The coding region of the human c-mos pseudogene contains Alu repeat insertions

We have determined the nucleotide sequence of an 841-bp fragment derived from a segment of the human genome previously cloned by Chumakov et al. [Gene 17 (1982) 19–26] and Zabarovsky et al. [Gene 23 (1983) 379–384] and containing regions homologous to the viral mos gene probe. This sequence displays homology with part of the coding region of the human and murine c-mos genes, contains several termination codons, and is interrupted by two Alu-family elements flanked by short direct repeats. Probably, the progenitor of the human c-mos gene was duplicated approximately at the time of mammalian divergence, was converted to a pseudogene, and acquired insertions of two Alu elements.     

A SINE-based dichotomous key for primate identification

For DNA samples or ‘divorced’ tissues, identifying the organism from which they were taken generally requires some type of analytical method. The ideal approach would be robust even in the hands of a novice, requiring minimal equipment, time, and effort. Genotyping SINEs (Short INterspersed Elements) is such an approach as it requires only PCR-related equipment, and the analysis consists solely of interpreting fragment sizes in agarose gels. Modern primate genomes are known to contain lineage-specific insertions of Alu elements (a primate-specific SINE); thus, to demonstrate the utility of this approach, we used members of the Alu family to identify DNA samples from evolutionarily divergent primate species. For each node of a combined phylogenetic tree (56 species; n = 8 [Hominids]; 11 [New World monkeys]; 21 [Old World monkeys]; 2 [Tarsiformes]; and, 14 [Strepsirrhines]), we tested loci (> 400 in total) from prior phylogenetic studies as well as newly identified elements for their ability to amplify in all 56 species. Ultimately, 195 loci were selected for inclusion in this Alu-based key for primate identification. This dichotomous SINE-based key is best used through hierarchical amplification, with the starting point determined by the level of initial uncertainty regarding sample origin. With newly emerging genome databases, finding informative retrotransposon insertions is becoming much more rapid; thus, the general principle of using SINEs to identify organisms is broadly applicable.     

Repetitive DNA in and around translocation breakpoints of the Philadelphia chromosome

This paper describes systematic sequence studies of repetitive DNA in and around translocation breakpoints on chromosomes 9 and 22, which are involved in the formation of the Philadelphia chromosome in acute leukemias. In addition to Alu repeats described in previous studies, the breakpoint regions appear to contain many other repetitive elements, including a member of a new repetitive family (MER34) reported in this paper. Identification of these repeats broadens current studies on the possible involvement of repetitive DNA in this intensely studied chromosomal translocation.     

Natural Selection and Functional Potentials of Human Noncoding Elements Revealed by Analysis of Next Generation Sequencing Data

Noncoding DNA sequences (NCS) have attracted much attention recently due to their functional potentials. Here we attempted to reveal the functional roles of noncoding sequences from the point of view of natural selection that typically indicates the functional potentials of certain genomic elements. We analyzed nearly 37 million single nucleotide polymorphisms (SNPs) of Phase I data of the 1000 Genomes Project. We estimated a series of key parameters of population genetics and molecular evolution to characterize sequence variations of the noncoding genome within and between populations, and identified the natural selection footprints in NCS in worldwide human populations. Our results showed that purifying selection is prevalent and there is substantial constraint of variations in NCS, while positive selectionis more likely to be specific to some particular genomic regions and regional populations. Intriguingly, we observed larger fraction of non-conserved NCS variants with lower derived allele frequency in the genome, indicating possible functional gain of non-conserved NCS. Notably, NCS elements are enriched for potentially functional markers such as eQTLs, TF motif, and DNase I footprints in the genome. More interestingly, some NCS variants associated with diseases such as Alzheimer''s disease, Type 1 diabetes, and immune-related bowel disorder (IBD) showed signatures of positive selection, although the majority of NCS variants, reported as risk alleles by genome-wide association studies, showed signatures of negative selection. Our analyses provided compelling evidence of natural selection forces on noncoding sequences in the human genome and advanced our understanding of their functional potentials that play important roles in disease etiology and human evolution.     

Characterization of pre-insertion loci of de novo L1 insertions

The human Long Interspersed Element-1 (LINE-1) and the Short Interspersed Element (SINE) Alu comprise 28% of the human genome. They share the same L1-encoded endonuclease for insertion, which recognizes an A+T-rich sequence. Under a simple model of insertion distribution, this nucleotide preference would lead to the prediction that the populations of both elements would be biased towards A+T-rich regions. Genomic L1 elements do show an A+T-rich bias. In contrast, Alu is biased towards G+C-rich regions when compared to the genome average. Several analyses have demonstrated that relatively recent insertions of both elements show less G+C content bias relative to older elements. We have analyzed the repetitive element and G+C composition of more than 100 pre-insertion loci derived from de novo L1 insertions in cultured human cancer cells, which should represent an evolutionarily unbiased set of insertions. An A+T-rich bias is observed in the 50 bp flanking the endonuclease target site, consistent with the known target site for the L1 endonuclease. The L1, Alu, and G+C content of 20 kb of the de novo pre-insertion loci shows a different set of biases than that observed for fixed L1s in the human genome. In contrast to the insertion sites of genomic L1s, the de novo L1 pre-insertion loci are relatively L1-poor, Alu-rich and G+C neutral. Finally, a statistically significant cluster of de novo L1 insertions was localized in the vicinity of the c-myc gene. These results suggest that the initial insertion preference of L1, while A+T-rich in the initial vicinity of the break site, can be influenced by the broader content of the flanking genomic region and have implications for understanding the dynamics of L1 and Alu distributions in the human genome.     

A dimorphic Alu Sb-like insertion in COL3A1 is ethnic-specific

Alu elements are a class of repetitive DNA sequences found throughout the human genome that are thought to be duplicated via an RNA intermediate in a process termed retroposition. Recently inserted Alu elements are closely related, suggesting that they are derived from a single source gene or closely related source genes. Analysis of the type III collagen gene (COL3A1) revealed a polymorphic Alu insertion in intron 8 of the gene. The Alu insertion in the COL3A1 gene had a high degree of nucleotide identity to the Sb family of Alu elements, a family of older Alu elements. The Alu sequence was less similar to the consensus sequence for the PV or Sb2 subfamilies, subfamilies of recently inserted Alu elements. These data support the observations that at least three source genes are active in the human genome, one of which is distinct from the PV and Sb2 subfamilies and predates either of these two subfamilies. Appearance of the Alu insertion in different ethnic populations suggests that the insertion may have occurred in the last 100,000 years. This Alu insert should be a useful marker for population studies and for marking COL3A1 alleles.     

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.     

Detection and frequency estimation of rare variants in pools of genomic DNA from large populations using mutational spectrometry

DNA variants underlying the inheritance of risk for common diseases are expected to have a wide range of population allele frequencies. The detection and scoring of the rare alleles (at frequencies of <0.01) presents significant practical problems, including the requirement for large sample sizes and the limitations inherent in current methodologies for allele discrimination. In the present report, we have applied mutational spectrometry based on constant denaturing capillary electrophoresis (CDCE) to DNA pools from large populations in order to improve the prospects of testing the role of rare variants in common diseases on a large scale. We conducted a pilot study of the cytotoxic T lymphocyte-associated antigen-4 gene (CTLA4) in type 1 diabetes (T1D). A total of 1228 bp, comprising 98% of the CTLA4 coding sequence, all adjacent intronic mRNA splice sites, and a 3′ UTR sequence were scanned for unknown point mutations in pools of genomic DNA from a control population of 10,464 young American adults and two T1D populations, one American (1799 individuals) and one from the United Kingdom (2102 individuals). The data suggest that it is unlikely that rare variants in the scanned regions of CTLA4 represent a significant proportion of T1D risk and illustrate that CDCE-based mutational spectrometry of DNA pools offers a feasible and cost-effective means of testing the role of rare variants in susceptibility to common diseases.     

Identification and characterization of novel polymorphic LINE-1 insertions through comparison of two human genome sequence assemblies

Mobile elements represent a relatively new class of markers for the study of human evolution. Long interspersed elements (LINEs) belong to a group of retrotransposons comprising approximately 21% of the human genome. Young LINE-1 (L1) elements that have integrated recently into the human genome can be polymorphic for insertion presence/absence in different human populations at particular chromosomal locations. To identify putative novel L1 insertion polymorphisms, we computationally compared two draft assemblies of the whole human genome (Public and Celera Human Genome assemblies). We identified a total of 148 potential polymorphic L1 insertion loci, among which 73 were candidates for novel polymorphic loci. Based on additional analyses we selected 34 loci for further experimental studies. PCR-based assays and DNA sequence analysis were performed for these 34 loci in 80 unrelated individuals from four diverse human populations: African-American, Asian, Caucasian, and South American. All but two of the selected loci were confirmed as polymorphic in our human population panel. Approximately 47% of the analyzed loci integrated into other repetitive elements, most commonly older L1s. One of the insertions was accompanied by a BC200 sequence. Collectively, these mobile elements represent a valuable source of genomic polymorphism for the study of human population genetics. Our results also suggest that the exhaustive identification of L1 insertion polymorphisms is far from complete, and new whole genome sequences are valuable sources for finding novel retrotransposon insertion polymorphisms.     

Genome‐wide variation in nucleotides and retrotransposons in alpine populations of Arabis alpina (Brassicaceae)

Advances in high‐throughput sequencing have promoted the collection of reference genomes and genome‐wide diversity. However, the assessment of genomic variation among populations has hitherto mainly been surveyed through single‐nucleotide polymorphisms (SNPs) and largely ignored the often major fraction of genomes represented by transposable elements (TEs). Despite accumulating evidence supporting the evolutionary significance of TEs, comprehensive surveys remain scarce. Here, we sequenced the full genomes of 304 individuals of Arabis alpina sampled from four nearby natural populations to genotype SNPs as well as polymorphic long terminal repeat retrotransposons (polymorphic TEs; i.e., presence/absence of TE insertions at specific loci). We identified 291,396 SNPs and 20,548 polymorphic TEs, comparing their contributions to genomic diversity and divergence across populations. Few SNPs were shared among populations and overall showed high population‐specific variation, whereas most polymorphic TEs segregated among populations. The genomic context of these two classes of variants further highlighted candidate adaptive loci having a putative impact on functional genes. In particular, 4.96% of the SNPs were identified as nonsynonymous or affecting start/stop codons. In contrast, 43% of the polymorphic TEs were present next to Arabis genes enriched in functional categories related to the regulation of reproduction and responses to biotic as well as abiotic stresses. This unprecedented data set, mapping variation gained from SNPs and complementary polymorphic TEs within and among populations, will serve as a rich resource for addressing microevolutionary processes shaping genome variation.     

Variability of GATA-microsatellite DNA in populations of the parthenogenetic species of lizard Lacerta unisexualis Darevsky

Multilocus DNA fingerprinting was used to analyze the genome variation of mini- and microsatellite DNA regions in parthenogenetic Caucasian rock lizard Lacerta unisexualis. The DNA fingerprints obtained with probe M13 were nearly identical in all populations examined (the average similarity index S = 0.992). The fingerprints obtained with probe (GATA)4 varied (S = 0.862). Polymorphic fragments were assumed to correspond to allelic variants of genetically unstable GATA loci. Comparison of the fingerprints of animals from four geographically isolated populations revealed several population-specific GATA microsatellite markers. Based on their distribution among the populations, the corresponding alleles were assumed to originate from a common ancestral allele.     

Sequence Variation within the KIV-2 Copy Number Polymorphism of the Human LPA Gene in African,Asian, and European Populations

Amazingly little sequence variation is reported for the kringle IV 2 copy number variation (KIV 2 CNV) in the human LPA gene. Apart from whole genome sequencing projects, this region has only been analyzed in some detail in samples of European populations. We have performed a systematic resequencing study of the exonic and flanking intron regions within the KIV 2 CNV in 90 alleles from Asian, European, and four different African populations. Alleles have been separated according to their CNV length by pulsed field gel electrophoresis prior to unbiased specific PCR amplification of the target regions. These amplicons covered all KIV 2 copies of an individual allele simultaneously. In addition, cloned amplicons from genomic DNA of an African individual were sequenced. Our data suggest that sequence variation in this genomic region may be higher than previously appreciated. Detection probability of variants appeared to depend on the KIV 2 copy number of the analyzed DNA and on the proportion of copies carrying the variant. Asians had a high frequency of so-called KIV 2 type B and type C (together 70% of alleles), which differ by three or two synonymous substitutions respectively from the reference type A. This is most likely explained by the strong bottleneck suggested to have occurred when modern humans migrated to East Asia. A higher frequency of variable sites was detected in the Africans. In particular, two previously unreported splice site variants were found. One was associated with non-detectable Lp(a). The other was observed at high population frequencies (10% to 40%). Like the KIV 2 type B and C variants, this latter variant was also found in a high proportion of KIV 2 repeats in the affected alleles and in alleles differing in copy numbers. Our findings may have implications for the interpretation of SNP analyses in other repetitive loci of the human genome.     

Molecular and genetic characterization of the allelic variants of Du215, Du281, Du323, and Du47G microsatellite loci in parthenogenetic lizard <Emphasis Type="Italic">Darevskia armeniaca</Emphasis> (Lacertidae)

A key issue in the study of unisexual (parthenogenetic) vertebrate species is the determination of their genetic and clonal diversity. In pursuing this aim, various markers of nuclear and mitochondrial genomes can be used. The most effective genetic markers include microsatellite DNA, characterized by high variability. The development and characterization of such markers is a necessary step in the genetic studies of parthenogenetic species. In the present study, using locus-specific PCR, for the first time, an analysis of allelic polymorphism of four microsatellite loci is performed in the populations of parthenogenetic species Darevskia armeniaca. In the studied populations, allelic variants of each locus are identified, and the nucleotide sequences of each allele are determined. It is demonstrated that allele differences are associated with the variation in the structure of microsatellite clusters and single nucleotide substitutions at fixed distances in flanking DNA regions. Structural allele variations form haplotype markers that are specific to each allele and are inherited from their parental bisexual species. It is established which of the parental alleles of each locus were inherited by the parthenogenetic species. The characteristics of the distribution and frequency of the alleles of microsatellite loci in the populations of D. armeniaca determining specific features of each population are obtained. The observed heterozygosity of the populations at the studied loci and the mutation rates in genome regions, as well as Nei’s genetic distances between the studied populations, are determined, and the phylogenetic relationships between them are established.     

Analysis and Optimization of Bulk DNA Sampling with Binary Scoring for Germplasm Characterization

The strategy of bulk DNA sampling has been a valuable method for studying large numbers of individuals through genetic markers. The application of this strategy for discrimination among germplasm sources was analyzed through information theory, considering the case of polymorphic alleles scored binarily for their presence or absence in DNA pools. We defined the informativeness of a set of marker loci in bulks as the mutual information between genotype and population identity, composed by two terms: diversity and noise. The first term is the entropy of bulk genotypes, whereas the noise term is measured through the conditional entropy of bulk genotypes given germplasm sources. Thus, optimizing marker information implies increasing diversity and reducing noise. Simple formulas were devised to estimate marker information per allele from a set of estimated allele frequencies across populations. As an example, they allowed optimization of bulk size for SSR genotyping in maize, from allele frequencies estimated in a sample of 56 maize populations. It was found that a sample of 30 plants from a random mating population is adequate for maize germplasm SSR characterization. We analyzed the use of divided bulks to overcome the allele dilution problem in DNA pools, and concluded that samples of 30 plants divided into three bulks of 10 plants are efficient to characterize maize germplasm sources through SSR with a good control of the dilution problem. We estimated the informativeness of 30 SSR loci from the estimated allele frequencies in maize populations, and found a wide variation of marker informativeness, which positively correlated with the number of alleles per locus.     

Recently integrated human Alu repeats: finding needles in the haystack

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

Polymorphic NumtS trace human population relationships

The human genome is constantly subjected to evolutionary forces which shape its architecture. Insertions of mitochondrial DNA sequences into nuclear genome (NumtS) have been described in several eukaryotic species, including Homo sapiens and other primates. The ongoing process of the generation of NumtS has made them valuable markers in primate phylogenetic studies, as well as potentially informative loci for reconstructing the genetic history of modern humans. Here, we report the identification of 53 human-specific NumtS by inspection of the UCSC genome browser, showing that they may be direct insertions of mitochondrial DNA into the human nuclear DNA after the human-chimpanzee split. In silico analyses allowed us to identify 14 NumtS which are polymorphic in terms of their presence/absence within the human genome in individuals of different ancestry. The allele frequencies of these polymorphic NumtS were calculated for 1000 Genomes Project sequence data from 13 populations worldwide, and principal components analysis and hierarchical clustering methods allowed the detection of strong signals of geographical structure related to the genetic diversity of these loci. All identified polymorphic human-specific NumtS together with a tandemly duplicated NumtS have also been validated by PCR amplification on a panel of 60 samples belonging to five native populations worldwide, confirming the expected NumtS variability. On the basis of these findings, we have succeeded in depicting the landscape of variation of a series of NumtS in several ethnic groups, making an advance in their identification as useful markers in the study on human population genetics.     

A tissue-specific decrease in the pre-mRNA level of L1-and Alu-containing alleles of human genes

LINE1 and Alu retroelements occupy approximately 17 and 13% of the human genome, respectively. They include the evolutionarily youngest element groups Ta-L1, AluYa5, and AluYb8, many inserts of which are polymorphic in the Homo sapiens population. Despite the data on the ability of L1 and Alu elements to cause various modifications of the genome, the effects of these retroelements on gene expression have yet not been studied. Using the RT PCR method, we analyzed the pre-mRNA (heterogeneous nuclear RNA) content of allele pairs of four genes in five human cell lines, heterozygous with respect to intronic inserts of L1 and Alu elements. We showed for the first time a tissue-specific decrease in the pre-mRNA content of the gene allele bearing L1 or Alu inserts relative to the other allele of the same gene lacking the retroelement.