamily. Among the loci showing sample-to-sample differences, 5 (AIP1) -8 (AIP2) were associated to known Alu polymorphisms. Moreover, we were able to confirm by PCR and DNA sequencing 4 new intragenic Alu elements, polymorphic in 10 additional individuals.     

Moving primate genomics beyond the chimpanzee genome

The comparative DNA sequence data that already exist on individual genomic loci depict the phylogenetic relationships of nearly all extant primate genera. Such a phylogenetic representation of the primates, validated by many sequenced primate genomes, and encompassing the full adaptive diversity of the order, is a prerequisite for identifying the genetic basis of humankind, and for testing the proposed human uniqueness of these traits. Some of these traits have been discovered recently, particularly in genes encoding proteins that are important for brain function.     

A map of the common chimpanzee genome

The completion of the chimpanzee genome will greatly help us determine which genetic changes are unique to humanity. Chimpanzees are our closest living relative, and a recent study has made considerable progress towards decoding the genome of our sister taxon.1 Over 75,000 common chimpanzee (Pan troglodytes) bacterial artificial chromosome end sequences were aligned and mapped to the human genome. This study shows the remarkable genetic similarity (98.77%) between humans and chimpanzees, while highlighting intriguing areas of potential difference. If we wish to understand the genetic basis of humankind, the completion of the chimpanzee genome deserves high priority.     

Human genomic deletions mediated by recombination between Alu elements

Recombination between Alu elements results in genomic deletions associated with many human genetic disorders. Here, we compare the reference human and chimpanzee genomes to determine the magnitude of this recombination process in the human lineage since the human-chimpanzee divergence approximately 6 million years ago. Combining computational data mining and wet-bench experimental verification, we identified 492 human-specific deletions (for a total of approximately 400 kb) attributable to this process, a significant component of the insertion/deletion spectrum of the human genome. The majority of the deletions (295 of 492) coincide with known or predicted genes (including 3 that deleted functional exons, as compared with orthologous chimpanzee genes), which implicates this process in creating a substantial portion of the genomic differences between humans and chimpanzees. Overall, we found that Alu recombination-mediated genomic deletion has had a much higher impact than was inferred from previously identified isolated events and that it continues to contribute to the dynamic nature of the human genome.     

Clustering and subfamily relationships of the Alu family in the human genome

Thirteen and 10 sequences of the Alu family of repeated DNA elements found within the human thymidine kinase and beta-tubulin genes, respectively, were compared. These genes have approximately five times the expected density of Alu family members. The consensus sequence that could be drawn from these 23 Alu family members would differ slightly from others drawn from random Alu family sequences but only at very heterogeneous positions. The different Alu family members do show different pairwise percentage identities, with approximately 15% (7 of 48 Alu family members analyzed) of them clearly representing a separate subfamily of sequences. This analysis also confirms the species- specific differences between human and the prosimian Galago crassicaudatus Alu family members. These data are consistent with both the origin of these sequences in primates less than 65-70 Myr ago and amplification since that time to their present 500,000 copies. The data do not show any special relationships among densely clustered Alu family members.      

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.     

Inverted Alu repeats unstable in yeast are excluded from the human genome

The nearly one million ALU: repeats in human chromosomes are a potential threat to genome integrity. ALU:s form dense clusters where they frequently appear as inverted repeats, a sequence motif known to cause DNA rearrangements in model organisms. Using a yeast recombination system, we found that inverted ALU: pairs can be strong initiators of genetic instability. The highly recombinagenic potential of inverted ALU: pairs was dependent on the distance between the repeats and the level of sequence divergence. Even inverted ALU:s that were 86% homologous could efficiently stimulate recombination when separated by <20 bp. This stimulation was independent of mismatch repair. Mutations in the DNA metabolic genes RAD27 (FEN1), POL3 (polymerase delta) and MMS19 destabilized widely separated and diverged inverted ALU:s. Having defined factors affecting inverted ALU: repeat stability in yeast, we analyzed the distribution of ALU: pairs in the human genome. Closely spaced, highly homologous inverted ALU:s are rare, suggesting that they are unstable in humans. ALU: pairs were identified that are potential sites of genetic change.     

A SINE in the genome of the cephalochordate amphioxus is an Alu element

Transposable elements of about 300 bp, termed "short interspersed nucleotide elements or SINEs are common in eukaryotes. However, Alu elements, SINEs containing restriction sites for the AluI enzyme, have been known only from primates. Here I report the first SINE found in the genome of the cephalochordate, amphioxus. It is an Alu element of 375 bp that does not share substantial identity with any genomic sequences in vertebrates. It was identified because it was located in the FoxD regulatory region in a cosmid derived from one individual, but absent from the two FoxD alleles of BACs from a second individual. However, searches of sequences of BACs and genomic traces from this second individual gave an estimate of 50-100 copies in the amphioxus genome. The finding of an Alu element in amphioxus raises the question of whether Alu elements in amphioxus and primates arose by convergent evolution or by inheritance from a common ancestor. Genome-wide analyses of transposable elements in amphioxus and other chordates such as tunicates, agnathans and cartilaginous fishes could well provide the answer.     

Generation of targeted deletions in the genome of Rhodothermus marinus

The aim of this work was to develop an approach for chromosomal engineering of the thermophile Rhodothermus marinus. A selection strategy for R. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpB strain with the R. marinus trpB gene. The current work identified an additional selective marker, purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp(+) selection was used during the deletion of purA from the R. marinus chromosome. The alternative Ade(+) selection was in turn used while deleting the endogenous trpB gene. Since both deletions are unmarked, the purA and trpB markers may be reused. Through the double deletant SB-62 (ΔtrpB ΔpurA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency in R. marinus strain SB-62 (ΔtrpB ΔpurA) was demonstrated by targeting putative carotenoid biosynthesis genes, crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5' and 3' homologous sequences, respectively. The resulting Trp(+) transformants were colorless rather than orange-red. The correct replacement of an internal crtBI fragment with the trpB marker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in the R. marinus chromosome can be readily replaced with linear molecules in a single step by double-crossover recombination.     

Some KpnI family members are associated with the Alu family in the human genome

The structures of the termini and their flanking regions of two human KpnI family members were investigated. The two differed in length, but the starting sequence at one terminal (defined as the 5' terminal) was found to be common to both members. The Alu family sequence was found in the 5' flanking regions. The KpnI family sequence started several base-pairs downstream from the 3' end of the Alu family sequence. In both cases, the Alu family sequence was not flanked by the direct repeat sequence common to the Alu family. These two members showed no sequence homology in 3' terminal regions. Interestingly, the Alu family plus the KpnI family unit was found to be flanked by a direct repeat sequence of several base-pair length. Based on these findings, relationship between the Alu family and KpnI family is discussed.     

Analysis of chimpanzee history based on genome sequence alignments

Population geneticists often study small numbers of carefully chosen loci, but it has become possible to obtain orders of magnitude for more data from overlaps of genome sequences. Here, we generate tens of millions of base pairs of multiple sequence alignments from combinations of three western chimpanzees, three central chimpanzees, an eastern chimpanzee, a bonobo, a human, an orangutan, and a macaque. Analysis provides a more precise understanding of demographic history than was previously available. We show that bonobos and common chimpanzees were separated ~1,290,000 years ago, western and other common chimpanzees ~510,000 years ago, and eastern and central chimpanzees at least 50,000 years ago. We infer that the central chimpanzee population size increased by at least a factor of 4 since its separation from western chimpanzees, while the western chimpanzee effective population size decreased. Surprisingly, in about one percent of the genome, the genetic relationships between humans, chimpanzees, and bonobos appear to be different from the species relationships. We used PCR-based resequencing to confirm 11 regions where chimpanzees and bonobos are not most closely related. Study of such loci should provide information about the period of time 5–7 million years ago when the ancestors of humans separated from those of the chimpanzees.     

Detection of deletions in the mitochondrial genome of Caenorhabditis elegans.

We have examined an aging population of Caenorhabditis elegans via a PCR assay to determine if deletions in the mitochondrial genome occur in the nematode. We detected eight such deletions, identified the breakpoints of four of these, and discovered direct repeats of 4-8 base pairs at the site of all four deletions. Six of the eight repeats involved in the deletions are located in or immediately adjacent to tRNAs. Without a biochemical bias, the probability of direct repeats being present at all four breakpoints was 4 x 10(-6).     

Analysis of repeat-mediated deletions in the mitochondrial genome of Saccharomyces cerevisiae

Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions.     

Sequencing the chimpanzee genome: insights into human evolution and disease

Large-scale sequencing of the chimpanzee genome is now imminent. Beyond the inherent fascination of comparing the sequence of the human genome with that of our closest living relative, this project is likely to yield tangible scientific benefits in two areas. First, the discovery of functionally important mutations that are specific to the human lineage offers a new path towards medical benefits. Second, chimpanzee-human comparisons are likely to yield molecular insights into how new biological characteristics evolve--findings that might be relevant throughout the tree of life.     

Site-specific deletions of the mitochondrial genome in the Pearson marrow-pancreas syndrome.

The Pearson marrow-pancreas syndrome is a fatal disorder involving the hematopoietic system and the exocrine pancreas in early infancy. We have previously shown that this disease results from a widespread defect of oxidative phosphorylation. Here, we describe deletions of the mitochondrial (mt) genome between repeated 8- to 13-bp sequences as consistent features of the disease. Studying a series of nine unrelated children, including the patient originally reported by H. Pearson, we found five different types of direct repeats at the boundaries of the mtDNA deletions and we provided evidence for conservation of the 3'-repeated sequence in the deletions. In addition, we found a certain degree of homology between the nucleotide composition of the direct repeats and several structures normally involved in mtDNA replication and mtRNA processing. These results are consistent either with the recognition and cleavage of a particular DNA sequence with a factor of still unknown origin or with a homologous recombination between direct-repeat mtDNA sequences in the Pearson syndrome.