High frequencies of short frameshifts in poly-CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12.

Slipped-strand mispairing (SSM) may play an major role in repetitive DNA sequence evolution by generating large numbers of short frameshift mutations within simple tandem repeats. Here we examine the frequency and size spectrum of frameshifts generated within poly-CA/TG sequences inserted into bacteriophage M13 in Escherichia coli hosts. The frequency of detectable frameshifts within a 40 bp tract of poly-CA/TG is greater than one percent and increases more than linearly with length, being lower by a factor of four in a 22 bp target sequence. The frequency increases more than 13-fold in mutL and mutS host cells, suggesting that a high proportion of frameshift events are normally repaired by methyl-directed mismatch repair. Of the 87 sequenced frameshifts in this study, 96% result from deletion or insertion of only or two 2 bp repeat units. The most frequent events are 2 bp deletions, 2 bp insertions, and 4 bp deletions, the relative frequencies of these events being about 18:6:1.     

Slipped-strand mispairing at noncontiguous repeats in Poecilia reticulata: a model for minisatellite birth

The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the "raw material" for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the "imperfect" or "short direct" repeats frequently observed adjacent to both mtDNA and nuclear VNTRs.     

Cross-hybridizing snake satellite, Drosophila, and mouse DNA sequences may have arisen independently

Previous reports have interpreted hybridization between snake satellite DNA and DNA clones from a variety of distant taxonomic groups as evidence for evolutionary conservation, which implies common ancestry (homology) and/or convergence (analogy) to produce the cross- hybridizing sequences. We have isolated 11 clones from a genomic library of Drosophila melanogaster, using a cloned 2.5-kb snake satellite probe of known nucleotide sequence. We have also analysed published sequence data from snakes, mice, and Drosophila. These data show that (1) all of the cross-hybridization between the snake, fly, and mouse clones can be accounted for by the presence of either of two tandem repeats, [GATA]n and [GACA]n and (2) these tandem repeats are organized differently among the different species. We find no evidence that these sequences are homologous apart from the existence of the simple repeat itself, although their divergence from a common ancestral sequence cannot be ruled out. The sequences contain a variety of homogeneous clusters of tandem repeats of CATA, GA, TA, and CA, as well as GATA and GACA. We suggest that these motifs may have arisen by a self-accelerating process involving slipped-strand mispairing of DNA. Homogeneity of the clusters might simply be the result of a rate of accumulation of tandem repeats that exceeds that of other mutations.      

Mitochondrial genome of the Komodo dragon: efficient sequencing method with reptile-oriented primers and novel gene rearrangements.

The mitochondrial genome of the Komodo dragon (Varanus komodoensis) was nearly completely sequenced, except for two highly repetitive noncoding regions. An efficient sequencing method for squamate mitochondrial genomes was established by combining the long polymerase chain reaction (PCR) technology and a set of reptile-oriented primers designed for nested PCR amplifications. It was found that the mitochondrial genome had novel gene arrangements in which genes from NADH dehydrogenase subunit 6 to proline tRNA were extensively shuffled with duplicate control regions. These control regions had 99% sequence similarity over 700 bp. Although snake mitochondrial genomes are also known to possess duplicate control regions with nearly identical sequences, the location of the second control region suggested independent occurrence of the duplication on lineages leading to snakes and the Komodo dragon. Another feature of the mitochondrial genome of the Komodo dragon was the considerable number of tandem repeats, including sequences with a strong secondary structure, as a possible site for the slipped-strand mispairing in replication. These observations are consistent with hypotheses that tandem duplications via the slipped-strand mispairing may induce mitochondrial gene rearrangements and may serve to maintain similar copies of the control region.     

鸮形目4种鸟类线粒体调控区全序列的测定与比较研究

利用Long-PCR和Primer Walking的方法对鸮形目的短耳鸮、长耳鸮、纵纹腹小鸮、灰林鸮4种鸟类的线粒体调控区进行了全序列测定。结果表明：短耳鸮的调控区跃度为3290bp;长耳鸮为2848bp;纵纹腹小鸮为2444bp;灰林鸮为1771bp。短耳鸮的调控区长度是4种鸮中最大的,并且是目前已知最大的鸟类线粒体调控区。这4种鸮类调控区的基本结构和其他鸟类相似,按照碱基变化速率的不同可以分为3个区：碱基变化速率较快的外围区域Ⅰ、Ⅲ和保守的中间区域Ⅱ。这4种鸟类调控区的3’端均存在大量的串联重复序列,短耳鸮为126bp单元重复7次和78bp单元重复14次;长耳鸮为127bp单元重复8次和78bp单几重复6次;纵纹腹小鸮有3个重复单元,分别为89bp单元重复3次、77bp单元重复4次和71bp单元重复6次;灰林鸮仅有1个单元的串联重复为78bp重复5次。调控区中串联重复序列可能是由链的滑动错配产生,另外这些重复序列都能形成热力学稳定的多重茎环二级结构,而且在重复序列中还发现一些保守基序,这说明重复序列可能具有一定的生理功能,影响调控区的调重控功能从而影响线粒体基因组的复制和转录。     

Does polyploidy lead to fewer and shorter microsatellites in Barbus (Teleostei: Cyprinidae)?

Screening of a hybrid Barbus barbus-B. meridionalis genome was performed for CA, GA, TAT, TCT, TAG, TGT, TATT, TACT, ATCT motifs, and simultaneously on another fish species, tilapia S. melanotheron . Sequences of positive clones were obtained for Barbus and revealed that repetitive structure significantly depends on the motif: most TAT and TATT repeats contain small numbers of repeats, and these repeats are highly heterogeneous, whereas other motifs (we mainly obtained CA and GATA repeats) form longer and much more homogeneous arrays. Polymorphism data from five loci in two different species of barbel show that perfectly repetitive loci are much more variable than imperfect loci (TAT and TATT). We compared the frequency of positive clones for different repeat motifs between barbel and tilapia. For dinucleotide repeats (CA and GA), the comparison was extended to additional fish species, trout and sea bass, which were screened in nearly identical conditions for these motifs. The most salient feature of these comparisons reveals that arrays of dinucleotide motifs are significantly under-represented and shorter in Barbus than in other fish species. We propose an explanation that can account for most features of microsatellites characterizing the genome of barbel. A bias toward deletion affecting slipped-strand mispairing events would lead to shortening and loss of microsatellite loci. Such a bias would represent an efficient way of eliminating useless DNA from polyploidized species with an excessive amount of DNA.     

DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences.

DNA tertiary structures are shown to be formed by denaturation and reannealing in vitro of molecularly-cloned DNA containing multiple tandem repeat sequences. Electron microscopy of homoduplex DNA molecules containing the human c-Harvey-ras gene revealed knot-like structures which mapped to the position of the 812 bp variable tandem repeat (VTR) sequence. We propose that the structures result from slipped-strand mispairing within the VTR and hybridisation of homologous repetitive sequences in the single-stranded loops so produced. Similar structures were also found in freshly-linearized supercoiled plasmids. More complex knot-like structures were found in homoduplexes of a 4 kb tandem array from the hypervariable region 3' to the human alpha-globin locus. Formation of such DNA tertiary structures in vitro also provides a practical method for identifying and mapping direct tandem repeat arrays that are at least 800 bp long.     

Complete mitochondrial genome of the seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae)

The complete mitochondrial genome (mitogenome) of the seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae), which is one of the best known insects capable of predation, is described with an emphasis on the noteworthy composition of the A+T-rich region. The C. septempunctata genome consists of 2 rRNAs, 22 tRNAs, 13 protein-coding genes, and 1 control region, designated as the A+T-rich region in insects. Along with an unusually long A+T-rich region (4469 bp), the 18,965-bp long C. septempunctata mitogenome was the largest in Coleoptera. The A+T-rich region is composed of a 2214-bp long non-repeat region composed of 80.17% A/T nucleotides and a 2256-bp long repeat region composed of 65.71% A/T nucleotides. The repeat region harbors 32 identical 70-bp long tandem repeats plus one 15-bp long incomplete first repeat. These repeat sequences may possibly have been caused by slipped-strand mispairing and unequal crossing-over events during DNA replication.     

Tandemly repeated sequences in the mitochondrial DNA control region and phylogeography of the Pike-Perches Stizostedion

DNA sequences from the mitochondrial DNA control region are used to test the phylogeographic relationships among the pike-perches, Stizostedion (Teleostei: Percidae) and to examine patterns of variation. Sequences reveal two types of variability: single nucleotide polymorphisms and 6 to 14 copies of 10- to 11-base-pair tandemly repeated sequences. Numbers of copies of the tandem repeats are found to evolve too rapidly to detect phylogenetic signal at any taxonomic level, even among populations. Sequence similarities of the tandem repeats among Stizostedion and other percids suggest concerted evolutionary processes. Predicted folding of the tandem repeats and their proximity to termination-associated sequences indicate that secondary structure mediates slipped-strand mispairing among the d-loop, heavy, and light strands. Neighbor-joining and maximum parsimony analyses of sequences indicate that the genus is divided into clades on the continents of North America and Eurasia. Calibrating genetic distances with divergence times supports the hypothesis that Stizostedion dispersed from Eurasia to North America across a North Pacific Beringial land bridge approximately 4 million years before present, near the beginning of the Pliocene Epoch. The North American S. vitreum and S. canadense appear separated by about 2.75 million years, and the Eurasian S. lucioperca and S. volgensis are diverged by about 1.8 million years, suggesting that speciation occurred during the late Pliocene Epoch.     

Expansion/contraction of mammalian mitochondrial DNA repeats in Escherichia coli mimics the mitochondrial heteroplasmy.

Length polymorphism due to tandem repeats is a common feature in animal mitochondrial DNA. The rabbit mitochondrial genome contains a 20 bp repeat domain, which generates a general heteroplasmic state. The observed polymorphic patterns suggest a dynamic equilibrium between gain and loss of units that maintains the copy number in the range 3-19 repeat units. In the apparent absence of recombination, slipped-strand mispairing during replication appears to be the primary cause of additions and deletions. To investigate this hypothesis we have set up a plasmid assay in Escherichia coli. A variable number of repeat units was inserted into a plasmid in both orientations relative to the colE1 origin of replication. Our data show that (i) a minimum unit number (>3) is necessary to generate length polymorphs, (ii) the number of events increases with the length tract, (iii) an excess of additions over deletions is found when the copy number is less than 10 and the trend is reversed when it is over 10, (iv) the frequency of deletions-additions is dependent on the orientation, (v) the polymorphism patterns are different according to the orientation. The length polymorphic pattern generated in the bacteria, in one orientation, mimics that observed in the mitochondria, suggesting that slipped mispairing between repeated sequences during DNA replication is responsible for the mitochondrial heteroplasmic state.     

Origin and Evolution of Tandem Repeats in the Mitochondrial DNA Control Region of Shrikes (<Emphasis Type="BoldItalic">Lanius</Emphasis> spp.)

The origin and evolution of a 128-bp tandem repeat in the mtDNA control region of shrikes (Lanius: Aves) were investigated. The tandem repeat is present in only two species, L. excubitor and L. ludovicianus. In contrast to the variation in repeat number in L. ludovicianus, all individuals of three subspecies of L. excubitor had three repeats. Comparative analysis suggests that a short direct repeat, and a secondary structure including the tandem repeat and a downstream inverted repeat, may be important in the origin of the tandem repeat by slipped-strand mispairing and its subsequent turnover. Homogenization of repeat sequences is most simply explained by expansion and contraction of the repeat array. Surprisingly, mtDNA sequences from L. excubitor were found to be paraphyletic with respect to L. ludovicianus. These results show the utility of a comparative analysis for insights into the evolutionary dynamics of mtDNA tandem repeats.[Reviewing Editor: Martin Kreitman]     

Variation among the dispersed (GATA)n sequences in Drosophila melanogaster

Five nonallelic copies of the dispersed (GATA)n repeated sequence of Drosophila melanogaster (referred to as GATA elements) have been sequenced and analysed. The GATA elements range in size from 111 to 444 bp, consisting predominantly of tandemly repeated GATAs, interspersed with variants of the subunit. The types and distributions of these variants are consistent with the hypothesis that they have arisen by a random accumulation of point mutations (substitutions, deletions, and insertions) in pure (GATA)n sequences. Duplications or deletions of the GATA subunit, and of GATA variants, have also probably occurred, as a result of either unequal crossing-over or slipped-strand mispairing. Evidence for duplication (deletion) has been obtained from a comparison of two allelic GATA elements isolated from different populations. GATA elements, in common with other dispersed, simple repeats, are probably highly variable in length.     

Tandem-repetitive noncoding DNA: forms and forces

A model of sequence-dependent, unequal crossing-over and gene amplification (slippage replication) has been stimulated in order to account for various structural features of tandemly repeated DNA sequences. It is shown that DNA whose sequence is not maintained by natural selection will exhibit repetitive patterns over a wide range of recombination rates as a result of the interaction of unequal crossing-over and slippage replication, processes that depend on sequence similarity. At high crossing-over frequencies, the nucleotide patterns generated in the simulations are simple and highly regular, with short, nearly identical sequences repeated in tandem. Decreasing recombination rates increase the tendency to longer and more-complex repeat units. Periodicities have been observed down to very low recombination rates (one or more orders of magnitude lower than mutation rate). At such low rates, most of the sequences contain repeats which have an extensive substructure and a high degree of heterogeneity among each other; often higher-order structures are superimposed on a tandem array. These results are compared with various structural properties of tandemly repeated DNAs known from eukaryotes, the spectrum ranging from simple-sequence DNAs, particularly the hypervariable mini-satellites, to the classical satellite DNAs, located in chromosomal regions of low recombination, e.g., heterochromatin.     

Tandemly Repeated Sequences in the Mitochondrial DNA Control Region and Phylogeography of the Pike-PerchesStizostedion

DNA sequences from the mitochondrial DNA control region are used to test the phylogeographic relationships among the pike-perches,Stizostedion(Teleostei: Percidae) and to examine patterns of variation. Sequences reveal two types of variability: single nucleotide polymorphisms and 6 to 14 copies of 10- to 11-base-pair tandemly repeated sequences. Numbers of copies of the tandem repeats are found to evolve too rapidly to detect phylogenetic signal at any taxonomic level, even among populations. Sequence similarities of the tandem repeats amongStizostedionand other percids suggest concerted evolutionary processes. Predicted folding of the tandem repeats and their proximity to termination-associated sequences indicate that secondary structure mediates slipped-strand mispairing among the d-loop, heavy, and light strands. Neighbor-joining and maximum parsimony analyses of sequences indicate that the genus is divided into clades on the continents of North America and Eurasia. Calibrating genetic distances with divergence times supports the hypothesis thatStizostediondispersed from Eurasia to North America across a North Pacific Beringial land bridge approximately 4 million years before present, near the beginning of the Pliocene Epoch. The North AmericanS. vitreumandS. canadenseappear separated by about 2.75 million years, and the EurasianS. luciopercaandS. volgensisare diverged by about 1.8 million years, suggesting that speciation occurred during the late Pliocene Epoch.     

Genomic organization of human 5 S rDNA and sequence of one tandem repeat

An organization of human 5 S rDNA repeats is inferred from Southern analyses of restriction digests of genomic DNA fractionated by pulsed-field and conventional gel electrophoreses. A single unit of 2.2 kb is repeated approximately 90 times within a 200-kb fragment (defined by enzymes that do not cleave within individual units, i.e., EcoR1, BglII, HindIII, and PvuII); a comparable number of 5 S sequences are scattered elsewhere in the genome. A lambda clone containing six complete 5 S repeats was obtained from a human placental DNA library. One repeat contains 2231 bp and includes poly(dG-dT).(dC-dA), tracts of polypyrimidine, and an Alu sequence in the spacer region. Also, 5-S-hybridizing clones, containing DNA inserts with an average size of 250 kb, have been obtained as yeast artificial chromosomes. Thus far, four clones have been partially characterized and shown to be 5 S sequences from loci separate from the tandem repeat units.     

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.     

On simple repeated GATCA sequences in animal genomes: a critical reappraisal

Simple tandemly organized GATCA sequences occurred in all eukaryotic genomes investigated. The amount and organization of individual GATCA sequences or derivatives thereof vary considerably in animal DNAs and can be assessed by simple but specific hybridization procedures with chemically pure oligonucleotide probes. In several animal species, including humans, GATCA sequences show extensive polymorphism, thus allowing individual-specific "DNA fingerprints." In selected rodents the sex-chromosomal organization of GATCA sequences is being studied extensively, revealing rapid evolutionary changes. In addition, insight can be expected into the sequences involved in obligatory meiotic crossing over between the X and Y chromosomes, into unequal crossing-over events, and into the linkage of GATCA elements to male-specific as well as to male-determining genes on the Y chromosome. The exact provenance of GATCA sequences in present-day eukaryotes cannot be pinpointed, but evolutionary conservation and several modes of de novo generation are discussed. Among these are unequal recombination, slipped strand mispairing, and other unspecified mechanisms. The latter include inherent properties that are responsible for the "selfish" or "ignorant" nature of simple repeats. Expression, if any, of GATCA sequences is critical to the overall significance of these ubiquitously interspersed simple repeats.     

Patterns of tandem repetition in plant whole genome assemblies

Tandem repeats often confound large genome assemblies. A survey of tandemly arrayed repetitive sequences was carried out in whole genome sequences of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the monocots rice and sorghum, and the dicots Arabidopsis thaliana, poplar, grapevine, and papaya, in order to test how these assemblies deal with this fraction of DNA. Our results suggest that plant genome assemblies preferentially include tandem repeats composed of shorter monomeric units (especially dinucleotide and 9–30-bp repeats), while higher repetitive units pose more difficulties to assemble. Nevertheless, notwithstanding that currently available sequencing technologies struggle with higher arrays of repeated DNA, major well-known repetitive elements including centromeric and telomeric repeats as well as high copy-number genes, were found to be reasonably well represented. A database including all tandem repeat sequences characterized here was created to benefit future comparative genomic analyses.