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1.
Jakupciak JP  Wells RD 《IUBMB life》2000,50(6):355-359
The expansion of triplet repeat sequences is an initial step in the disease etiology of a number of hereditary neurological disorders in humans. Diseases such as myotonic dystrophy, Huntington's, several spinocerebellar ataxias, fragile X syndrome, and Friedreich's ataxia are caused by the expansions of CTG.CAG, CGG.CCG, or GAA.TTC repeats. The mechanisms of the expansion process have been investigated intensely in E. coli, yeast, transgenic mice, mammalian cell culture, and in human clinical cases. Whereas studies from 1994-1999 have implicated DNA replication and repair at the paused synthesis sites due to the unusual conformations of the triplet repeat sequences, recent work has shown that homologous recombination (gene conversion) is a powerful mechanism for generating massive expansions, in addition to, or in concert with, replication and repair.  相似文献   

2.
Trinucleotide repeat (TNR) instability in humans is governed by unique cis-elements. One element is a threshold, or minimal repeat length, conferring frequent mutations. Since thresholds have not been directly demonstrated in model systems, their molecular nature remains uncertain. Another element is sequence specificity. Unstable TNR sequences are almost always CNG, whose hairpin-forming ability is thought to promote instability by inhibiting DNA repair. To understand these cis-elements further, TNR expansions and contractions were monitored by yeast genetic assays. A threshold of approximately 15--17 repeats was observed for CTG expansions and contractions, indicating that thresholds function in organisms besides humans. Mutants lacking the flap endonuclease Rad27p showed little change in the expansion threshold, suggesting that this element is not altered by the presence or absence of flap processing. CNG or GNC sequences yielded frequent mutations, whereas A-T rich sequences were substantially more stable. This sequence analysis further supports a hairpin-mediated mechanism of TNR instability. Expansions and contractions occurred at comparable rates for CTG tract lengths between 15 and 25 repeats, indicating that expansions can comprise a significant fraction of mutations in yeast. These results indicate that several unique cis-elements of human TNR instability are functional in yeast.  相似文献   

3.
The human cancer susceptibility gene, BRCA2, functions in double-strand break repair by homologous recombination, and it appears to function via interaction of a repetitive region (“BRC repeats”) with RAD-51. A putatively simpler homolog, dmbrca2, was identified in Drosophila melanogaster recently and also affects mitotic and meiotic double-strand break repair. In this study, we examined patterns of repeat variation both within Drosophila pseudoobscura and among available Drosophila genome sequences. We identified extensive variation within and among closely related Drosophila species in BRC repeat number, to the extent that variation within this genus recapitulates the extent of variation found across the entire animal kingdom. We describe patterns of evolution across species by documenting recent repeat expansions (sometimes in tandem arrays) and homogenizations within available genome sequences. Overall, we have documented patterns and modes of evolution in a new model system of a gene which is important to human health.  相似文献   

4.
Expansions of DNA trinucleotide repeats cause at least 17 inherited neurodegenerative diseases, such as Huntington''s disease. Expansions can occur at frequencies approaching 100% in affected families and in transgenic mice, suggesting that specific cellular proteins actively promote (favor) expansions. The inference is that expansions arise due to the presence of these promoting proteins, not their absence, and that interfering with these proteins can suppress expansions. The goal of this study was to identify novel factors that promote expansions. We discovered that specific histone deacetylase complexes (HDACs) promote CTG•CAG repeat expansions in budding yeast and human cells. Mutation or inhibition of yeast Rpd3L or Hda1 suppressed up to 90% of expansions. In cultured human astrocytes, expansions were suppressed by 75% upon inhibition or knockdown of HDAC3, whereas siRNA against the histone acetyltransferases CBP/p300 stimulated expansions. Genetic and molecular analysis both indicated that HDACs act at a distance from the triplet repeat to promote expansions. Expansion assays with nuclease mutants indicated that Sae2 is one of the relevant factors regulated by Rpd3L and Hda1. The causal relationship between HDACs and expansions indicates that HDACs can promote mutagenesis at some DNA sequences. This relationship further implies that HDAC3 inhibitors being tested for relief of expansion-associated gene silencing may also suppress somatic expansions that contribute to disease progression.  相似文献   

5.
Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases. The mechanism and timing of the expansion events are poorly understood, however. We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable. The instability is 6 to 10 times more pronounced in meiosis than during mitotic division. The influence of meiosis on instability is 4.4 times greater when the second YAC with a repeat tract is not present. Most of the changes we observed in trinucleotide repeat tracts are large contractions of 21 to 50 repeats. The orientation of the insert with the repeats has no effect on the frequency and distribution of the contractions. In our experiments, expansions were found almost exclusively during gametogenesis. Genetic analysis of segregating markers among meiotic progeny excluded unequal crossover as the mechanism for instability. These unique patterns have novel implications for possible mechanisms of repeat instability.  相似文献   

6.
Trinucleotide repeat (TNR) expansion is the causative mutation for at least 17 inherited neurological diseases. An important question in the field is which proteins drive the expansion process. This study reports that the multi-functional protein Sem1 is a novel driver of TNR expansions in budding yeast. Mutants of SEM1 suppress up to 90% of expansions. Subsequent analysis showed that Sem1 facilitates expansions via its function in the 26S proteasome, a highly conserved multi-subunit complex with both proteolytic and non-proteolytic functions. The proteolytic function of the 26S proteasome is relevant to expansions, as mutation of additional proteasome components or treatment of yeast with a proteasome inhibitor suppressed CTG•CAG expansions. The 26S proteasome also drives expansions in human cells. In a human astrocytic cell line, siRNA-mediated knockdown of 26S proteasome subunits PSMC5 or PSMB3 reduced expansions. This expansion phenotype, both in yeast and human cells, is dependent on the proteolytic activity of the proteasome rather than a stress response owing to depletion of free ubiquitin. Thus, the 26S proteasome is a novel factor that drives expansions in both yeast and human cells by a mechanism involving protein degradation.  相似文献   

7.
Trinucleotide repeats undergo contractions and expansions in humans, leading in some cases to fatal neurological disorders. The mechanism responsible for these large size variations is unknown, but replication-slippage events are often suggested as a possible source of instability. We constructed a genetic screen that allowed us to detect spontaneous expansions/contractions of a short trinucleotide repeat in yeast. We show that deletion of RAD27, a gene involved in the processing of Okazaki fragments, increases the frequency of contractions tenfold. Repair of a chromosomal double-strand break (DSB) using a trinucleotide repeat-containing template induces rearrangements of the repeat with a frequency 60 times higher than the natural rate of instability of the same repeat. Our data suggest that both gene conversion and single-strand annealing are major sources of trinucleotide repeat rearrangements. Received: 8 January 1999 / Accepted: 17 March 1999  相似文献   

8.
9.
The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5’ UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.  相似文献   

10.
Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in modulating both genomic and phenotypic diversity.  相似文献   

11.
Two component sensor-response regulator systems (TCSs) are very common in the genomes of the Streptomyces species that have been fully sequenced to date. It has been suggested that this large number is an evolutionary response to the variable environment that Streptomyces encounter in soil. Notwithstanding this, TCSs are also more common in the sequenced genomes of other Actinomycetales when these are compared to the genomes of most other eubacteria. In this study, we have used DNA/DNA genome microarray analysis to compare 14 Streptomyces species and one closely related genus to Streptomyces coelicolor in order to identify a core group of such systems. This core group is compared to the syntenous and non-syntenous TCSs present in the genome sequences of other Actinomycetales in order to separate the systems into those present in Actinomycetales in general, the Streptomyces specific systems and the species specific systems. Horizontal transfer does not seem to play a very important role in the evolution of the TCS complement analyzed in this study. However, cognate pairs do not necessarily seem to evolve at the same pace, which may indicate the evolutionary responses to environmental variation may be reflected differently in sequence changes within the two components of the TCSs. The overall analysis allowed subclassification of the orphan TCSs and the TCS cognate pairs and identification of possible targets for further study using gene knockouts, gene overexpression, reporter genes and yeast two hybrid analysis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

12.
Trinucleotide repeat expansions are now a well-established mutational mechanism in human genetic disease. An unstable CAG repeat is known to be responsible for three neurodegenerative disorders: Huntington's disease, spinal and bulbar musclar atrophy and spinocerebellar ataxia type 1. Similarities in the genetics of these diseases, the size of the repeat expansions and the position of the unstable repeat within the gene (when known) suggest a common basis to the observed phenotypes. The cloning of two regions at which chromosome breakage can be induced (FRAXA and FRAXE) has in each case uncovered an unstable CG-rich triplet repeat which becomes methylated when fully expanded. In addition to these two classes of mutation, the presence of an expanded CTG repeat in the 3′ untranslated region of a protein kinase causes myotonic dystrophy. The size of the respective expansions, repeat stability, mutational origins and possible mechanisms of action are discussed.  相似文献   

13.
Aldose reductase (AR) is considered a potential mediator of diabetic complications and is a drug target for inhibitors of diabetic retinopathy and neuropathy in clinical trials. However, the physiological role of this enzyme still has not been established. Since effective inhibition of diabetic complications will require early intervention, it is important to delineate whether AR fulfills a physiological role that cannot be compensated by an alternate aldo–keto reductase. Functional genomics provides a variety of powerful new tools to probe the physiological roles of individual genes, especially those comprising gene families. Several eucaryotic genomes have been sequenced and annotated, including yeast, nematode and fly. To probe the function of AR, we have chosen to utilize the budding yeast Saccharomyces cerevisiae as a potential model system. Unlike Caenorhabditis elegans and D. melanogaster, yeast provides a more desirable system for our studies because its genome is manipulated more readily and is able to sustain multiple gene deletions in the presence of either drug or auxotrophic selectable markers. Using BLAST searches against the human AR gene sequence, we identified six genes in the complete S. cerevisiae genome with strong homology to AR. In all cases, amino acids thought to play important catalytic roles in human AR are conserved in the yeast AR-like genes. All six yeast AR-like open reading frames (ORFs) have been cloned into plasmid expression vectors. Substrate and AR inhibitor specificities have been surveyed on four of the enzyme forms to identify, which are the most functionally similar to human AR. Our data reveal that two of the enzymes (YDR368Wp and YHR104Wp) are notable for their similarity to human AR in terms of activity with aldoses and substituted aromatic aldehydes. Ongoing studies are aimed at characterizing the phenotypes of yeast strains containing single and multiple knockouts of the AR-like genes.  相似文献   

14.
Single nucleotide polymorphism (SNP) markers have become a genetic technology of choice because of their automation and high precision of allele calls. In this study, our goal was to develop 94 SNPs and test them across well-chosen common bean (Phaseolus vulgaris L.) germplasm. We validated and accessed SNP diversity at 84 gene-based and 10 non-genic loci using KASPar technology in a panel of 70 genotypes that have been used as parents of mapping populations and have been previously evaluated for SSRs. SNPs exhibited high levels of genetic diversity, an excess of middle frequency polymorphism, and a within-genepool mismatch distribution as expected for populations affected by sudden demographic expansions after domestication bottlenecks. This set of markers was useful for distinguishing Andean and Mesoamerican genotypes but less useful for distinguishing within each gene pool. In summary, slightly greater polymorphism and race structure was found within the Andean gene pool than within the Mesoamerican gene pool but polymorphism rate between genotypes was consistent with genepool and race identity. Our survey results represent a baseline for the choice of SNP markers for future applications because gene-associated SNPs could themselves be causative SNPs for traits. Finally, we discuss that the ideal genetic marker combination with which to carry out diversity, mapping and association studies in common bean should consider a mix of both SNP and SSR markers.  相似文献   

15.
Friedreich ataxia (FRDA) is primarily caused by an unstable GAA repeat-expansion mutation within intron 1 of the FRDA gene. However, the exact mechanisms leading to this expansion and its consequences are not fully understood. To study the dynamics of this mutation, we have generated two lines of human FRDA YAC transgenic mice that contain GAA repeat expansions within the appropriate genomic context. We have detected intergenerational instability and age-related somatic instability in both lines, with pronounced expansions found in the cerebellum. The dynamic nature of our transgenic GAA repeats is comparable with previous FRDA patient somatic tissue data. However, there is a difference between our FRDA YAC transgenic mice and other trinucleotide-repeat mouse models, which do not show pronounced repeat instability in the cerebellum. This represents the first mouse model of FRDA GAA repeat instability that will help to dissect the mechanism of this repeat.  相似文献   

16.
C K Cemal  C Huxley  S Chamberlain 《Gene》1999,236(1):53-61
Machado-Joseph disease or spinocerebellar ataxia 3 (SCA3) is a progressive neurodegenerative disorder caused by pathological expansion of a trinucleotide repeat motif present within exon 4 of the MJD1 gene. Previous attempts to create a transgenic animal model have failed to produce a neurological deficit truly representative of the disease phenotype. This appears to be the result of inappropriate expression of the mutant protein in neuronal populations generally spared in the disease state. Introduction of a human disease gene in the context of a yeast artificial chromosome clone containing endogenous regulatory elements would enhance the potential for correct tissue/cell-specific expression at physiological levels. We report the introduction of expanded CAG repeat motifs into a 250kb yeast artificial chromosome clone spanning the MJD1 locus using two rounds of homologous recombination. Transformants exhibited both expansions and contractions of the motif with alleles ranging in size from 48 to 84 repeat units. The availability of these clones for modelling of the disease in transgenic animals should allow elucidation of the role of repeat length in the phenotypic spectrum of the disease.  相似文献   

17.
Summary We describe the structure of a gene expressed in the salivary gland cells of the dipteranChironomus tentans and show that it encodes 1 of the approximately 15 secretory proteins exported by the gland cells. This sp115,140 gene consists of approximately 65 copies of a 42-bp sequence in a central uninterrupted core block, surrounded by short nonrepetitive regions. The repeats within the gene are highly similar to each other, but divergent repeats are present in a pattern which suggests that the repeat structure has been remodeled during evolution. The 42-bp repeat in the gene is a simple variant of the more complex repeat unit present in the Balbiani ring genes, encoding four of the other secretory proteins. The structure of the sp115,140 gene suggests that related repeat structures have evolved from a common origin and resulted in the set of genes whose secretory proteins interact in the assembly of the secreted protein fibers.  相似文献   

18.
Aldose reductase (AR) is considered a potential mediator of diabetic complications and is a drug target for inhibitors of diabetic retinopathy and neuropathy in clinical trials. However, the physiological role of this enzyme still has not been established. Since effective inhibition of diabetic complications will require early intervention, it is important to delineate whether AR fulfills a physiological role that cannot be compensated by an alternate aldo-keto reductase. Functional genomics provides a variety of powerful new tools to probe the physiological roles of individual genes, especially those comprising gene families. Several eucaryotic genomes have been sequenced and annotated, including yeast, nematode and fly. To probe the function of AR, we have chosen to utilize the budding yeast Saccharomyces cerevisiae as a potential model system. Unlike Caenorhabditis elegans and D. melanogaster, yeast provides a more desirable system for our studies because its genome is manipulated more readily and is able to sustain multiple gene deletions in the presence of either drug or auxotrophic selectable markers. Using BLAST searches against the human AR gene sequence, we identified six genes in the complete S. cerevisiae genome with strong homology to AR. In all cases, amino acids thought to play important catalytic roles in human AR are conserved in the yeast AR-like genes. All six yeast AR-like open reading frames (ORFs) have been cloned into plasmid expression vectors. Substrate and AR inhibitor specificities have been surveyed on four of the enzyme forms to identify, which are the most functionally similar to human AR. Our data reveal that two of the enzymes (YDR368Wp and YHR104Wp) are notable for their similarity to human AR in terms of activity with aldoses and substituted aromatic aldehydes. Ongoing studies are aimed at characterizing the phenotypes of yeast strains containing single and multiple knockouts of the AR-like genes.  相似文献   

19.
Sopher BL  Myrick SB  Hong JY  Smith AC  La Spada AR 《Gene》2000,261(2):383-390
Production of mouse models of inherited neurodegenerative diseases is an important step towards understanding the mechanism of neurotoxicity and for testing potential therapies. We are interested in creating a mouse model for X-linked spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by expansion of a CAG repeat within the androgen receptor (AR) gene. To permit generation of mice that will show a SBMA phenotype within their life span, we decided to obtain a yeast artificial chromosome (YAC) carrying the AR gene and introduce CAG repeat mutations numbering 100 or more triplets. SBMA patients with more than 70 CAGs have never been observed; therefore, we chose to expand a 59 CAG repeat tract in vivo in Escherichia coli. Although we set out to expand this repeat tract using a recombination paradigm involving two plasmid co-propagation, we did not observe large expansions. We were instead able to incrementally generate repeat tracts from 100 to 200 CAGs in a yeast integrating plasmid vector by taking advantage of replication instability. In the course of our experiments that yielded these CAG repeat tracts, we evaluated the role of repeat orientation, vector co-propagation, and recA function on the expansion process. We then used one of the yeast integrating vectors to successfully produce an AR YAC construct carrying 100 CAG repeats. AR YAC CAG100 will serve as a valuable reagent for the production of a SBMA mouse.  相似文献   

20.
Siyanova  E. Yu.  Mirkin  S. M. 《Molecular Biology》2001,35(2):168-182
This review describes a novel type of genome instability, expansion of trinucleotide repeats. Originally discovered in 1991 upon cloning the gene responsible for the fragile X syndrome, it has proved to be a general phenomenon responsible for a growing number of human neurological disorders. Besides apparent medical importance, the discovery of trinucleotide repeat expansion unraveled a fundamental problem of human genetics: a non-Mendelian type of inheritance called anticipation. Understanding the mechanisms of repeat expansion and the molecular pathways leading from these expansions to human diseases became a formidable task for modern biology and one of its spectacular achievements. Here we discuss the major breakthroughs in this field made during the last decade, with an emphasis on molecular models of repeat expansion.  相似文献   

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