The myotonic dystrophy type 1 triplet repeat sequence induces gross deletions and inversions

The capacity of (CTG.CAG)n and (GAA.TTC)n repeat tracts in plasmids to induce mutations in DNA flanking regions was evaluated in Escherichia coli. Long repeats of these sequences are involved in the etiology of myotonic dystrophy type 1 and Friedreich's ataxia, respectively. Long (CTG.CAG)n (where n = 98 and 175) caused the deletion of most, or all, of the repeats and the flanking GFP gene. Deletions of 0.6-1.8 kbp were found as well as inversions. Shorter repeat tracts (where n = 0 or 17) were essentially inert, as observed for the (GAA.TTC)176-containing plasmid. The orientation of the triplet repeat sequence (TRS) relative to the unidirectional origin of replication had a pronounced effect, signaling the participation of replication and/or repair systems. Also, when the TRS was transcribed, the level of deletions was greatly elevated. Under certain conditions, 30-50% of the products contained gross deletions. DNA sequence analyses of the breakpoint junctions in 47 deletions revealed the presence of 1-8-bp direct or inverted homologies in all cases. Also, the presence of non-B folded conformations (i.e. slipped structures, cruciforms, or triplexes) at or near the breakpoints was predicted in all cases. This genetic behavior, which was previously unrecognized for a TRS, may provide the basis for a new type of instability of the myotonic dystrophy protein kinase (DMPK) gene in patients with a full mutation.     

Instability of CAG and CTG trinucleotide repeats in Saccharomyces cerevisiae.

A quantitative genetic assay was developed to monitor alterations in tract lengths of trinucleotide repeat sequences in Saccharomyces cerevisiae. Insertion of (CAG)50 or (CTG)50 repeats into a promoter that drives expression of the reporter gene ADE8 results in loss of expression and white colony color. Contractions within the trinucleotide sequences to repeat lengths of 8 to 38 restore functional expression of the reporter, leading to red colony color. Reporter constructs including (CAG)50 or (CTG)50 repeat sequences were integrated into the yeast genome, and the rate of red colony formation was measured. Both orientations yielded high rates of instability (4 x 10(-4) to 18 x 10(-4) per cell generation). Instability depended on repeat sequences, as a control harboring a randomized (C,A,G)50 sequence was at least 100-fold more stable. PCR analysis of the trinucleotide repeat region indicated an excellent correlation between change in color phenotype and reduction in length of the repeat tracts. No preferential product sizes were observed. Strains containing disruptions of the mismatch repair gene MSH2, MSH3, or PMS1 or the recombination gene RAD52 showed little or no difference in rates of instability or distributions of products, suggesting that neither mismatch repair nor recombination plays an important role in large contractions of trinucleotide repeats in yeast.     

Yeast telomere repeat sequence (TRS) improves circular plasmid segregation, and TRS plasmid segregation involves the RAP1 gene product.

Telomere repeat sequences (TRSs) can dramatically improve the segregation of unstable circular autonomously replicating sequence (ARS) plasmids in Saccharomyces cerevisiae. Deletion analysis demonstrated that yeast TRSs, which conform to the general sequence (C(1-3)A)n, are able to stabilize circular ARS plasmids. A number of TRS clones of different primary sequence and C(1-3)A tract length confer the plasmid stabilization phenotype. TRS sequences do not appear to improve plasmid replication efficiency, as determined by plasmid copy number analysis and functional assays for ARS activity. Pedigree analysis confirms that TRS-containing plasmids are missegregated at low frequency and that missegregated TRS-containing plasmids, like ARS plasmids, are preferentially retained by the mother cell. Plasmids stabilized by TRSs have properties that distinguish them from centromere-containing plasmids and 2 microns-based recombinant plasmids. Linear ARS plasmids, which include two TRS tracts at their termini, segregate inefficiently, while circular plasmids with one or two TRS tracts segregate efficiently, suggesting that plasmid topology or TRS accessibility interferes with TRS segregation function on linear plasmids. In strains carrying the temperature-sensitive mutant alleles rap1grc4 and rap1-5, TRS plasmids are not stable at the semipermissive temperature, suggesting that RAP1 protein is involved in TRS plasmid stability. In Schizosaccharomyces pombe, an ARS plasmid was stabilized by the addition of S. pombe telomere sequence, suggesting that the ability to improve the segregation of ARS plasmids is a general property of telomere repeats.     

SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS- strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences.     

Tandem duplication. A novel type of triplet repeat instability

Triplet repeat sequence (TRS) inserts containing (CTG.CAG)(n) (17-175 units in length) were tandemly duplicated when propagated in plasmids in Escherichia coli. The products of this novel type of TRS genetic instability are tracts of as many as 34 multiple units, which contain the entire TRS as well as 129 base pairs of nonrepetitive flanking sequence. The duplication process required the presence of two or more TRS-containing units. Close proximity (170 base pairs) of the TRS to the R6K gamma origin of replication of the pUTminiTn5Cm-derived constructs stimulated the tandem duplication process. These events are proposed to occur due to secondary structure formation, stalling of DNA synthesis, and slippage-mediated misalignment of the complementary strands relative to each other during DNA replication. This mechanism may account for the TRS-associated duplications in protein kinase and metalloprotease genes in neuroblastomas and melanomas, as well as the massive repeat expansions in type II triplet repeat neurological diseases.     

Fidelity of primate cell repair of a double-strand break within a (CTG).(CAG) tract. Effect of slipped DNA structures

At least 15 human diseases are caused by the instability of gene-specific (CTG).(CAG) repeats. The precise mechanism of instability remains unknown, though bacterial and yeast models have suggested a role for aberrant repair of double-strand breaks (DSBs). Using an established primate DSB repair system, we have investigated the fidelity of repair of a DSB within a (CTG).(CAG) repeat tract. DSB repair substrates were generated from plasmids that are stably replicated in their circular form, permitting us to highlight the effects of DSB repair on repeat stability and minimize the contribution of replication. DSBs were introduced into repeat-containing plasmids using a unique BsmI site, such that the entire repeat tract comprised one free end of the linearized plasmid. Substrates containing 17, 47, and 79 repeats, in either their linear duplex form or containing slipped structures (out-of-register interstrand mispairings at repeat sequences), were transiently transfected into primate cells. Linearized plasmids with repeats were repaired with mildly reduced efficiency, while the presence of slipped structures considerably reduced repair efficiency. The repaired products were characterized for alterations within the repeat tract and flanking sequence. DSB repair induced predominantly repeat deletions. Notably, a polarized/directional deletion effect was observed, in that the repetitive end of the DSB was preferentially removed. This phenomenon was dramatically enhanced when slipped structures were present within the repeat tract, providing the first evidence for error-prone processing of slipped-strand structures. These results suggest the existence of primate nuclease activities that are specific for (CTG).(CAG) repeats and the structures they form.     

DNA elements important for CAG*CTG repeat thresholds in Saccharomyces cerevisiae

Trinucleotide repeat (TNR) instability is of interest because of its central role in human diseases such as Huntington’s and its unique genetic features. One distinctive characteristic of TNR instability is a threshold, defined as a minimal repeat length that confers frequent mutations. While thresholds are well established, important risk determinants for disease-causing mutations, their mechanistic analysis has been delayed by the lack of suitably tractable experimental systems. In this study, we directly compared for the first time three DNA elements—TNR sequence, purity and flanking sequence—all of which are suggested in the literature to contribute to thresholds. In a yeast model system, we find that CAG repeats require a substantially longer threshold to contract than CTG tracts, indicating that the lagging template repeat sequence helps determine the threshold. In contrast, ATG interruptions within a CTG run do not inhibit contractions via a threshold mechanism, but by altering the likelihood of forming a hairpin intermediate. The presence of a GC-rich flanking sequence, similar to a haplotype found in some Huntington’s patients, does not detectably alter expansions of Okazaki fragment CTG tracts, suggesting no role for this flanking sequence on thresholds. Together these results help better define TNR thresholds by delineating sequence elements that modulate instability.     

Involvement of the nucleotide excision repair protein UvrA in instability of CAG*CTG repeat sequences in Escherichia coli.

Several human genetic diseases have been associated with the genetic instability, specifically expansion, of trinucleotide repeat sequences such as (CTG)(n).(CAG)(n). Molecular models of repeat instability imply replication slippage and the formation of loops and imperfect hairpins in single strands. Subsequently, these loops or hairpins may be recognized and processed by DNA repair systems. To evaluate the potential role of nucleotide excision repair in repeat instability, we measured the rates of repeat deletion in wild type and excision repair-deficient Escherichia coli strains (using a genetic assay for deletions). The rate of triplet repeat deletion decreased in an E. coli strain deficient in the damage recognition protein UvrA. Moreover, loops containing 23 CTG repeats were less efficiently excised from heteroduplex plasmids after their transformation into the uvrA(-) strain. As a result, an increased proportion of plasmids containing the full-length repeat were recovered after the replication of heteroduplex plasmids containing unrepaired loops. In biochemical experiments, UvrA bound to heteroduplex substrates containing repeat loops of 1, 2, or 17 CAG repeats with a K(d) of about 10-20 nm, which is an affinity about 2 orders of magnitude higher than that of UvrA bound to the control substrates containing (CTG)(n).(CAG)(n) in the linear form. These results suggest that UvrA is involved in triplet repeat instability in cells. Specifically, UvrA may bind to loops formed during replication slippage or in slipped strand DNA and initiate DNA repair events that result in repeat deletion. These results imply a more comprehensive role for UvrA, in addition to the recognition of DNA damage, in maintaining the integrity of the genome.     

The contribution of cis-elements to disease-associated repeat instability: clinical and experimental evidence

Alterations in the length (instability) of gene-specific microsatellites and minisatellites are associated with at least 35 human diseases. This review will discuss the various cis-elements that contribute to repeat instability, primarily through examination of the most abundant disease-associated repetitive element, trinucleotide repeats. For the purpose of this review, we define cis-elements to include the sequence of the repeat units, the length and purity of the repeat tracts, the sequences flanking the repeat, as well as the surrounding epigenetic environment, including DNA methylation and chromatin structure. Gender-, tissue-, developmental- and locus-specific cis-elements in conjunction with trans-factors may facilitate instability through the processes of DNA replication, repair and/or recombination. Here we review the available human data that supports the involvement of cis-elements in repeat instability with limited reference to model systems. In diverse tissues at different developmental times and at specific loci, repetitive elements display variable levels of instability, suggesting vastly different mechanisms may be responsible for repeat instability amongst the disease loci and between various tissues.     

Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats

A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT)(n).(AGAAT)(n) from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electrophoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT)(n).(AGAAT)(n) repeat forms an unpaired region, which further extends into adjacent A+T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT)(5).(AGAAT)(5) occurred while 2D gel analysis and chemical probing indicate greater unpairing in A+T-rich sequences in other regions of the plasmid. The observed experimental results are consistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A+T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high superhelical densities, DNA strands within the presumably compact A+T-rich region were accessible to small chemicals and oligonucleotide hybridization. Thus, DNA strands in this "collapsed structure" remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation.     

Generation of long tracts of disease-associated DNA repeats

The generation of long uninterrupted DNA repeats is important for the study of repeat instability associated with several human genetic diseases, including myotonic dystrophy type 1. However, obtaining defined lengths of long repeats in vitro has been problematic. Strand slippage and/or DNA secondary structure formation may prevent efficient ligation. For example, a purified (CTG)140.(CAG)140 repeat fragment containing 4-bp AGCA/TGCT overhanging ends ligated poorly using T4 or Escherichia coli DNA ligase, although limited repeat ligation occurred using thermostable DNA ligase. Here we describe a general procedure for ligating multimers of DNA repeats. Multimers are efficiently ligated when slippage is prevented or when DNA repeats contain a single G/C overhang. A cloning vector is designed from which pure repeat fragments containing a G/C overhang can be generated for further ligation. (CAG)n.(CTG)n DNA molecules longer than 800 bp were generated using this approach. This approach also worked for (GAA)n.(TTC)n, (CCTG)n-(CAGG)n, and (ATTCT)n.(AGAAT)n tracts associated with Friedreich ataxia, DM2, and spinocerebellar ataxia type 10, respectively.     

(dT-dC)n and (dG-dA)n tracts arrest single stranded DNA replication in vitro.

Previous in vivo studies have indicated that (dT-dC)n.(dG-dA)n tracts (referred to here as (TC)n.(GA)n), which are widely dispersed in vertebrate genomes, may serve as pause or arrest signals for DNA replication and amplification. To determine whether these repeat elements act as stop signals for DNA replication in vitro, single stranded DNAs including (TC)n or (GA)n tracts of various lengths, were prepared by cloning such tracts into phage M13 vectors, and were replicated with the Klenow fragment of the E. coli DNA polymerase I, or with the calf thymus DNA polymerase alpha, by extension of an M13 primer. Gel electrophoresis of the reaction products revealed that the replication was specifically arrested around the middle of both (TC)n and (GA)n tracts of n greater than or equal to 16. However, whereas in the (TC)n tracts the arrests were less prominent at pH = 8.0 than at pH = 6.5-7.5, and were completely eliminated at pH = 8.5, the arrests in the (GA)n tracts were stronger at the higher pH values. These results, and previous data, suggest that the arrests were caused by formation of unusual DNA structures, possibly triple helices between partially replicated (TC)n or (GA)n tracts, and unreplicated portions of these sequences.     

Isolation and Characterization of Microsatellites in Snap Bean

The objectives of this study were to isolate and characterize microsatellites from a heat tolerant variety of snap bean (Phaseolus vulgaris L.) in order to generate polymorphic genetic markers linked to quantitative trait loci for heat tolerance. A genomic library contained 400-800 bp inserts was constructed and screened for the presence of (GA/CT)n and (CA/GT)n repeats. The proportion of positive clones yielded estimated of 3.72×10 4 such dinucleotide repeats per genome, roughly comparable to the abundance reported in other eukaryotic genomes. Twenty-six positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT)n motif was much more abundant than the (CA/GT)n motif in these clones. The (GA/CT)n repeats also showed longer average repeat length (mean n=10.4 versus 6.5), suggesting that they are better candidates for yielding polymorphic genetic markers in the snap bean genome.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属(Eucalyptus)的10 000条EST序列进行分析, 在其中的1 499条序列上共发现1 775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外, 还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关; 微卫星的丰度与重复单元长度也呈负相关(三碱基重复微卫星除外)。在桉树EST序列中, 重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面, 桉树EST序列与杨树(Populus trichocarpa)基因组注释的转录序列随重复单元长度的变化呈现出相同的规律, 但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低, 推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计, 并对设计的引物进行PCR检测, 结果表明所设计的引物具有极高的扩增成功率。     

Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.)

 Ninety-four newly developed microsatellite markers were integrated into existing RFLP framework maps of four rice populations, including two doubled haploid, a recombinant inbred, and an interspecific backcross population. These simple sequence repeats (SSR) were predominantly poly(GA) motifs, targetted because of their abundance in rice. They were isolated from a previously described sheared library and a newly constructed enzyme-digested library. Differences in the average length of poly(GA) tracts were observed for clones isolated from the two libraries. The length of GA motifs averaged 21 repeat units for clones isolated from the Tsp-509-digested library, while motifs averaged 17 units for clones from the sheared library. There was no evidence of clustering of microsatellite markers near centromeres or telomeres. Mapping of the 94 newly developed markers as well as of 27 previously reported microsatellites provided genome-wide coverage of the 12 chromosomes, with an average distance of 1 SSLP (simple sequence repeat polymorphism) per 16–20 cM. Received: 13 February 1997/Accepted: 28 February 1997     

文蛤微卫星DNA的筛选及其特性分析

采用磁珠富集分离法从文蛤(Meretrix meretrix)的基因组中筛选得到49条微卫星DNA序列,其中两碱基重复有3种类型36个序列,四碱基重复有14种类型26个序列.重复次数在5～30之间的序列占75.6%,30次以上重复的序列占24.4%,最高重复次数为100.根据重复单元的排列特点,完美型、非完美型及混合型序列所占比例分别为61.2%、14.5%和24.5%.本研究中构建的文蛤微卫星文库将在文蛤种质资源评价及分子遗传学研究中发挥作用.