Segmental Duplications in the Human Genome

The review considers the structure, evolution, and possible mechanisms of formation and spreading of intrachromosomal and interchromosomal segmental duplications (SD), which account for more than 5% of the human genome. Most SD consist of multiple modules, which occur in several copies in different genome regions. SD are preferentially located in pericentric and subtelomeric regions, which are least studied on the human chromosomes. Homologous recombination between SD results in various chromosome rearrangements, contributing to the genome instability and the origin of several human hereditary disorders.     

Segmental Duplications in Subtelomeric Regions of Human Chromosome 13

Owing to a great progress in studying the human genome, its euchromatic portion is almost completely sequenced; the complete sequence is still unknown only for pericentric and telomeric regions and short arms of acrocentric chromosomes. Extended satellite blocks and segmental duplications located in these regions substantially hinder the joining of the sequenced fragments and construction of the full-length genome map. The sequence was established for a 1.5-kb human chromosome 13 subtelomeric region, which is about 10 kb away from the rDNA cluster, and deposited in GenBank under accession no. AF478540. The region showed 83–84% homology to the pericentric region of human chromosome 19, and contained short fragments homologous to the pericentric region of human chromosome 13. The results may contribute to the current revision of genome evolution concepts in view of numerous segmental duplications revealed.     

The Power of the Methods for Detecting Interlocus Gene Conversion

Interlocus gene conversion can homogenize DNA sequences of duplicated regions with high homology. Such nonvertical events sometimes cause a misleading evolutionary interpretation of data when the effect of gene conversion is ignored. To avoid this problem, it is crucial to test the data for the presence of gene conversion. Here, we performed extensive simulations to compare four major methods to detect gene conversion. One might expect that the power increases with increase of the gene conversion rate. However, we found this is true for only two methods. For the other two, limited power is expected when gene conversion is too frequent. We suggest using multiple methods to minimize the chance of missing the footprint of gene conversion.INTERLOCUS (ectopic or nonallelic) gene conversion occurs between paralogous regions such that their DNA sequences are shuffled and homogenized (Petes and Hill 1988; Harris et al. 1993; Goldman and Lichten 1996). As a consequence, the DNA sequences of paralogous genes become similar (i.e., concerted evolution, Ohta 1980; Dover 1982; Arnheim 1983). This homogenizing effect of gene conversion sometimes causes problems in the inference of the evolutionary history of duplicated genes or multigene family. Common misleading inferences include an underestimation of the age of duplicated genes (Gao and Innan 2004; Teshima and Innan 2004). This is largely because the concept of the molecular clock is automatically incorporated in most software of phylogenetic analyses, and those software are frequently applied to multigene families without careful consideration of the potential effect of gene conversion.To understand the evolutionary roles of gene duplication, it is crucial to date each duplication event. To do this, we first need to know precisely the action of gene conversion among the gene family of interest. There have been a number of methods for detecting gene conversion, but their power has not been fully explored. Here, we systematically compare their performance by simulations to provide a guideline on which method works best under what condition. Our simulations show that some methods have a serious problem that causes a misleading interpretation: they do not detect any evidence for gene conversion when the gene conversion rate is too high. Thus, as is always true, lack of evidence is no evidence for absence, and we must be very careful about this effect when analyzing data with those tests, as is demonstrated below.There seem to be four major ideas behind the methods for detecting gene conversion, which are summarized below. A number of methods have been developed to detect interlocus gene conversion, and they belong to one of these four broad categories.

1. Incompatibility between an estimated gene tree and the true duplication history: Figure 1A illustrates a simple situation of a pair of duplicated genes, X and Y, that arose before the speciation event of species A and B. The upper tree of Figure 1A shows a tree representing the true history. When a gene tree is estimated from their DNA sequences, it should be consistent with the true tree when genes X and Y have accumulated mutations independently. Gene conversion potentially violates this relationship. When genes X and Y are subject to frequent gene conversion, the two paralogous genes in each species should be more closely related, resulting in a gene tree illustrated in the bottom tree in Figure 1A. Thus, incongruence between the real tree and an inferred gene tree can provide strong evidence for gene conversion (unless there is no lineage sorting or misinference of the gene tree).Open in a separate windowFigure 1.—Summary of the simulations in the two-species two-locus model. (A) Illustration of the model. (B–E) The power of the four approaches. The average gene conversion tract length (1/q) is assumed to be 100 bp. See Figure S1 for the results with 1/q = 1000 bp.It should be noted that a single gene conversion event usually transfers a short fragment. Consequently, it occasionally happens that incongruence is detected only in a part of the duplicated region. Thus, searching local regions of incongruence has been a well-recognized method for detecting nonvertical evolutionary events such as recombination, gene conversion, and horizontal gene transfer (Farris 1971; Brown et al. 1972), and some computational methods based on this idea have been developed (Balding et al. 1992).
2. Incompatibility of gene trees in different subregions: The idea of (i) can work even without knowing the real history. As mentioned above, incompatibility in the tree shape between different subregions can be evidence for local gene conversion because those subregions should have different histories of gene conversion (Sneath et al. 1975; Stephens 1985). A number of statistical algorithms incorporate this idea (e.g., Jakobsen et al. 1997; McGuire et al. 1997; Weiller 1998).
3. GENECONV: A local gene conversion also leaves its trace in the alignment of sequences. GENECONV is a software developed by Sawyer (1989) to detect such signatures (http://www.math.wustl.edu/∼sawyer/geneconv/). GENECONV looks at an alignment of multiple sequences in a pairwise manner and searches unusually long regions of high identity between the focal pair conditional on the pattern of variable sites in the other sequences, which are candidates of recent gene conversion (a similar idea is also seen in Sneath et al. 1975). The statistical significance is determined by random shuffling of variable sites in the alignment.
4. Shared polymorphism: Suppose polymorphism data are available in both of the duplicated genes. Then, with gene conversion, there could be polymorphisms shared by the two genes, which can be evidence for gene conversion (Innan 2003a). It should be noted that parallel mutations can create shared polymorphism even without gene conversion, but the chance should be very low when the point mutation rate is usually very low. Polymorphism data usually have tremendous amounts of information on very recent events and can be a powerful means to detect gene conversion (e.g., Stephens 1985; Betrán et al. 1997; Innan 2002).

In this study, we investigate and compare the performance of the methods based on these four ideas with simple settings. It should be noted that because our primary focus is on interlocus gene conversion, we ignore methods that can be used for detecting only allelic gene conversion, such as Fearnhead and Donnelly (2001), Hudson (2001), and Gay et al. (2007).     

水稻基因组中的节段重复

利用１３个多拷贝探针,研究水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）基因组第８、９、１１和１２染色体上的节段重复。由同一探针检测到的多拷贝位点通常位于不同染色体的相同部位。不同探针检测到的多拷贝位点在不同染色体上的位置顺序相同。第８种９染色体上的相同多拷贝位点的线性排列,提示这两条染色体在进行上可能来源于同一原始染色体。而第９染色体上的一个节段与前人报道的以及本研究进一步证实的第１１和第１２染色体短臂     

Segmental Duplications Are Common in Rice Genome

Segmental duplications on rice (Oryza sativa L.) chromosomes 8, 9, 11, and 12 were studied by examining the distributions of sequences resolved by 13 probes detecting multiple copies of DNA sequences. Four of the hybridization bands detected by a repetitive sequence probe, rTRS, were mapped to the ends of all the four chromosomes. Two or three of the bands detected by each of the other 12 probes were also mapped to different chromosomes. The bands detected by the same probe usually occurred in similar locations of different chromosomes. Loci detected by different DNA probes were often similarly arranged on different chromosomes. Chromosomes 8 and 9 showed colinearity of marker loci arrangement indicating a possible common origin. A segment on chromosome 9 was also very similar to the previously reported duplicated fragments on the ends of chromosomes 11 and 12 which were also detected in this study, indicating a likely common origin. Moreover, the various degrees of distributional similarity of the segments suggest a complex relationship among the chromosomes in the evolution of the rice genome. These results support the proposition that chromosome duplication and diversification may be a mechanism for the origin and evolution of the chromosomes in the rice genome.     

Tandem and Interspersed Repeats Contribute to the Mosaic Structure of Segmental Duplications in the Human Genome

Intrachromosomal and interchromosomal segmental duplications account for more than 5% of the human genome. To analyze the processes resulting in the complex mosaic structure of duplicons, a draft human genome sequence was searched for duplicated segments of a genomic fragment of the pericentric region of the chromosome 21 short arm. The duplicons found consist of modules having paralogs in various genome regions. Module ends are flanked with various tandem or interspersed repeats, which are more unstable as compared with unique sequences. In most cases, the boundaries of duplicated segments exactly coincide with or are in close proximity to hot spots of various rearrangements within repeats or boundaries between repeats and unique sequences or between two different repeats. Homologous recombination between repetitive elements was assumed to be the major mechanism contributing to the mosaic structure of duplicons.     

Occurrence of Repeat Induced Point Mutation in Long Segmental Duplications of Neurospora

Previous studies of repeat induced point mutation (RIP) have typically involved gene-size duplications resulting from insertion of transforming DNA at ectopic chromosomal positions. To ascertain whether genes in larger duplications are subject to RIP, progeny were examined from crosses heterozygous for long segmental duplications obtained using insertional or quasiterminal translocations. Of 17 distinct mutations from crossing 11 different duplications, 13 mapped within the segment that was duplicated in the parent, one was closely linked, and three were unlinked. Half of the mutations in duplicated segments were at previously unknown loci. The mutations were recessive and were expressed both in haploid and in duplication progeny from Duplication X Normal, suggesting that both copies of the wild-type gene had undergone RIP. Seven transition mutations characteristic of RIP were found in 395 base pairs (bp) examined in one ro-11 allele from these crosses and three were found in ~750 bp of another. A single chain-terminating C to T mutation was found in 800 bp of arg-6. RIP is thus responsible. These results are consistent with the idea that the impaired fertility that is characteristic of segmental duplications is due to inactivation by RIP of genes needed for progression through the sexual cycle.     

Mis-Assembled "Segmental Duplications" in Two Versions of the Bos taurus Genome

We analyzed the whole genome sequence coverage in two versions of the Bos taurus genome and identified all regions longer than five kilobases (Kbp) that are duplicated within chromosomes with >99% sequence fidelity in both copies. We call these regions High Fidelity Duplications (HFDs). The two assemblies were Btau 4.2, produced by the Human Genome Sequencing Center at Baylor College of Medicine, and UMD Bos taurus 3.1 (UMD 3.1), produced by our group at the University of Maryland. We found that Btau 4.2 has a far greater number of HFDs, 3111 versus only 69 in UMD 3.1. Read coverage analysis shows that 39 million base pairs (Mbp) of sequence in HFDs in Btau 4.2 appear to be a result of a mis-assembly and therefore cannot be qualified as segmental duplications. UMD 3.1 has only 0.41 Mbp of sequence in HFDs that are due to a mis-assembly.     

Pancreatic-Type Ribonuclease 1 Gene Duplications in Rat Species

Mammalian pancreatic-type ribonucleases (RNases) 1 represent single-copy genes in the genome of most investigated mammalian species, including Mus musculus and other murid rodents. However, in six species belonging to the genus Rattus and closely related taxa, several paralogous gene products were identified by Southern blotting and PCR amplifications of genomic sequences. Phylogenies of nucleotide and derived amino acid sequences were reconstructed by several procedures, with three Mus species as outgroup. Duplications of the RNase 1 occurred after the divergence of Niviventer cremoriventer and Leopoldamys edwardsi from the other investigated species. Four groups of paralogous genes could be identified from specific amino acid sequence features in each of them. Low ratios of nonsynonymous-to-synonymous substitutions and the paucity of pseudogene features suggest functional gene products. One of the RNase 1 genes of R. norvegicus is expressed in the pancreas. RNases 1 were isolated from pancreatic tissues of R. rattus and R. exulans and submitted to N-terminal amino acid sequence analysis. In R. rattus, the orthologue of the expressed gene of R. norvegicus was identified, but in R. exulans, two paralogous gene products were found. The gene encoding for one of these had not yet been found by PCR amplification of genomic DNA. A well-defined group of orthologous sequences found in five investigated species codes for very basic RNases. Northern blot analysis showed expression of messenger RNA for this RNase in the spleen of R. norvegicus, but the protein product could not be identified. Evolutionary rates of RNase 1, expressed as nucleotide substitutions per site per 10(3) million years (Myr), vary between 5 and 9 in the lines leading to Mus, Niviventer, and Lepoldamys (on the basis of an ancestral date of mouse/rat divergence of 12.2 Myr) and between 20 and 50 in the lines to the other sequences after divergence from Niviventer and Leopoldamys (5.5 Myr).     

Genome Resilience and Prevalence of Segmental Duplications Following Fast Neutron Irradiation of Soybean

Fast neutron radiation has been used as a mutagen to develop extensive mutant collections. However, the genome-wide structural consequences of fast neutron radiation are not well understood. Here, we examine the genome-wide structural variants observed among 264 soybean [Glycine max (L.) Merrill] plants sampled from a large fast neutron-mutagenized population. While deletion rates were similar to previous reports, surprisingly high rates of segmental duplication were also found throughout the genome. Duplication coverage extended across entire chromosomes and often prevailed at chromosome ends. High-throughput resequencing analysis of selected mutants resolved specific chromosomal events, including the rearrangement junctions for a large deletion, a tandem duplication, and a translocation. Genetic mapping associated a large deletion on chromosome 10 with a quantitative change in seed composition for one mutant. A tandem duplication event, located on chromosome 17 in a second mutant, was found to cosegregate with a short petiole mutant phenotype, and thus may serve as an example of a morphological change attributable to a DNA copy number gain. Overall, this study provides insight into the resilience of the soybean genome, the patterns of structural variation resulting from fast neutron mutagenesis, and the utility of fast neutron-irradiated mutants as a source of novel genetic losses and gains.     

Gene Duplications and Evolution of Vertebrate Voltage-Gated Sodium Channels

Voltage-gated sodium channels underlie action potential generation in excitable tissue. To establish the evolutionary mechanisms that shaped the vertebrate sodium channel α-subunit (SCNA) gene family and their encoded Na_v1 proteins, we identified all SCNA genes in several teleost species. Molecular cloning revealed that teleosts have eight SCNA genes, compared to ten in another vertebrate lineage, mammals. Prior phylogenetic analyses have indicated that the genomes of both teleosts and tetrapods contain four monophyletic groups of SCNA genes, and that tandem duplications expanded the number of genes in two of the four mammalian groups. However, the number of genes in each group varies between teleosts and tetrapods, suggesting different evolutionary histories in the two vertebrate lineages. Our findings from phylogenetic analysis and chromosomal mapping of Danio rerio genes indicate that tandem duplications are an unlikely mechanism for generation of the extant teleost SCNA genes. Instead, analyses of other closely mapped genes in D. rerio as well as of SCNA genes from several teleost species all support the hypothesis that a whole-genome duplication was involved in expansion of the SCNA gene family in teleosts. Interestingly, despite their different evolutionary histories, mRNA analyses demonstrated a conservation of expression patterns for SCNA orthologues in teleosts and tetrapods, suggesting functional conservation. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Axel Meyer]     

A Model-Based Analysis of GC-Biased Gene Conversion in the Human and Chimpanzee Genomes

GC-biased gene conversion (gBGC) is a recombination-associated process that favors the fixation of G/C alleles over A/T alleles. In mammals, gBGC is hypothesized to contribute to variation in GC content, rapidly evolving sequences, and the fixation of deleterious mutations, but its prevalence and general functional consequences remain poorly understood. gBGC is difficult to incorporate into models of molecular evolution and so far has primarily been studied using summary statistics from genomic comparisons. Here, we introduce a new probabilistic model that captures the joint effects of natural selection and gBGC on nucleotide substitution patterns, while allowing for correlations along the genome in these effects. We implemented our model in a computer program, called phastBias, that can accurately detect gBGC tracts about 1 kilobase or longer in simulated sequence alignments. When applied to real primate genome sequences, phastBias predicts gBGC tracts that cover roughly 0.3% of the human and chimpanzee genomes and account for 1.2% of human-chimpanzee nucleotide differences. These tracts fall in clusters, particularly in subtelomeric regions; they are enriched for recombination hotspots and fast-evolving sequences; and they display an ongoing fixation preference for G and C alleles. They are also significantly enriched for disease-associated polymorphisms, suggesting that they contribute to the fixation of deleterious alleles. The gBGC tracts provide a unique window into historical recombination processes along the human and chimpanzee lineages. They supply additional evidence of long-term conservation of megabase-scale recombination rates accompanied by rapid turnover of hotspots. Together, these findings shed new light on the evolutionary, functional, and disease implications of gBGC. The phastBias program and our predicted tracts are freely available.     

The Origin of Trypsin: Evidence for Multiple Gene Duplications in Trypsins

The trypsin family of serine proteases is one of the most studied protein families, with a wealth of amino acid sequence information available in public databases. Since trypsin-like enzymes are widely distributed in living organisms in nature, likely evolutionary scenarios have been proposed. A novel methodology for Fourier transformation of biological sequences (FOTOBIS) is presented. The methodology is well suited for the identification of the size and extent of short repeats in protein sequences. In the present paper the trypsin family of enzymes is analyzed with FOTOBIS and strong evidence for tandem gene duplication is found. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15–18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which the contemporary trypsins have developed. Received: 22 November 1997 / Accepted: 26 January 1998     

Metallothionein Gene Duplications and Metal Tolerance in Natural Populations of Drosophila melanogaster

A search for duplications of the Drosophila melanogaster metallothionein gene (Mtn) yielded numerous examples of this type of chromosomal rearrangement. These duplications are distributed widely--we found them in samples from four continents, and they are functional--larvae carrying Mtn duplications produce more Mtn RNA and tolerate increased cadmium and copper concentrations. Six different duplication types were characterized by restriction-enzyme analyses using probes from the Mtn region. The restriction maps show that in four cases the sequences, ranging in size between 2.2 and 6.0 kb, are arranged as direct, tandem repeats; in two other cases, this basic pattern is modified by the insertion of a putative transposable element into one of the repeated units. Duplications of the D. melanogaster metallothionein gene such as those that we found in natural populations may represent early stages in the evolution of a gene family.     

Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken V_L and V_H loci were knocked out in DT40 cells and replaced with human V_K and V_H genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human V_K and V_H regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens.     

Single and Coincident Intragenic Mutations Attributable to Gene Conversion in a Human Cell Line

Two polymorphic sites are located within the heterozygous TK1 locus in the human lymphoblastoid cell line TK6: an inactivating frameshift in exon 4 of the nonfunctional allele and a phenotypically silent frameshift in exon 7 of the functional allele. Through the use of these intragenic polymorphisms and microsatellite markers that flank TK1, we demonstrate that partial gene conversion accounts for 3/75 (0.04) spontaneous and 9/163 (0.06) X-ray-induced TK1(-) mutants, thus comprising a significant component of forward mutations at this locus. In all cases, the conversion tract is <1 cM, rendering double exchange a remote alternate explanation for these results. Sequence analysis of full length TK1 cDNA provides rigorous exclusion of deletion events as a mechanism for generation of these allelotypes. Detailed examination of allelotypes in TK1(-) mutants identified two mechanisms for the generation of coincident sequence alterations that sometimes accompanied gene conversions. Mutations within the conversion tract were attributed to either error-prone gap filling synthesis during recombinational repair or mismatch repair within a heteroduplex region following branch migration. These findings suggest that a proportion of point mutations may not be targeted to sites of DNA base damage, but rather may arise as secondary consequences from the repair of DNA strand breaks.