Mutations in Haemophilus influenzae mismatch repair genes increase mutation rates of dinucleotide repeat tracts but not dinucleotide repeat-driven pilin phase variation rates

High-frequency, reversible switches in expression of surface antigens, referred to as phase variation (PV), are characteristic of Haemophilus influenzae. PV enables this bacterial species, an obligate commensal and pathogen of the human upper respiratory tract, to adapt to changes in the host environment. Phase-variable hemagglutinating pili are expressed by many H. influenzae isolates. PV involves alterations in the number of 5' TA repeats located between the -10 and -35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate biosynthesis and assembly of pili. Dinucleotide repeat tracts are destabilized by mismatch repair (MMR) mutations in Escherichia coli. The influence of mutations in MMR genes of H. influenzae strain Rd on dinucleotide repeat-mediated PV rates was investigated by using reporter constructs containing 20 5' AT repeats. Mutations in mutS, mutL, and mutH elevated rates approximately 30-fold, while rates in dam and uvrD mutants were increased 14- and 3-fold, respectively. PV rates of constructs containing 10 to 12 5' AT repeats were significantly elevated in mutS mutants of H. influenzae strains Rd and Eagan. An intact hif locus was found in 14 and 12% of representative nontypeable H. influenzae isolates associated with either otitis media or carriage, respectively. Nine or more tandem 5' TA repeats were present in the promoter region. Surprisingly, inactivation of mutS in two serotype b H. influenzae strains did not alter pilin PV rates. Thus, although functionally analogous to the E. coli MMR pathway and active on dinucleotide repeat tracts, defects in H. influenzae MMR do not affect 5' TA-mediated pilin PV.     

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.     

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.      

The distribution and frequency of microsatellite loci in Drosophila melanogaster

We report the results of a comprehensive search of Drosophila melanogaster DNA sequences in GenBank for di-, tri-, and tetranucleotide repeats of more than four repeat units, and a DNA library screen for dinucleotide repeats. Dinucleotide repeats are more abundant (66%) than tri- (30%) or tetranucleotide (4%) repeats. We estimate that 1917 dinucleotide repeats with 10 or more repeat units are present in the euchromatic D. melanogaster genome and, on average, they occur once every 60 kb. Relative to many other animals, dinucleotide repeats in D. melanogaster are short. Tri- and tetranucleotide repeats have even fewer repeat units on average than dinucleotide repeats. Our WorldWide Web site (http://www.bio.cornell.edu/genetics/aquadro/aquadro.html) posts the complete list of 1298 microsatellites (≥ five repeat units) identified from the GenBank search. We also summarize assay conditions for 70 D. melanogaster microsatellites characterized in previous studies and an additional 56 newly characterized markers.     

Isolation and characterization of microsatellite markers in the Serra Spanish mackerel, Scomberomorus brasiliensis

Thirteen nuclear-encoded microsatellites from a genomic DNA library of Serra Spanish mackerel, Scomberomorus brasiliensis, were isolated and characterized. The microsatellites include 10 perfect repeats (eight tetranucleotide and two dinucleotide) and three imperfect repeats (two tetranucleotide and one dinucleotide). An additional five microsatellites, isolated originally from two congeneric species (S. cavalla and S. niphonius), were characterized in S. brasiliensis. Serra Spanish mackerel support artisanal fisheries along the Caribbean and Atlantic coasts of Central and South America, from Belize to Brazil.     

Characterization of rabbit DNA micros extracted from the EMBL nucleotide sequence database

Microsatellite polymorphisms are invaluable for mapping vertebrate genomes. In order to estimate the occurrence of microsatellites in the rabbit genome and to assess their feasibility as markers in rabbit genetics, a survey on the presence of all types of mononucleotide, dinucleotide, trinucleotide and tetranucleotide repeats, with a length of about 20 bp or more, was conducted by searching the published rabbit DNA sequences in the EMBL nucleotide database (version 32). A total of 181 rabbit microsatellites could be extracted from the present database. The estimated frequency of microsatellites in the rabbit genome was one microsatellite for every 2–3 kb of DNA. Dinucleotide repeats constituted the prevailing class of microsatellites, followed by trinucleotide, mononucleotide and tetranucleotide repeats, respectively. The average length of the microsatellites, as found in the database, was 26, 23, 23 and 22 bp for mono-, di-, tri- and tetranucleotide repeats, respectively. The most common repeat motif was AG, followed by A, AC, AGG and CCG. This group comprised about 70% of all extracted rabbit microsatellites. About 61% of the microsatellites were found in non-coding regions of genes, whereas 15% resided in (protein) coding regions. A significant fraction of rabbit microsatellites (about 22%) was found within interspersed repetitive DNA sequences.     

Eucalyptus microsatellites mined in silico: survey and evaluation

Eucalyptus is an important short rotation pulpy woody plant, grown widely in the tropics. Recently, many genomic programmes are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. These sequences can be utilized for analysis of simple sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) available in the transcribed genes. In this study, in silico analysis of 15,285 sequences representing partial and full-length mRNA from Eucalyptus species for their use in developing SSRs or microsatellites were carried out. A total of 875 EST-SSRs were identified from 772 SSR containing ESTs. Motif size of 6 for dinucleotide and 5 for trinucleotide, tetranucleotide, and pentanucleotides were considered in locating the microsatellites. The average frequency of identified SSRs was 12.9%. The dinucleotide repeats were the most abundant among the dinucleotide, trinucleotide and tetranucleotide motifs and accounted for 50.9% of the Eucalyptus genome. Primer designing analysis showed that 571 sequences with SSRs had sufficient flanking regions for polymerase chain reaction (PCR) primer synthesis. Evaluation of the usefulness of the SSRs showed that EST-derived SSRs can generate polymorphic markers as all the primers showed allelic diversity among the 16 provenances of E. tereticornis.     

Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes.

We examined the stability of microsatellites of different repeat unit lengths in Saccharomyces cerevisiae strains deficient in DNA mismatch repair. The msh2 and msh3 mutations destabilized microsatellites with repeat units of 1, 2, 4, 5, and 8 bp; a poly(G) tract of 18 bp was destabilized several thousand-fold by the msh2 mutation and about 100-fold by msh3. The msh6 mutations destabilized microsatellites with repeat units of 1 and 2 bp but had no effect on microsatellites with larger repeats. These results argue that coding sequences containing repetitive DNA tracts will be preferred target sites for mutations in human tumors with mismatch repair defects. We find that the DNA mismatch repair genes destabilize microsatellites with repeat units from 1 to 13 bp but have no effect on the stability of minisatellites with repeat units of 16 or 20 bp. Our data also suggest that displaced loops on the nascent strand, resulting from DNA polymerase slippage, are repaired differently than loops on the template strand.     

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.     

Control of GT repeat stability in Schizosaccharomyces pombe by mismatch repair factors

The mismatch repair (MMR) system ensures genome integrity by removing mispaired and unpaired bases that originate during replication. A major source of mutational changes is strand slippage in repetitive DNA sequences without concomitant repair. We established a genetic assay that allows measuring the stability of GT repeats in the ade6 gene of Schizosaccharomyces pombe. In repair-proficient strains most of the repeat variations were insertions, with addition of two nucleotides being the most frequent event. GT repeats were highly destabilized in strains defective in msh2 or pms1. In these backgrounds, mainly 2-bp insertions and 2-bp deletions occurred. Surprisingly, essentially the same high mutation rate was found with mutants defective in msh6. In contrast, a defect in swi4 (a homologue of Msh3) caused only slight effects, and instability was not further increased in msh6 swi4 double mutants. Also inactivation of exo1, which encodes an exonuclease that has an MMR-dependent function in repair of base-base mismatches, caused only slightly increased repeat instability. We conclude that Msh2, Msh6, and Pms1 have an important role in preventing tract length variations in dinucleotide repeats. Exo1 and Swi4 have a minor function, which is at least partially independent of MMR.     

An unusually low microsatellite mutation rate in Dictyostelium discoideum, an organism with unusually abundant microsatellites

The genome of the social amoeba Dictyostelium discoideum is known to have a very high density of microsatellite repeats, including thousands of triplet microsatellite repeats in coding regions that apparently code for long runs of single amino acids. We used a mutation accumulation study to see if unusually high microsatellite mutation rates contribute to this pattern. There was a modest bias toward mutations that increase repeat number, but because upward mutations were smaller than downward ones, this did not lead to a net average increase in size. Longer microsatellites had higher mutation rates than shorter ones, but did not show greater directional bias. The most striking finding is that the overall mutation rate is the lowest reported for microsatellites: approximately 1 x 10(-6) for 10 dinucleotide loci and 6 x 10(-6) for 52 trinucleotide loci (which were longer). High microsatellite mutation rates therefore do not explain the high incidence of microsatellites. The causal relation may in fact be reversed, with low mutation rates evolving to protect against deleterious fitness effects of mutation at the numerous microsatellites.     

Twelve novel polymorphic microsatellites in a marine fish species,yellow croaker Larimichthys polyactis

We isolated 12 polymorphic microsatellites from an important marine food fish Larimichthys polyactis and characterized them in 32 unrelated individuals. Among the 12 microsatellites, four were tetranucleotide repeats and eight were dinucleotide repeats. The allele number ranged from five to 25 with an average of 15.4/locus; average expected heterozygosity was 0.81, ranging from 0.57 to 0.95, whereas the observed heterozygosity ranged from 0.34 to 1.00 (average: 0.78). Nine of the 12 markers conformed to Hardy–Weinberg equilibrium and showed no sign of linkage. These microsatellites will be useful for population genetic studies and selective breeding programs of this species.     

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.     

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     

赤拟谷盗全基因组和EST中微卫星的丰度

微卫星是近年大力开发的一种分子标记,为了推进赤拟谷盗Tribolium castaneum(Herbst)遗传学相关研究,对赤拟谷盗全基因组和EST中由1~6个碱基重复单元组成的简单序列重复进行分析,进而对其微卫星的丰度和分布进行比较分析。微卫星在赤拟谷盗EST中的分布频率为1/0.87kb,其中单碱基重复序列占71.25%,是最丰富的重复单元,而六、三、四、二,五碱基重复单元序列分别占23.93%,2.94%,1.56%,0.17%,0.15%。全基因组中微卫星的分布频率为1/3.65kb,其中六碱基重复序列占61.96%,是最丰富的重复单元,而三,四,一,五,二碱基重复单元序列分别占14.35%,13.75%,4.68%,3.60%,1.69%。同时发现富含A和T碱基的微卫星占主导地位,富含G和C碱基的微卫星数量较少。进一步的分析显示,微卫星在每条染色体上的丰度存在很大的相似性。     

Mutation rate variation at human dinucleotide microsatellites

Mutation is the ultimate source of genetic variation, and mutation rate is thus an important parameter governing the extent of genetic variation. Microsatellites are highly informative genetic markers that have been widely used in genetic studies. While previous studies showed that the mutation rate differs in di-, tri-, and tetranucleotide repeats, how mutation rate distributes within each class of repeat is poorly understood. This study first revealed the pattern of the mutation rate variation within the dinucleotide repeats. Two data sets were used. The first is the allele frequency data from 115 microsatellites with dinucleotide repeats distributed along the human genome in 10 worldwide populations. The second data set is much larger, consisting of the allele frequency of 5252 dinucleotide repeats from the Genome Database. Mutation rate for each locus is estimated through a new homozygosity-based estimator, which has been shown to be unbiased and highly efficient and is reasonably robust against deviations from the single-step model. The mutation rates among loci can be approximated well by a gamma distribution and its shape parameter can be accurately estimated with this approach. This result provides the basic guidelines for analyzing the large-scale genomic data from microsatellite loci.     

Bioinformatic Mining of Type I Microsatellites from Expressed Sequence Tags of Channel Catfish (<Emphasis Type="Italic">Ictalurus punctatus</Emphasis>)

Gene-derived markers are pivotal to the analysis of genome structure, organization, and evolution and necessary for comparative genomics. However, gene-derived markers are relatively difficult to develop. This project utilized the genomic resources of channel catfish expressed sequence tags (ESTs) to identify simple sequence repeats (SSRs), or microsatellites. It took the advantage of ESTs for the establishment of gene identities, and of microsatellites for the acquisition of high polymorphism. When microsatellites are tagged to genes, the microsatellites can then be used as gene markers. A bioinformatic analysis of 43,033 ESTs identified 4855 ESTs containing microsatellites. Cluster analysis indicated that 1312 of these ESTs fell into 569 contigs, and the remaining 3534 ESTs were singletons. A total of 4103 unique microsatellite-containing genes were identified. The dinucleotide CA/TG and GA/TC pairs were the most abundant microsatellites. AT-rich microsatellite types were predominant among trinucleotide and tetranucleotide microsatellites, consistent with our earlier estimation that the catfish genome is highly AT-rich. Our preliminary results indicated that the majority of the identified microsatellites were polymorphic and, therefore, useful for genetic linkage mapping of catfish. Mapping of these gene-derived markers is under way, which will set the foundation for comparative genome analysis in catfish.     

Microsatellite DNA markers for population genetic studies and parentage assignment in cobia,Rachycentron canadum

Twenty nuclear‐encoded microsatellites from a genomic DNA library of cobia, Rachycentron canadum, were isolated and characterized. The microsatellites include two tetranucleotide, one trinucleotide, three combination tetranucleotide/dinucleotide, nine dinucleotide, and five imperfect (dinucleotide) repeat motifs. Gene diversity ranged between zero to 0.910; the number of alleles among a sample of 24 fish ranged from one to 15. Cobia support an important recreational fishery in the southeastern United States and recently have become of interest to aquaculture. The microsatellites developed will be useful tools for studying both population genetics (e.g. stock structure, effective population size) and inheritance of traits important to aquaculture.     

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli

The influence of mutations in the 3' to 5' exonucleolytic proofreading epsilon-subunit of Escherichia coli DNA polymerase III on the genetic instabilities of the CGG.CCG and the CTG.CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49(ts) and the mutD5 mutations destabilize the CGG.CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG.CAG repeats were destabilized by the dnaQ49(ts) mutation by a process mediated by long hairpin loop structures (>/=5 repeats). The mutD5 mutator strain stabilized the (CTG.CAG)(175) tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in the mutD5 strain, presumably due to the greater stability of long CTG.CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5 strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG.CAG and CGG.CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.