Development of a yeast-based assay system for monitoring microsatellite instability

Simple sequence repeats (microsatellites) are found in all eukaryotic genomes. Instabilities within these sequences have been associated with several human disorders including Huntington's chorea and myotonic dystrophy. Further studies have identified links between microsatellite instability, faulty mismatch repair and certain human cancers, in particular a form of hereditary colorectal cancer. The assay system described here consists of a congenic set of yeast strains mutated in DNA replication and mismatch repair genes and assay plasmids with which it is possible to measure differences in microsatellite stability in the range of 5-850-fold. The development of this technology will allow monitoring of environmental and dietary influences on the genomic stability in the context of human disease.     

Microsatellite Instability in Yeast: Dependence on the Length of the Microsatellite

One of the most common microsatellites in eukaryotes consists of tandem arrays [usually 15-50 base pairs (bp) in length] of the dinucleotide GT. We examined the rates of instability for poly GT tracts of 15, 33, 51, 99 and 105 bp in wild-type and mismatch repair-deficient strains of Saccharomyces cerevisiae. Rates of instability increased more than two orders of magnitude as tracts increased in size from 15 to 99 bp in both wild-type and msh2 strains. The types of alterations observed in long and short tracts in wild-type strains were different in two ways. First, tracts >/=51 bp had significantly more large deletions than tracts </=33 bp. Second, for the 99- and 105-bp tracts, almost all events involving single repeats were additions; for the smaller tracts, both additions and deletions of single repeats were common.     

Rate and pattern of mutation at microsatellite loci in maize

Microsatellites are important tools for plant breeding, genetics, and evolution, but few studies have analyzed their mutation pattern in plants. In this study, we estimated the mutation rate for 142 microsatellite loci in maize (Zea mays subsp. mays) in two different experiments of mutation accumulation. The mutation rate per generation was estimated to be 7.7 x 10(-4) for microsatellites with dinucleotide repeat motifs, with a 95% confidence interval from 5.2 x 10(-4) to 1.1 x 10(-3). For microsatellites with repeat motifs of more than 2 bp in length, no mutations were detected; so we could only estimate the upper 95% confidence limit of 5.1 x 10(-5) for the mutation rate. For dinucleotide repeat microsatellites, we also determined that the variance of change in the number of repeats (sigma(m)2) is 3.2. We sequenced 55 of the 73 observed mutations, and all mutations proved to be changes in the number of repeats in the microsatellite or in mononucleotide tracts flanking the microsatellite. There is a higher probability to mutate to an allele of larger size. There is heterogeneity in the mutation rate among dinucleotide microsatellites and a positive correlation between the number of repeats in the progenitor allele and the mutation rate. The microsatellite-based estimate of the effective population size of maize is more than an order of magnitude less than previously reported values based on nucleotide sequence variation.     

Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines

At least nine human genetic diseases, including myotonic dystrophy (DM) and fragile X syndrome have been associated with the expansion of CTG or CGG trinucleotide repeats within the disease loci. Little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. Mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats. The effect of mutations in two mismatch repair genes on the size of trinucleotide repeats in the DM and FRAXA loci has been analyzed. PCR and Southern analysis of the triplet repeat regions of the DM and fragile X mental retardation (FRAXA) loci in cell lines HTC116 and LoVo, which contain mutations in both alleles of the hMLH1 and hMSH2 genes, respectively, indicated that the size of the endogenous (CTG)_n and (CGG)_n tracts fall within the range observed in the normal population. This suggests that mutations in hMLH1 or hMSH2 do not result in the instability of CTG or CGG tracts to the levels observed in individuals with myotonic dystrophy or fragile X syndrome. Received: 4 December 1995 / Revised: 29 January 1996, 7 March 1996     

Mutation rates in the complex microsatellite MYCL1 and related simple repeats in cultured human cells

Microsatellite instability is a phenotype observed in tumors cells that have defects in DNA mismatch repair (MMR). Most markers used for detecting microsatellite instability are mono- and dinucleotide repeats, but one tetranucleotide repeat (MYCL1) has been reported to be useful for this purpose. The MYCL1 repeat is actually a complex repeat, made up of approximately 14 GAAA tetranucleotides plus various other GA-rich repeats. In order to determine the nature of the instability of the this sequence, we have used a frameshift-reversion assay in MMR-proficient and -deficient human cells to compare the mutation rates and the types of mutation of MYCL1 to those of the related simple repeats (GAAA)17, (GA)17, and (CA)17. We found that the complex repeat was the most stable of the repeats examined in cells deficient in MMR; the tetranucleotide was less stable, while the dinucleotides were the least stable. In MMR-proficient cells, the relative rates were reversed; the MYCL1 repeat was the least stable, the tetranucleotide was more stable, and the dinucleotides were the most stable. These results suggest that MYCL1 and the pure tetranucleotide have relatively low rates of errors during replication, but that the errors in these repeats are corrected less efficiently than those in the smaller repeats. Because of their high rate of instability in MMR-proficient cells, MYCL1 and other tetranucleotide repeats appear to lack specificity for detection of tumors with defective MMR.     

Distribution of dinucleotide microsatellites in the Drosophila melanogaster genome.

Microsatellites, a special class of repetitive DNA, have become one of the most popular genetic markers. The progress of various genome projects has made it possible to study the genomic distribution of microsatellites and to evaluate the potential influence of several parameters on their genesis. We report the distribution of dinucleotide microsatellites in the genome of Drosophila melanogaster. When considering only microsatellites with five or more repeat units, the average length of dinucleotide repeats in D. melanogaster is 6.7 repeats. We tested a wide range of parameters which could potentially influence microsatellite density, and we did not detect a significant influence of recombination rate, number of exons, or total length of coding sequence. In concordance with the neutral expectation for the origin of microsatellites, a significant positive correlation between AT content and (AT/TA)n microsatellite density was detected. While this pattern may indicate that microsatellite genesis is a random process, we also found evidence for a nonrandom distribution of microsatellites. Average microsatellite density was higher on the X chromosome, but extreme heterogeneity was observed between different genomic regions. Such a clumping of microsatellites was also evident on a more local scale, as 38.9% of the contiguous sequences analyzed showed a deviation from a random distribution of microsatellites.     

Simple sequences

Simple sequences (or microsatellites) are stretches of monotonous repetitions of short (1–5 bp) nucleotide motifs that are distributed across the whole genome in eukaryotes. They are probably generated by slippage during replication and their primary mutation rate seems to be controlled predominantly by the efficiency of the mismatch repair system. Although most mutations in simple sequence loci appear to be neutral, some mutations in particular stretches have been implicated as having a role in human genetic diseases.     

Mismatch repair-driven mutational bias in D. melanogaster

We have used microsatellite sequences to evaluate the influence of the mismatch repair system on mutation bias in D. melanogaster. While mismatch-proficient cells have the highest mutation rate at (GT)(n) repeats, (AT)(n) repeats were the least stable ones in spel1(-/-) flies lacking functional mismatch repair. Furthermore, the mutation spectrum of long microsatellite alleles in spel1(-/-) was slightly upward biased, resulting in a gain of repeats, whereas wild-type flies have a strong downward bias. Interestingly, this mismatch repair-mediated downward mutation bias is reflected in the genome composition of D. melanogaster. When compared to other species, D. melanogaster has significantly shorter microsatellites. Our results suggest that the mismatch repair system may have an important role in shaping genome composition.     

Low abundance of Escherichia coli microsatellites is associated with an extremely low mutation rate

It is widely assumed that microsatellites are generated by replication slippage, a mutation process specific to repetitive DNA. Consistent with their high mutation rate, microsatellites are highly abundant in most eukaryotic genomes. In Escherichia coli, however, microsatellites are rare and short despite the fact that a high microsatellite mutation rate was described. We show that this high microsatellite instability depends on the presence of the F-plasmid. E. coli cells lacking the F-plasmid have extremely low microsatellite mutation rates. This result provides a possible explanation for the genome-wide low density of microsatellites in E. coli. Furthermore, we show that the F-plasmid induced microsatellite instability is independent of the mismatch repair pathway.     

Artiodactyl retroposons: association with microsatellites and use in SINEmorph detection by PCR.

During a search of polymorphic microsatellites for bovine genome mapping, we found that microsatellites often occur as tails of artiodactyl C-A retroposon elements. In this element, C (85bp) is a tRNA derivative, while A (117bp) is of unknown origin. The A element also occurs as dimer element with a connecting 27bp linker sequence comprising hexanucleotide CACTTT repeats. In 10 clones (45% of those selected deliberately for dinucleotide repeats), the microsatellite motif is associated with the C-A retroposon. In 50% of 44 database artiodactyl C-A sequences, the element also has a microsatellite tail. The microsatellite is usually a simple (CA)n repeat, but in some cases it is an apparent derivative of the linker sequence CACTTT. All but one of 33 database dimer elements have trinucleotide repeat tails (AGC)n, n = 1-9. Microsatellites, retroposons, and their truncated versions (C and/or A) often occur as clusters. We derived the consensus sequence (202bp) of the C-A element, and designed four primers for inter-SINE amplification with the aim of finding SINEmorph polymorphisms. The method is potentially powerful for rapidly producing polymorphic markers for artiodactyl genome mapping.     

Transcribed dinucleotide microsatellites and their associated genes from channel catfish Ictalurus punctatus.

The presence of trinucleotide microsatellites within genes is a well-known cause for a number of genetic diseases. However, the precise distribution of dinucleotide microsatellites within genes is less well documented. Here we report 15 unique cDNAs containing dinucleotide repeats from the channel catfish Ictalurus punctatus. Gene identities of nine of the 15 cDNAs were determined, of which three encode structural genes, and six encode regulatory proteins. Five cDNAs harbored dinucleotide repeats in the 5' untranslated region (5'-NTR), nine in the 3'-NTR, and one in the coding region. The presence of these transcribed dinucleotide repeats and their potential expansion in size within coding regions could lead to disruption of the original protein and/or formation of new genes by frame shift. The low number of dinucleotide repeats within coding regions suggests that they were strongly selected against. All the transcribed microsatellite loci examined were polymorphic making them useful for gene mapping in catfish.     

Mononucleotide repeats represent an important source of polymorphic microsatellite markers in Aspergillus nidulans

In fungi, microsatellites occur less frequently throughout the genome and tend to be less polymorphic compared with other organisms. Most studies that develop microsatellites for fungi focus on dinucleotide and trinucleotide repeats, and thus mononucleotide repeats, which are much more abundant in fungal genomes, may represent an overlooked resource. This study examined the relative probabilities of polymorphism in mononucleotide, dinucleotide and trinucleotide repeats in Aspergillus nidulans. As previously found, the probability of polymorphism increased with increasing number of repeating units. Dinucleotide and trinucleotide repeats had higher probabilities of polymorphism than mononucleotide repeats, but this was offset by the presence of numerous long mononucleotide repeats within the genome. Mononucleotide microsatellites with 20 or more repeating units have a probability of polymorphism similar to dinucleotide and trinucleotide microsatellites, and therefore, consideration of mononucleotide repeats will substantially increase the number of potential markers available.     

Genetic recombination destabilizes (CTG)n.(CAG)n repeats in E. coli

The expansion of trinucleotide repeats has been implicated in 17 neurological diseases to date. Factors leading to the instability of trinucleotide repeat sequences have thus been an area of intense interest. Certain genes involved in mismatch repair, recombination, nucleotide excision repair, and replication influence the instability of trinucleotide repeats in both Escherichia coli and yeast. Using a genetic assay for repeat deletion in E. coli, the effect of mutations in the recA, recB, and lexA genes on the rate of deletion of (CTG)n.(CAG)n repeats of varying lengths were examined. The results indicate that mutations in recA and recB, which decrease the rate of recombination, had a stabilizing effect on (CAG)n.(CTG)n repeats decreasing the high rates of deletion seen in recombination proficient cells. Thus, recombination proficiency correlates with high rates of genetic instability in triplet repeats. Induction of the SOS system, however, did not appear to play a significant role in repeat instability, nor did the presence of triplet repeats in cells turn on the SOS response. A model is suggested where deletion during exponential growth may result from attempts to restart replication when paused at triplet repeats.     

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.     

Long tomato microsatellites are predominantly associated with centromeric regions.

Microsatellites as genetic markers are used in many crop plants. Major criteria for their usability as molecular markers include that they are highly polymorphic and evenly spread throughout a genome. In tomato, it has been reported that long arrays of tetranucleotide microsatellites containing the motif GATA are highly clustered around the centromeres of all chromosomes. In this study, we have isolated tomato microsatellites containing long arrays (> 20 repeats) of the dinucleotide motifs GA, GT, AT, as well as GATA, assessed their variability within Lycopersicon esculentum varieties and mapped them onto a genetic map of tomato. The investigated microsatellite markers exhibited between 1 and 5 alleles in a diverse set of L. esculentum lines. Mapping of the microsatellites onto the genetic map of tomato demonstrates that, as previously shown, GATA microsatellites are highly clustered in the regions of the tomato centromeres. Interestingly, the same centromeric location was now found for long dinucleotide microsatellite markers. Because of this uneven distribution, genetic mapping of the entire tomato genome using long dinucleotide microsatellites will be very difficult to achieve and microsatellite markers with shorter arrays of microsatellites could be more suitable for mapping experiments albeit their lower level of polymorphism. Some microsatellite markers described in this study might provide a useful tool to study the molecular structure of tomato centromeric regions and for variety identification.     

有尾噬菌体目中微卫星和复合型微卫星的发生及分析

简单重复序列亦称微卫星,被成功应用于许多真核生物、原核生物和病毒的基因组和进化研究,但是噬菌体中的微卫星目前很少被研究。因此对60条尾病毒目基因组中的微卫星和和复合型微卫星(由两个或两个以上直接相邻的微卫星组成)做综合性分析,在这60个基因组中总共观察到11 874个微卫星和449个复合型微卫星。相关性分析表明微卫星个数与基因组大小成正线性相关(ρ=0.899, P<0.01)。参考序列中的微卫星个数少于对应的随机序列中微卫星个数,这种反常现象主要是因为参考序列含有较少的单核苷酸和二核苷酸重复。A/T和AT/TA重复是单核苷酸和二核苷酸重复中最主要的类型,因此单核苷酸重复中的GC含量明显低于相应的序列中的GC含量;相比之下,微卫星中的二核苷酸和三核苷酸重复的GC含量与对应的参考序列的GC含量无明显区别。尾病毒目基因组中的这些结果与其它生物体基因组存在一定的差别。有助于了解尾病毒目中微卫星的分布、进化和生物学功能。     

Fragile X gene instability: anchoring AGGs and linked microsatellites.

Interspersed AGGs within the FMR1 gene CGG repeat region may anchor the sequence and prevent slippage during replication. In order to detect the AGG position variations, we developed a method employing partial MnlI restriction analysis and analyzed X chromosomes from 187 males, including 133 normal controls (117 with 20-34 and 16 with 35-52 repeats), plus 54 fragile X premutations with 56-180 repeats. Among controls, the interspersed AGG positions were highly polymorphic, with a heterozygosity of 91%. Among the control samples, 1.5% had no AGG positions, 25% had one, 71% had two, and 3% had three. Among the fragile X premutation samples, 63% had no AGG, while 37% had only one AGG. Analysis of premutation samples within fragile X families showed that variation occurred only within the 3' end of the region. Thus, the instability was polar. Controls with > or = 15 pure CGG repeats were associated with the longest alleles of two nearby microsatellites, FRAXAC1 with 20-21 repeats and DXS548 with 202-206 bp and with increased microsatellite heterozygosity. The association of long pure CGG regions, as with fragile X chromosomes, with the longer and more heterozygous microsatellite alleles suggests they may be related mechanistically. Further, our results do not support a recent suggestion that the frequency of fragile X alleles may be increasing. Finally, analysis of a set of nonhuman primate samples showed that long pure CGG tracks are variable in size and are located within the 3' region, which suggests that polar instability within FMR1 is evolutionarily quite old.     

Drosophila virilis has long and highly polymorphic microsatellites

Comparative genomics is a powerful approach to inference of the dynamics of genome evolution. Most information about the evolution of microsatellites in the genus Drosophila has been obtained from Drosophila melanogaster. For comparison, we collected microsatellite data for the distantly related species Drosophila virilis. Screening about 0.5 Mb of nonredundant genomic sequence from GenBank, we identified 239 dinucleotide microsatellites. On average, D. virilis dinucleotides were significantly longer than D. melanogaster microsatellites (7.69 repeats vs. 6.75 repeats). Similarly, direct cloning of microsatellites resulted in a higher mean repeat number in D. virilis than in D. melanogaster (12.7 repeats vs. 12.2 repeats). Characterization of 11 microsatellite loci mapping to division 40-49 on the fourth chromosome of D. virilis indicated that D. virilis microsatellites are more variable than those of D. melanogaster.     

Mutations in polI but not mutSLH destabilize Haemophilus influenzae tetranucleotide repeats

Haemophilus influenzae (Hi), an obligate upper respiratory tract commensal/pathogen, uses phase variation (PV) to adapt to host environment changes. Switching occurs by slippage of nucleotide repeats (microsatellites) within genes coding for virulence molecules. Most such microsatellites in Hi are tetranucleotide repeats, but an exception is the dinucleotide repeats in the pilin locus. To investigate the effects on PV rates of mutations in genes for mismatch repair (MMR), insertion/deletion mutations of mutS, mutL, mutH, dam, polI, uvrD, mfd and recA were constructed in Hi strain Rd. Only inactivation of polI destabilized tetranucleotide (5'AGTC) repeat tracts of chromosomally located reporter constructs, whereas inactivation of mutS, but not polI, destabilized dinucleotide (5'AT) repeats. Deletions of repeats were predominant in polI mutants, which we propose are due to end-joining occurring without DNA polymerization during polI-deficient Okazaki fragment processing. The high prevalence of tetranucleotides mediating PV is an exceptional feature of the Hi genome. The refractoriness to MMR of hypermutation in Hi tetranucleotides facilitates adaptive switching without the deleterious increase in global mutation rates that accompanies a mutator genotype.     

GenBank数据库中鳜鱼微卫星位点的筛选及特征分析

微卫星(Microsatellite)是一类由2-6个核苷酸经多次单位串联组成的高度变异重复DNA序列(Schlotterer and Tautz,1992)。它具有按照孟德尔方式分离、突变快、多态信息含量丰富、呈共显性遗传等特点,其核心序列在同一物种中具有保守性,因此,可以根据微卫星的侧翼序列设计合适的引