Highly Specific Contractions of a Single CAG/CTG Trinucleotide Repeat by TALEN in Yeast

Trinucleotide repeat expansions are responsible for more than two dozens severe neurological disorders in humans. A double-strand break between two short CAG/CTG trinucleotide repeats was formerly shown to induce a high frequency of repeat contractions in yeast. Here, using a dedicated TALEN, we show that induction of a double-strand break into a CAG/CTG trinucleotide repeat in heterozygous yeast diploid cells results in gene conversion of the repeat tract with near 100% efficacy, deleting the repeat tract. Induction of the same TALEN in homozygous yeast diploids leads to contractions of both repeats to a final length of 3–13 triplets, with 100% efficacy in cells that survived the double-strand breaks. Whole-genome sequencing of surviving yeast cells shows that the TALEN does not increase mutation rate. No other CAG/CTG repeat of the yeast genome showed any length alteration or mutation. No large genomic rearrangement such as aneuploidy, segmental duplication or translocation was detected. It is the first demonstration that induction of a TALEN in an eukaryotic cell leads to shortening of trinucleotide repeat tracts to lengths below pathological thresholds in humans, with 100% efficacy and very high specificity.     

Distribution and abundance of microsatellites in the yeast genome can Be explained by a balance between slippage events and point mutations

We fit a Markov chain model of microsatellite evolution introduced by Kruglyak et al. to data on all di-, tri-, and tetranucleotide repeats in the yeast genome. Our results suggest that many features of the distribution of abundance and length of microsatellites can be explained by this simple model, which incorporates a competition between slippage events and base pair substitutions, with no need to invoke selection or constraints on the lengths. Our results provide some new information on slippage rates for individual repeat motifs, which suggest that AT-rich trinucleotide repeats have higher slippage rates. As our model predicts, we found that many repeats were adjacent to shorter repeats of the same motif. However, we also found a significant tendency of microsatellites of different motifs to cluster.     

Expansion of GAA trinucleotide repeats in mammals

We have previously shown that GAA trinucleotide repeats have undergone significant expansion in the human genome. Here we present the analysis of the length distribution of all 10 nonredundant trinucleotide repeat motifs in 20 complete eukaryotic genomes (6 mammalian, 2 nonmammalian vertebrates, 4 arthropods, 4 fungi, and 1 each of nematode, amoebozoa, alveolate, and plant), which showed that the abundance of large expansions of GAA trinucleotide repeats is specific to mammals. Analysis of human-chimpanzee-gorilla orthologs revealed that loci with large expansions are species-specific and have occurred after divergence from the common ancestor. PCR analysis of human controls revealed large expansions at multiple human (GAA)(30+) loci; nine loci showed expanded alleles containing >65 triplets, analogous to disease-causing expansions in Friedreich ataxia, including two that are in introns of genes of unknown function. The abundance of long GAA trinucleotide repeat tracts in mammalian genomes represents a significant mutation potential and source of interindividual variability.     

Differential distribution of simple sequence repeats in eukaryotic genome sequences.

Complete chromosome/genome sequences available from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Saccharomyces cerevisiae were analyzed for the occurrence of mono-, di-, tri-, and tetranucleotide repeats. In all of the genomes studied, dinucleotide repeat stretches tended to be longer than other repeats. Additionally, tetranucleotide repeats in humans and trinucleotide repeats in Drosophila also seemed to be longer. Although the trends for different repeats are similar between different chromosomes within a genome, the density of repeats may vary between different chromosomes of the same species. The abundance or rarity of various di- and trinucleotide repeats in different genomes cannot be explained by nucleotide composition of a sequence or potential of repeated motifs to form alternative DNA structures. This suggests that in addition to nucleotide composition of repeat motifs, characteristic DNA replication/repair/recombination machinery might play an important role in the genesis of repeats. Moreover, analysis of complete genome coding DNA sequences of Drosophila, C. elegans, and yeast indicated that expansions of codon repeats corresponding to small hydrophilic amino acids are tolerated more, while strong selection pressures probably eliminate codon repeats encoding hydrophobic and basic amino acids. The locations and sequences of all of the repeat loci detected in genome sequences and coding DNA sequences are available at http://www.ncl-india.org/ssr and could be useful for further studies.     

Comparison of simple sequence repeats in 19 Archaea

All organisms that have been studied until now have been found to have differential distribution of simple sequence repeats (SSRs), with more SSRs in intergenic than in coding sequences. SSR distribution was investigated in Archaea genomes where complete chromosome sequences of 19 Archaea were analyzed with the program SPUTNIK to find di- to penta-nucleotide repeats. The number of repeats was determined for the complete chromosome sequences and for the coding and non-coding sequences. Different from what has been found for other groups of organisms, there is an abundance of SSRs in coding regions of the genome of some Archaea. Dinucleotide repeats were rare and CG repeats were found in only two Archaea. In general, trinucleotide repeats are the most abundant SSR motifs; however, pentanucleotide repeats are abundant in some Archaea. Some of the tetranucleotide and pentanucleotide repeat motifs are organism specific. In general, repeats are short and CG-rich repeats are present in Archaea having a CG-rich genome. Among the 19 Archaea, SSR density was not correlated with genome size or with optimum growth temperature. Pentanucleotide density had an inverse correlation with the CG content of the genome.     

Distribution and evolution of short tandem repeats in closely related bacterial genomes

Simultaneous identification and comparison of perfect and imperfect microsatellites within a genome is a valuable tool both to overcome the lack of a consensus definition of SSRs and to assess repeat history. Detailed analysis of the overall distribution of perfect and imperfect microsatellites in closely related bacterial taxa is expected to give new insight into the evolution of prokaryotic genomes. We have performed a genome-wide analysis of microsatellite distribution in four Escherichia coli and seven Chlamydial strains. Chlamydial strains generally have a higher density of SSRs and show greater intra-group differences of SSR distribution patterns than E. coli genomes. In most investigated genomes the distribution of the total lengths of matching perfect and imperfect trinucleotide repeats are highly similar, with the notable exception of C. muridarum. Closely related strains show more similar repeat distribution patterns than strains separated by a longer divergence time. The discrepancy between the preferred classes of perfect and imperfect repeats in C. muridarum implies accelerated evolution of SSRs in this particular strain. Our results suggest that microsatellites, although considerably less abundant than in eukaryotic genomes, may nevertheless play an important role in the evolution of prokaryotic genomes and several gene families.     

Mining and survey of simple sequence repeats in wheat rust Puccinia sp

The abundance and inherent potential for extensive allelic variations in simple sequence repeats (SSRs) or microsatellites resulted in valuable source for genetic markers in eukaryotes. In this study, we analyzed and compared the abundance and organisation of SSR in the genome of two important fungal pathogens of wheat, brown or leaf rust (Puccinia triticina) and black or stem rust (Puccinia graminis f. sp. tritici). P. triticina genome with two fold genome size as compared to P. graminis tritici has lower relative abundance and SSR density. The distribution pattern of different SSR motifs provides the evidence of greater accumulation of dinucleotide followed by trinucleotide repeats. More than two-hundred different types of repeat motifs were observed in the genomes. The longest SSR motifs varied in both genomes and some of the repeat motifs are found in higher frequency. The information about survey of relative abundance, relative density, length and frequency of different repeat motifs in Puccinia sp. will be useful for developing SSR markers that could find several applications in analysis of fungal genome such as genetic diversity, population genetics, race identification and acquisition of new virulence.     

Features of trinucleotide repeat instability in vivo

Unstable repeats are associated with various types of cancer and have been implicated in more than 40 neurode-generative disorders. Trinucleotide repeats are located in non-coding and coding regions of the genome. Studies of bacteria, yeast, mice and man have helped to unravel some features of the mechanism of trinucleotide expansion. Looped DNA structures comprising trinucleotide repeats are processed during replication and/or repair to generate deletions or expansions. Most in vivo data are consistent with a model in which expansion and deletion occur by different mechanisms. In mammals, microsatellite instability is complex and appears to be influenced by genetic, epigenetic and developmental factors.     

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.     

蜜蜂全基因组中微卫星的丰度及其分布

查找出蜜蜂基因组中由1～6个碱基重复单元组成的简单序列重复,分析蜜蜂基因组中微卫星的分布频率,并比较其在各染色体中的分布频率。微卫星在蜜蜂基因组中的分布频率为1/0·804kb,其中二碱基重复序列占26·86%,是最丰富的重复单元,而六、一、三、四、五碱基重复单元序列分别占24·74%,22·19%,13·65%,10·98%,2·59%。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。第4,1,3条染色体微卫星分布频率较高,而第11,14,12条染色体微卫星分布频率较低。     

TRbase: a database relating tandem repeats to disease genes for the human genome

MOTIVATION: Tandem repeats are associated with disease genes, play an important role in evolution and are important in genomic organization and function. Although much research has been done on short perfect patterns of repeats, there has been less focus on imperfect repeats. Thus, there is an acute need for a tandem repeats database that provides reliable and up to date information on both perfect and imperfect tandem repeats in the human genome and relates these to disease genes. RESULTS: This paper presents a web-accessible relational tandem repeats database that relates tandem repeats to gene locations and disease genes of the human genome. In contrast to other available databases, this database identifies both perfect and imperfect repeats of 1-2000 bp unit lengths. The utility of this database has been illustrated by analysing these repeats for their distribution and frequencies across chromosomes and genomic locations and between protein-coding and non-coding regions. The applicability of this database to identify diseases associated with previously uncharacterized tandem repeats is demonstrated.     

High GC content of simple sequence repeats in Herpes simplex virus type 1 genome

The presence, locations and composition of simple sequence repeats (SSRs) in Herpes simplex virus type 1 (HSV-1) genome were extracted and analyzed by using the software Imperfect Microsatellite Extractor (IMEx). There were 663 mon-, 502 di-, 184 tri-, 20 tetra-, 4 penta- and 4 hexanucleotide SSRs that were observed in different distribution between coding and noncoding regions in the HSV-1 genome. G/C, GC/CG, and (GGC)(n) were predominant in mononucleotide, dinucletide, trinucleotide repeats respectively. Indeed, the results showed that GC content in simple sequence repeats was notably higher than that in entire HSV-1 genome. Our data might be helpful for studying the pathogenesis, genome structure and evolution of HSV-1.     

Analysis of distribution and significance of simple sequence repeats in enteric bacteria Shigella dysenteriae SD197

We have explored the possible role of SSR density in genome to generate biological information. In our study, we have checked the SSR (simple sequence repeats) status in virulent and non virulent genes of enteric bacteria to see whether the SSRs distribution contributes to virulence. The genome, plasmid and virulent genes sequences in fasta format were downloaded from NCBI GenBank and VFDB. The sequences were subjected to SSR analysis using software tool ssr.exe. The resulting data was pasted in excel sheet and further analyzed for percentage of each type of SSR. Higher nucleotide repeats have been observed in our study. Overall high density of SSRs can enhance antigenic variance of the pathogen population in a strategy that counteracts the host immune response. Frequency of A and T repeats is higher in the chromosome, plasmid and the virulence genes. However, in dinucleotide repeats the frequencies of GC/CG repeats are higher in genome, whereas plasmid has more of AT/TA repeats. Genome has trinucleotide repeats having predominantly G and C whereas plasmid has trinucleotide repeats having predominantly A and T. The repeat number obtained and percentage of repeats is higher in virulence genes as compared to other gene families. Due to the presence of this large number of SSRs, the organism has an enormous potential for generating this genomic and phenotypic diversity.     

Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations

Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR–Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)₃₃. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.     

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.     

Genome-wide comparative analysis of simple sequence coding repeats among 25 insect species

We present a detailed genome-scale comparative analysis of simple sequence repeats within protein coding regions among 25 insect genomes. The repetitive sequences in the coding regions primarily represented single codon repeats and codon pair repeats. The CAG triplet is highly repetitive in the coding regions of insect genomes. It is frequently paired with the synonymous codon CAA to code for polyglutamine repeats. The codon pairs that are least repetitive code for polyalanine repeats. The frequency of hexanucleotide and dinucleotide motifs of codon pair repeats is significantly (p<0.001) different in the Drosophila species compared to the non-Drosophila species. However, the frequency of synonymous and non-synonymous codon pair repeats varies in a correlated manner (r(2)=0.79) among all the species. Results further show that perfect and imperfect repeats have significant association with the trinucleotide and hexanucleotide coding repeats in most of these insects. However, only select species show significant association between the numbers of perfect/imperfect hexamers and repeat coding for single amino acid/amino acid pair runs. Our data further suggests that genes containing simple sequence coding repeats may be under negative selection as they tend to be poorly conserved across species. The sequences of coding repeats of orthologous genes vary according to the known phylogeny among the species. In conclusion, the study shows that simple sequence coding repeats are important features of genome diversity among insects.     

Haploinsufficiency of yeast FEN1 causes instability of expanded CAG/CTG tracts in a length-dependent manner

Trinucleotide repeat diseases, such as Huntington's disease, are caused by the expansion of trinucleotide repeats above a threshold of about 35 repeats. Once expanded, the repeats are unstable and tend to expand further both in somatic cells and during transmission, resulting in a more severe disease phenotype. Flap endonuclease 1 (Fen1), has an endonuclease activity specific for 5' flap structures and is involved in Okazaki fragment processing and base excision repair. Fen1 also plays an important role in preventing instability of CAG/CTG trinucleotide repeat sequences, as the expansion frequency of CAG/CTG repeats is increased in FEN1 mutants in vitro and in yeast cells defective for the yeast homolog, RAD27. Here we have tested whether one copy of yeast FEN1 is enough to maintain CAG/CTG tract stability in diploid yeast cells. We found that CAG/CTG repeats are stable in RAD27 +/- cells if the tract is 70 repeats long and exhibit a slightly increased expansion frequency if the tract is 85 or 130 repeats long. However for CAG-155 tracts, the repeat expansion frequency in RAD27 +/- cells is significantly higher than in RAD27 +/+ cells. This data indicates that cells containing longer CAG/CTG repeats need more Fen1 protein to maintain tract stability and that maintenance of long CAG/CTG repeats is particularly sensitive to Fen1 levels. Our results may explain the relatively small effects seen in the Huntington's disease (HD) FEN1 +/- heterozygous mice and myotonic dystrophy type 1 (DM1) FEN1 +/- heterozygous mice, and suggest that inefficient flap processing by Fen1 could play a role in the continued expansions seen in humans with trinucleotide repeat expansion diseases.     

Increased Instability of Human CTG Repeat Tracts on Yeast Artificial Chromosomes during Gametogenesis

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.