Slipped-strand mispairing: a major mechanism for DNA sequence evolution

Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following several features characterize such sequences: (1) they typically consist of a variety of repeated motifs of 1-10 bases--but may include much larger repeats as well; (2) larger repeat units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped-strand mispairing events, in concert with unequal crossing- over, can readily account for all of these features. The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to result from nonrandom patterns of nucleotide substitution. We argue that the intrahelical process of slipped-strand mispairing is much more likely to be the major factor in the initial expansion of short repeated motifs and that, after initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossing-over or other interhelical events because of their propensity to mispair. Evidence is presented that single-base repeats (the shortest possible motifs) are represented by longer runs in mammalian introns than would be expected on a random basis, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Simple repetitive sequences may therefore represent a natural ground state of DNA unselected for coding functions.      

Microevolution of pandemic Vibrio parahaemolyticus assessed by the number of repeat units in short sequence tandem repeat regions

The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10(-4) mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered.     

Genome-wide prediction of human VNTRs

Polymorphic minisatellites, also known as variable number of tandem repeats (VNTRs), are tandem repeat regions that show variation in the number of repeat units among chromosomes in a population. Currently, there are no general methods for predicting which minisatellites have a high probability of being polymorphic, given their sequence characteristics. An earlier approach has focused on potentially highly polymorphic and hypervariable minisatellites, which make up only a small fraction of all minisatellites in the human genome. We have developed a model, based on available minisatellite and VNTR sequence data, that predicts the probability that a minisatellite (unit size > or = 6 bp) identified by the computer program Tandem Repeats Finder is polymorphic (VNTR). According to the model, minisatellites with high copy number and high degree of sequence similarity are most likely to be VNTRs. This approach was used to scan the draft sequence of the human genome for VNTRs. A total of 157,549 minisatellite repeats were found, of which 29,224 are predicted to be VNTRs. Contrary to previous results, VNTRs appear to be widespread and abundant throughout the human genome, with an estimated density of 9.1 VNTRs/Mb.     

Sequence rearrangement in the AT-rich minisatellite of the novel rice transposable element Basho.

In the process of characterizing a rice wx deletion mutant, an AT-rich minisatellite sequence that consisted of units of approximately 80 bp was detected about 2.3 kb downstream of the wx gene. This AT-rich minisatellite was a multiple-copy element (1 x 10(3) to 2 x 10(3) copies per haploid genome) and interspersed in the rice genome. By BLAST homology search it was indicated that not only the tandem repeat but also both flanking sequences were conserved among copies. According to the characteristics of the termini (5'-CHH ... CTAG-3') and a target site preference for T, this AT-rich minisatellite accompanying the flanking sequences was classified into a novel transposon, Basho. The results of direct amplification of Basho showed that relatively large variation in size existed in the Basho family. We estimate the variation to be generated by not only alteration of the number of units in the minisatellite but also by duplications of larger blocks including the conserved flanking sequences caused by single-strand mispairing (SSM) at noncontiguous repeats. Because the AT-rich minisatellite contained in Basho possessed several motifs of the matrix attachment region (MAR) in its repeat unit, the functional role as MAR in the rice genome was discussed.     

Genetic associations between ADHD and dopaminergic genes (<Emphasis Type="Italic">DAT1</Emphasis> and <Emphasis Type="Italic">DRD4</Emphasis>) VNTRs in Korean children

It is well known that dopaminergic genes affect the development of attention deficit hyperactivity disorder (ADHD) in various populations. Many studies have shown that variable number tandem repeats (VNTRs) located within the 3′-untranslated region of DAT1 and in exon 3 of DRD4 are associated with ADHD development; however, these results were inconsistent. Therefore, we investigated the genetic association between two VNTRs and ADHD in Korean children. We determined the VNTRs using PCR. We examined genotype and allele frequency differences between the experimental and control groups, along with the odds ratios, using Chi square and exact tests. We observed a significant association between the children with ADHD and the control group in the 10R/10R genotype of DAT1 VNTRs (p?=?0.025). In addition, the 11R allele of DAT1 VNTRs showed a higher frequency in the control group than in the ADHD group (p?=?0.023). Also, the short repeat (without 11R) and long repeat alleles (including 11R) were associated with ADHD (p?<?0.05). The analysis of DRD4 VNTRs revealed that the 2R allele is associated with ADHD (p?=?0.025). A significant result was also observed in long and short repeats (p?<?0.05). Additionally, ADHD subtypes showed that the DRD4 VNTRs are associated with combined and hyperactive-impulsive subtype groups (p?<?0.05). Therefore, our results suggest that DAT1 VNTRs and DRD4 VNTRs play a role in the genetic etiology of ADHD in Korean children.     

Five polymorphic microsatellite VNTRs on the human X chromosome

The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT.dG/dA.dC)n, where n = approximately 10-60. We and others have found that several of these repeats have variable lengths in different individuals, with allelic fragments varying in size by multiples of 2 bp. These "microsatellite" variable number of tandem repeats (VNTRs) may be scored by PCR, using unique flanking primers to amplify the repeat-containing regions and resolving the products on DNA sequencing gels. Since few VNTRs have been found on the X chromosome, we screened a flow-sorted X chromosome-specific genomic library for microsatellites. Approximately 25% of the phage clones hybridized to a poly (dT-dG).poly(dA-dC) probe. Of seven X-linked microsatellites present in positive phages, five are polymorphic and three have both eight or more alleles and heterozygosities exceeding 75%. Using PCR to amplify genomic DNAs from hybrid cell panels, we confirmed the X localization of these VNTRs and regionally mapped four of them. The fifth VNTR was regionally mapped by virtue of its tight linkage to DXS87 in Centre du Polymorphisme Humain families. We conclude that whatever factors limit the occurrence of "classical" VNTRs and RFLPs on the X chromosome do not appear to operate in the case of microsatellite VNTRs.     

Molecular evolution of a plastid tandem repeat locus in an orchid lineage

The molecular evolution of a chloroplast minisatellite locus in the Anacamptis palustris (Orchidaceae) lineage and haplotype variation in two Italian A. palustris populations were investigated. A phylogenetic analyses of the chloroplast tRNA(LEU) intron, where the minisatellite locus is located, revealed that a deletion in the ancestor of the A. palustris lineage led to the formation of two noncontiguous, complementary sequence motifs. We propose a model to explain the initial formation of the minisatellite repeat motif, starting with the two noncontiguous, complementary sequence motifs. A survey of minisatellite variation in four species of the A. palustris lineage revealed several haplotypes that differed not only in repeat number, but also in repeat organization. A haplotype network suggests that three different minisatellite loci evolved independently at the same position in the tRNA(LEU) intron. A secondary structure model revealed that the A. palustris minisatellite repeat forms a stem region of the tRNA(LEU) intron, which allows its notable expansion without negatively affecting splicing. Minisatellite variation was high in the two examined A. palustris populations where 20 haplotypes were detected, whereas no length variation was detected in a neighboring poly (A) microsatellite locus. We estimated a chloroplast minisatellite mutation rate of 3.2 x 10(-3) mutations per generation. Southern blot analyses did not find evidence for chloroplast heteroplasmy. Based on the analysis of the largest known, extant A. palustris population, a stepwise mutation model (SMM) was inferred.     

The role of introns in repeat protein gene formation

Genes composed of tandem repetitive sequence motifs are abundant in nature and are enriched in eukaryotes. To investigate repeat protein gene formation mechanisms, we have conducted a large-scale analysis of their introns and exons. We find that a wide variety of repeat motifs exhibit a striking conservation of intron position and phase, and are composed of exons that encode one or two complete repeats. These results suggest a simple model of repeat protein gene formation from local duplications. This model is corroborated by amino acid sequence similarity patterns among neighboring repeats from various repeat protein genes. The distribution of one- and two-repeat exons indicates that intron-facilitated repeat motif duplication, in which the start and end points of duplication are located in consecutive intronic regions, significantly exceeds intron-independent duplication. These results suggest that introns have contributed to the greater abundance of repeat protein genes in eukaryotic versus prokaryotic organisms, a conclusion that is supported by taxonomic analysis.     

Identification and characterization of tandem repeats in exon III of dopamine receptor D4 (DRD4) genes from different mammalian species

In this study we have identified and characterized dopamine receptor D4 (DRD4) exon III tandem repeats in 33 public available nucleotide sequences from different mammalian species. We found that the tandem repeat in canids could be described in a novel and simple way, namely, as a structure composed of 15- and 12- bp modules. Tandem repeats composed of 18-bp modules were found in sequences from the horse, zebra, onager, and donkey, Asiatic bear, polar bear, common raccoon, dolphin, harbor porpoise, and domestic cat. Several of these sequences have been analyzed previously without a tandem repeat being found. In the domestic cow and gray seal we identified tandem repeats composed of 36-bp modules, each consisting of two closely related 18-bp basic units. A tandem repeat consisting of 9-bp modules was identified in sequences from mink and ferret. In the European otter we detected an 18-bp tandem repeat, while a tandem repeat consisting of 27-bp modules was identified in a sequence from European badger. Both these tandem repeats were composed of 9-bp basic units, which were closely related with the 9-bp repeat modules identified in the mink and ferret. Tandem repeats could not be identified in sequences from rodents. All tandem repeats possessed a high GC content with a strong bias for C. On phylogenetic analysis of the tandem repeats evolutionary related species were clustered into the same groups. The degree of conservation of the tandem repeats varied significantly between species. The deduced amino acid sequences of most of the tandem repeats exhibited a high propensity for disorder. This was also the case with an amino acid sequence of the human DRD4 exon III tandem repeat, which was included in the study for comparative purposes. We identified proline-containing motifs for SH3 and WW domain binding proteins, potential phosphorylation sites, PDZ domain binding motifs, and FHA domain binding motifs in the amino acid sequences of the tandem repeats. The numbers of potential functional sites varied pronouncedly between species. Our observations provide a platform for future studies of the architecture and evolution of the DRD4 exon III tandem repeat, and they suggest that differences in the structure of this tandem repeat contribute to specialization and generation of diversity in receptor function.     

Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation

Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.     

Detection of species-specific long VNTRs in mitochondrial control region and their application to identifying sympatric Hong Kong grouper (Epinephelus akaara) and yellow grouper (Epinephelus awoara)

Length variation and heteroplasmy were observed in PCR products of the first half of mtDNA control region of both Hong Kong grouper (Epinephelus akaara) and yellow grouper (Epinephelus awoara). DNA sequencing unveiled the phenomena were caused by the presence of species-specific long variable number tandem repeats (VNTRs). This is the first report on the mtDNA VNTRs and their heteroplasmy in groupers. Moreover, these VNTRs are also the longest such structure found in teleost fish. Thereafter, we designed two species-specific PCR reverse primers according to the 3' end sequences of the VNTRs and successfully established assays for the identification of these two sympatric grouper species.     

Variable Numbers of Tandem Repeats in Plasmodium falciparum Genes

Genome variation studies in Plasmodium falciparum have focused on SNPs and, more recently, large-scale copy number polymorphisms and ectopic rearrangements. Here, we examine another source of variation: variable number tandem repeats (VNTRs). Interspersed low complexity features, including the well-studied P. falciparum microsatellite sequences, are commonly classified as VNTRs; however, this study is focused on longer coding VNTR polymorphisms, a small class of copy number variations. Selection against frameshift mutation is a main constraint on tandem repeats (TRs) in coding regions, while limited propagation of TRs longer than 975 nt total length is a minor restriction in coding regions. Comparative analysis of three P. falciparum genomes reveals that more than 9% of all P. falciparum ORFs harbor VNTRs, much more than has been reported for any other species. Moreover, genotyping of VNTR loci in a drug-selected line, progeny of a genetic cross, and 334 field isolates demonstrates broad variability in these sequences. Functional enrichment analysis of ORFs harboring VNTRs identifies stress and DNA damage responses along with chromatin modification activities, suggesting an influence on genome mutability and functional variation. Analysis of the repeat units and their flanking regions in both P. falciparum and Plasmodium reichenowi sequences implicates a replication slippage mechanism in the generation of TRs from an initially unrepeated sequence. VNTRs can contribute to rapid adaptation by localized sequence duplication. They also can confound SNP-typing microarrays or mapping short-sequence reads and therefore must be accounted for in such analyses.     

甜菜属厚皮菜SWTY-1细胞质VNTRs多态性分析

利用数目可变串联重复序列(Variable Number of Tandem Repeats,VNTRs)微卫星标记方法,对重庆厚皮菜甜菜材料SWTY-1群体中100个单株的细胞质线粒体DNA片段中TR2位点VNTRs片段多态性进行分析。结果显示97个单株线粒体TR2位点微卫星串联重复序列均为3拷贝,与普通糖甜菜一致;3个单株线粒体TR2位点微卫星串联重复序列为6拷贝,发现甜菜属厚皮菜细胞质TR2位点VNTRs存在多态性,在该群体中发现了不同于甜菜栽培种新的细胞质单株。对该群体材料100个单株的抽薹及结籽进行观测,结果显示微卫星串联重复序列为6拷贝的变异植株中2个单株花期未抽苔开花,1株抽苔晚未形成正常种子;细胞质TR2位点VNTRs片段拷贝数为3的植株中2个单株未能正常抽薹,其他植株均正常抽薹结籽。     

A stochastic mechanism for biofilm formation by Mycoplasma pulmonis

Bacterial biofilms are communities of bacteria that are enclosed in an extracellular matrix. Within a biofilm the bacteria are protected from antimicrobials, environmental stresses, and immune responses from the host. Biofilms are often believed to have a highly developed organization that is derived from differential regulation of the genes that direct the synthesis of the extracellular matrix and the attachment to surfaces. The mycoplasmas have the smallest of the prokaryotic genomes and apparently lack complex gene-regulatory systems. We examined biofilm formation by Mycoplasma pulmonis and found it to be dependent on the length of the tandem repeat region of the variable surface antigen (Vsa) protein. Mycoplasmas that produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and glass. Mycoplasmas that produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the medium. The biofilms and the microcolonies contained an extracellular matrix which contained Vsa protein, lipid, DNA, and saccharide. As variation in the number of Vsa tandem repeats occurs by slipped-strand mispairing, the ability of the mycoplasmas to form a biofilm switches stochastically.     

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.     

The Evolution of Tandemly Repetitive DNA: Recombination Rules

Variable numbers of tandem repeats (VNTRs), which include hypervariable regions, minisatellites and microsatellites, can be assigned together with satellite DNAs to define a class of noncoding tandemly repetitive DNA (TR-DNA). The evolution of TR-DNA is assumed to be driven by an unbiased recombinational process. A simulation model of unequal exchange is presented and used to investigate the evolutionary persistence of single TR-DNA lineages. Three different recombination rules are specified to govern the expansion and contraction of a TR-DNA lineage from an initial array of two repeats to, finally, a single repeat allele, which cannot participate in a misalignment and exchange process. In the absence of amplification or selection acting to bias array evolution toward expansion, the probability of attaining a target array size is a function only of the initial number of repeats. We show that the proportions of lineages attaining a targeted array size are the same irrespective of recombination rule and rate, demonstrating that our simulation model is well behaved. The time taken to attain a target array size, the persistence of the target array, and the total persistence time of repetitive array structure, are functions of the initial number of repeats, the rate of recombination, and the rules of misalignment preceding recombinational exchange. These relationships are investigated using our simulation model. While misalignment constraint is probably greatest for satellite DNA it also seems important in accounting for the evolution of VNTR loci including minisatellites. This conclusion is consistent with the observed nonrandom distributions of VNTRs and other TR-DNAs in the human genome.     

VNTRseek—a computational tool to detect tandem repeat variants in high-throughput sequencing data

DNA tandem repeats (TRs) are ubiquitous genomic features which consist of two or more adjacent copies of an underlying pattern sequence. The copies may be identical or approximate. Variable number of tandem repeats or VNTRs are polymorphic TR loci in which the number of pattern copies is variable. In this paper we describe VNTRseek, our software for discovery of minisatellite VNTRs (pattern size ≥ 7 nucleotides) using whole genome sequencing data. VNTRseek maps sequencing reads to a set of reference TRs and then identifies putative VNTRs based on a discrepancy between the copy number of a reference and its mapped reads. VNTRseek was used to analyze the Watson and Khoisan genomes (454 technology) and two 1000 Genomes family trios (Illumina). In the Watson genome, we identified 752 VNTRs with pattern sizes ranging from 7 to 84 nt. In the Khoisan genome, we identified 2572 VNTRs with pattern sizes ranging from 7 to 105 nt. In the trios, we identified between 2660 and 3822 VNTRs per individual and found nearly 100% consistency with Mendelian inheritance. VNTRseek is, to the best of our knowledge, the first software for genome-wide detection of minisatellite VNTRs. It is available at http://orca.bu.edu/vntrseek/.     

Construction of silk fiber core in lepidoptera

The formation and properties of lepidopteran silk fibers depend on amino acid repeats in the principal protein, heavy chain fibroin (H-fibroin). In H-fibroins of the "bombycoid" type, concatenations of alanine or of the GAGAGS crystalline motifs (1st tier repeats) and adjacent sequences breaking periodicity make 2nd tier repeats. Two to six such repeats comprise a 3rd tier assembly, and 12 assemblies, linked by an amorphous sequence, constitute the repetitive H-fibroin region. Heterogeneity in the repeat length and intercalation of amorphous regions prevent excessive crystallization. In the "pyraloid" H-fibroins, iterations of simple motifs are absent and assemblies of several complex motifs constitute highly regular repeats that are organized in about 12 highest order reiterations without specific spacers. Repeat homogeneity appears crucial for the alignment and interaction of the disjunct motifs that must be registered precisely to form crystallites; repeat heterogeneity is associated with decreased fiber strength. Both H-fibroin types are typically hydrophobic, and their secretion requires disulfide linkage to light chain fibroin and participation of another protein, P25. These auxiliary proteins are absent in saturniid moths with amphiphilic H-fibroin repeats. The selection at nucleic acid and protein levels and the availability of nutrients play roles in H-fibroin evolution.     

A Computer Simulation Study of Vntr Population Genetics: Constrained Recombination Rules Out the Infinite Alleles Model

Extensive allelic diversity in variable numbers of tandem repeats (VNTRs) has been discovered in the human genome. For population genetic studies of VNTRs, such as forensic applications, it is important to know whether a neutral mutation-drift balance of VNTR polymorphism can be represented by the infinite alleles model. The assumption of the infinite alleles model that each new mutant is unique is very likely to be violated by unequal sister chromatid exchange (USCE), the primary process believed to generate VNTR mutants. We show that increasing both mutation rates and misalignment constraint for intrachromosomal recombination in a computer simulation model reduces simulated VNTR diversity below the expectations of the infinite alleles model. Maximal constraint, represented as slippage of single repeats, reduces simulated VNTR diversity to levels expected from the stepwise mutation model. Although misalignment rule is the more important variable, mutation rate also has an effect. At moderate rates of USCE, simulated VNTR diversity fluctuates around infinite alleles expectation. However, if rates of USCE are high, as for hypervariable VNTRs, simulated VNTR diversity is consistently lower than predicted by the infinite alleles model. This has been observed for many VNTRs and accounted for by technical problems in distinguishing alleles of neighboring size classes. We use sampling theory to confirm the intrinsically poor fit to the infinite alleles model of both simulated VNTR diversity and observed VNTR polymorphisms sampled from two Papua New Guinean populations.