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.     

Characterization of a set of variable number of tandem repeat markers conserved in bovidae.

Screening purpose-built libraries with minisatellite probes, we have isolated 36 bovine variable number of tandem repeat markers (VNTRs) characterized by a mean heterozygosity of 59.3 within the American Holstein breed. Matching probabilities and exclusion powers were estimated by Monte-Carlo simulation, showing that the top 5 to 10 markers could be used as a very efficient DNA-based system for individual identification and paternity diagnosis. The isolated VNTR systems should contribute significantly to the establishment of a bovine primary DNA marker map. Linkage analysis, use of somatic cell hybrids, and in situ hybridization demonstrate that these bovine VNTRs are scattered throughout the bovine genome, without evidence for proterminal confinement as in the human, and that at least some of them are organized as clusters. Moreover, Southern blot analysis and in situ hybridization demonstrate conservation of sequence and map location of minisatellites within Bovidae.     

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.     

Hypermutable minisatellites,a human affair?

Minisatellites are a class of highly polymorphic GC-rich tandem repeats. They include some of the most variable loci in the human genome, with mutation rates ranging from 0.5% to >20% per generation. Structurally, they consist of 10- to 100-bp intermingled variant repeats, making them ideal tools for dissecting mechanisms of instability at tandem repeats. Distinct mutation processes generate rare intra-allelic somatic events and frequent complex conversion-like germline mutations in these repeats. Furthermore, turnover of repeats at human minisatellites is controlled by intense recombinational activity in DNA flanking the repeat array. Surprisingly, whereas other mammalian genomes possess minisatellite-like sequences, hypermutable loci have not been identified that suggest human-specific turnover processes at minisatellite arrays. Attempts to transfer minisatellite germline instability to the mouse have failed. However, yeast models are now revealing valuable information regarding the mechanisms regulating instability at these tandem repeats. Finally, minisatellites and tandem repeats provide exquisitely sensitive molecular tools to detect genomic insults such as ionizing radiation exposure. Surprisingly, by a mechanism that remains elusive, there are transgenerational increases in minisatellite instability.     

Relationship between micro- and minisatellites in the human genome

Micro- and minisatellites constitute an essential part of DNA with a low sequence complexity and carry several important functions. A search for tandem repeats in the human genome with a length of a repeat unit of up to 70 bp, including repeats with a great number of nucleotide substitutions, has been performed using the TaadeaSWAN program. It was shown that, for a considerable number of minisatellites with the length of the repeating unit of less than 25 nt, a shorter repeating motif can be distinguished in the sequence of this repeat, which often is similar to the sequence of minisatellites widely occurring in the human genome. A model of hierarchic origination of minisatellites in the human genome is suggested.     

Polymers of random short oligonucleotides detect polymorphic loci in the human genome.

Polymers of random 14 mer oligonucleotides are shown to detect discrete loci in the human genome. Eighteen different synthetic tandem repeats of random 14 base-pair units (STRs) have been generated and all of them turn out to detect polymorphic loci on southern blots of human DNA samples, presumably corresponding to a variable number of tandem repeats (VNTR). This finding suggests that minisatellites are a major component of the human genome and are strongly associated with the generation of genetic variability. In addition, it should open new strategies to make new polymorphic probes available.     

The human factor VII gene is polymorphic due to variation in repeat copy number in a minisatellite

The gene coding for human factor VII, a vitamin K-dependent coagulation factor, contains five minisatellite imperfect tandem repeats with monomer element lengths ranging from 14 to 37 bp, and copy numbers ranging from 6 to 52. Three of these repeats are entirely within introns, one is entirely in an untranslated portion of an exon, and one spans an exon-intron border and contains coding sequence. A consensus sequence derived from a comparison of the monomers is similar to a core sequence found in other minisatellites. All of the minisatellites display higher-order periodicities. At least one of these minisatellites is polymorphic. A variation in repeat copy number has been observed in a tandem-repeat region in the seventh factor-VII intron.     

Minisatellite polymorphism as a tool to distinguish closely related Lactococcus lactis strains

Genome plasticity is considered as a means for bacteria to adapt to their environment. Plasticity in tandem repeat sequences on bacterial genomes has been recently exploited to trace the epidemiology of pathogens. Here, we examine the utility of minisatellite (i.e., a repeat unit of six nucleotides or more) typing in non-pathogenic food bacteria of the species Lactococcus lactis. Thirty-four minisatellites identified on the sequenced L. lactis ssp. lactis strain IL1403 genome were first analyzed in 10 closely related ssp. lactis strains, as determined by randomly amplified polymorphic DNA (RAPD). The selected tandem repeats varied in length, percent identity between repeats, and locations. We showed that: (i) the greatest polymorphism was in orfs encoding exported proteins or in intergenic regions; (ii) two thirds of minisatellites were little- or non-variable, despite as much as 90% identity between tandem repeats; and (iii) dendrograms based on either RAPD or minisatellite analyses were similar. Seven minisatellites identified in this study are potentially useful for lactococcal typing. We then asked whether tandem repeats in L. lactis were stable upon very long-term (up to two years) storage. Despite large rearrangements previously reported in derivative strains, just one of 10 minisatellites tested underwent an alteration, suggesting that tandem repeat rearrangements probably occur during active DNA replication. We conclude that multiple locus minisatellite analysis can be a valuable tool to follow lactococcal strain diversity.     

Mathematical Modelling of Mycobacterium tuberculosis VNTR Loci Estimates a Very Slow Mutation Rate for the Repeats

Minisatellites are highly variable tandem repeats used for over 20 years in humans for DNA fingerprinting. In prokaryotes fingerprinting techniques exploiting VNTR (variable number of tandem repeats) polymorphisms have become widely used recently in bacterial typing. However although many investigations into the mechanisms underlying minisatellite variation in humans have been performed, relatively little is known about the processes that mediate bacterial minisatellite polymorphism. An understanding of this is important since it will influence how the results from VNTR experiments are interpreted. The minisatellites of Mycobacterium tuberculosis are well characterized since they are some of the few polymorphic loci in what is otherwise a very homogeneous organism. Using VNTR results from a well-defined and characterized set of M. tuberculosis strains we show that the repeats at a locus are likely to evolve by stepwise contraction or expansion in the number of repeats. A stochastic continuous-time population mathematical model was developed to simulate the evolution of the repeats. This allowed estimation of the tendency of the repeats to increase or decrease and the rate at which they change. The majority of loci tend to lose rather than gain repeats. All of the loci mutate extremely slowly, with an average rate of 2.3 x 10(-8), which is 350 times slower than that of a set of VNTR repeats with similar diversity observed experimentally in Escherichia coli. This suggests that the VNTR profile of a strain of M. tuberculosis will be indicative of its clonal lineage and will be unlikely to vary in epidemiologically-related strains.     

Relationship of micro-and minisatellites in the human genome

Micro-and minisatellites constitute an essential part of DNA with low sequence complexity and perform a number of important functions. The TandemSWAN program was used to search the human genome for tandem repeats with a length of a repeated unit to 70 bp, including repeats with a large number of nucleotide substitutions. It was shown that, for a significant fraction of the program-found minisatellites with a repeat unit length less than 25 bp, a shorter repeated motif can be discerned in this sequence, which is often similar to the sequence of microsatellites occurring widely in the human genome. A model of hierarchical origin of minisatellites in the human genome was proposed.     

Stable minihairpin structures forming at minisatellite DNA isolated from yellow fin sea bream Acanthopagrus latus

The lengths of simple repeat sequences are generally unstable or polymorphic (highly variable with respect to the numbers of tandem repeats). Previously we have isolated a family of minisatellite DNA (GenBank accession AF422186) that appears specifically and abundantly in the genome of yellow fin sea bream Acanthopagrus latus but not in closely-related red sea bream Pagrus major, and found that the numbers of tandem arrays in the homologous loci are polymorphic. This means that the minisatellite sequence has appeared and propagated in A. latus genome after speciation. In order to understand what makes the minisatellite widespread within the A. latus genome and what causes the polymorphic nature of the number of tandem repeats, the structural features of single-stranded polynucleotides were analyzed by electrophoresis, chemical modification, circular dichroism (CD), differential scanning calorimetry (DSC) and electron microscopy. The results suggest that a portion of the repeat unit forms a stable minihairpin structure, and it can cause polymerase pausing within the minisatellite DNA.     

Vntr Allele Frequency Distributions under the Stepwise Mutation Model: A Computer Simulation Approach

Variable numbers of tandem repeats (VNTRs) are a class of highly informative and widely dispersed genetic markers. Despite their wide application in biological science, little is known about their mutational mechanisms or population dynamics. The objective of this work was to investigate four summary measures of VNTR allele frequency distributions: number of alleles, number of modes, range in allele size and heterozygosity, using computer simulations of the one-step stepwise mutation model (SMM). We estimated these measures and their probability distributions for a wide range of mutation rates and compared the simulation results with predictions from analytical formulations of the one-step SMM. The average heterozygosity from the simulations agreed with the analytical expectation under the SMM. The average number of alleles, however, was larger in the simulations than the analytical expectation of the SMM. We then compared our simulation expectations with actual data reported in the literature. We used the sample size and observed heterozygosity to determine the expected value, 5th and 95th percentiles for the other three summary measures, allelic size range, number of modes and number of alleles. The loci analyzed were classified into three groups based on the size of the repeat unit: microsatellites (1-2 base pair (bp) repeat unit), short tandem repeats [(STR) 3-5 bp repeat unit], and minisatellites (15-70 bp repeat unit). In general, STR loci were most similar to the simulation results under the SMM for the three summary measures (number of alleles, number of modes and range in allele size), followed by the microsatellite loci and then by the minisatellite loci, which showed deviations in the direction of the infinite allele model (IAM). Based on these differences, we hypothesize that these three classes of loci are subject to different mutational forces.     

Isolation and Characterization of Mouse Minisatellites

Minisatellites provide the most informative system for analyzing processes of tandem repeat turnover in humans. However, little is known about minisatellites and the mechanisms by which they mutate in other species. To this end, we have isolated and characterized 76 endogenous mouse VNTRs. Fifty-one loci have been localized on mouse chromosomes and, unlike in humans, show no clustering in proterminal regions. Sequence analysis of 25 loci revealed the majority to be authentic minisatellites with GC-rich repeat units ranging from 14 to 47 bp in length. We have further characterized 3 of the most polymorphic loci both inMus musculussubspecies and in inbred strains by using minisatellite variant repeat mapping (MVR) by PCR to gain insight into allelic diversity and turnover processes. MVR data suggest that mouse minisatellites mutate mainly by intra-allelic nonpolar events at a rate well below 10⁻³per gamete, in contrast to the high-frequency complex meiotic gene conversion-like events seen in humans. These results may indicate a fundamental difference in mechanisms of minisatellite mutation and genome turnover between mice and humans.     

Analysis of the novel VNTR polymorphisms ofMUC8 gene

Analysis of mucin genes has identified the presence of several features that may represent important functional domains in mucin glycoproteins. In the central region of each mucin, there are a variable number of tandem repeats (VNTR; minisatellites). However, their genomic levels are unclear because of complex genomic properties. We report here the distribution of VNTR and polymorphic analysis ofMUC8. We searched for VNTR ofMUC8 using the Tandem Repeat Finder program and found nine VNTR motif. Six (MUC8 MS1～MS6) among the nine VNTRs were evaluated in this study. Each VNTR inMUC8 region was analyzed in genomic DNA obtained from 200 unrelated individuals and multi-generational families. All VNTRs (MUC8 MS1, -MS2, -MS3, -MS4,-MS5 and -MS6) were genotyped as polymorphic. The degree of polymorphism within theMUC8-MS5 showed the highest heterozygosity (h = 0.786) in theMUC8 region. In order to perform a segregation analysis of the VNTRs inMUC8, we analyzed genomic DNA obtained from two generations of five families and from three generations of two families. Six of the polymorphic VNTRs were transmitted through meiosis following a Mendelian inheritance, which suggests that polymorphic VNTRs could be useful markers for paternity mapping and DNA fingerprinting.     

Analysis of VNTRs in the solute carrier family 6, member 18 (SLC6A18) and lack of association with hypertension

We report here the distribution of VNTRs (variable number of tandem repeats; minisatellites) and polymorphic analysis of SLC6A18, which is a member of the SLC6 Na(+)- and Cl(-)-dependent neurotransmitter transporter family. In this study, DNA was obtained from 300 unrelated individuals and 205 patients with essential hypertension (EH). We then analyzed the VNTRs in the genomic DNA by searching for minisatellites of SLC6A18 using the Tandem Repeat Finder program. Eight novel VNTRs were identified: five of which were polymorphic minisatellites (SLC6A18-MS1, -MS2, -MS4, -MS5, and -MS6) and three of which were monomorphic minisatellites (SLC6A18-MS3, -MS7, and -MS8). Next, we investigated the relationship between EH and four of the polymorphic minisatellites (SLC6A18-MS1, -MS2, -MS4, and -MS6). We excluded SLC6A18-MS5 from the common/rare allele analysis, because most individuals were heterozygous and hypervariable for this locus. There were no significant differences observed in the overall distribution of these minisatellites, which indicates that these polymorphisms are not responsible for EH susceptibility in the Korean population. A segregation analysis of the minisatellites in SLC6A18 was then conducted by analyzing genomic DNA obtained from two generations of five families and from three generations of two families. The five polymorphic minisatellites were transmitted through meiosis following Mendelian inheritance, which suggests that polymorphic minisatellites could be useful markers for paternity mapping and DNA fingerprinting. In summary, we discovered five novel VNTR polymorphisms in SLC6A18; however, these variations were not associated with EH.     

Intrinsic polymorphism of variable number tandem repeat loci in the human genome.

In the human genome, short tandem repetitive (STR) DNA sequences often show restriction fragment length polymorphisms (RFLPs) due to variation in the number of copies of the repeat unit. For a subset of these sequences known as minisatellites or variable number tandem repeat loci (VNTR), it has been proposed that a homologous "core" sequence of 10-12 nucleotides is involved in the mechanism(s) generating the polymorphism. In our present study we have prepared oligonucleotide probes complementary to one or two repeat units of several VNTR loci. Under stringent hybridization and wash conditions these probes hybridize locus specifically thus allowing the evaluation of the intrinsic polymorphism of individual loci. Our results indicate that not all of the loci having STR DNA sequences are polymorphic despite the fact that they share the "core" sequence. This suggests that more than the DNA sequence of the locus is involved in the mechanism(s) generating the polymorphism.     

A GGCAGG motif in minisatellites affecting their germline instability

Mouse and human genomes contain hypervariable DNA regions consisting of tandem repeats of a short sequence referred to as minisatellites. This variation is thought to arise through processes such as unequal crossover or replication slippage. A mo-1 minisatellite probe comprising a 14-base pair repeat sequence reveals many polymorphic fragments even in DNA of BALB/c sublines. Oligonucleotide probes with single base substitution in the mo-1 have been synthesized and used for assessing sequence involved in generation of polymorphisms. The results indicate that the loci containing mo-1 homologues with mutation in the GGCAGG sequence are monomorphic despite the other mutants showing polymorphism. Reciprocally, locus-specific polymorphic clones, Pc-1 and Pc-2, have been isolated with hybridization to mo-1, and both are shown to contain repeated sequence comprising the GGCAGG sequence. They reveal high mutation rates of 8.8% and 3.3% per gamete, respectively. These results strongly suggest that the motif contributes to the germline instability of minisatellites.     

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.     

Conservation of intronic minisatellite polymorphisms in the SCK1/SHC2 gene of Hominidae

The neuronally expressed Shc adaptor homolog SCK1/SHC2 gene contains an unusually high number of minisatellites. In humans, twelve different minisatellite sequences are located in introns of SCK1/SHC2 and ten of them are highly polymorphic. Here we used primers developed for humans to screen ten intronic loci of SCK1/SHC2 in chimpanzee and gorilla, and undertook a comprehensive analysis of the genomic sequence to address the evolutionary events driving these variable repeats. All ten loci amplified in chimpanzee and gorilla contained hypervariable and low-variability minisatellites. The human polymorphic locus TR1 was monomorphic in chimpanzee and gorilla, but we detected polymorphic alleles in these apes for the human monomorphic TR7 locus. When we examined the repeat size among these hominoids, there was no consistent variation by length from humans to great apes. In spite of the inconsistent evolutionary dynamics in repeat length variation, exon 16 was highly conserved between humans and great apes. These results suggest that non-coding intronic minisatellites do not show a consistent evolutionary paradigm but evolved with different patterns among each minisatellite locus. These findings provide important insight for minisatellite conservation during hominoid evolution.