Microsatellite Nucleotide Research Articles

The nucleotide composition of microsatellites impacts both replication fidelity and mismatch repair in human colorectal cells

Introduction: Microsatellite instability is a key mechanism of colon carcinogenesis. We have previously studied mutations within a (CA)13 microsatellite using an EGFP-based reporter-assay that allows the distinction of replication errors and mismatch repair activity. Here we utilize this assay to compare mutations of mono- and dinucleotide repeats in human colorectal cells. Methods: HCT116 and HCT116+chr3 cells were stably transfected with EGFP-based plasmids harboring A10, G10, G16, (CA)13, and (CA)26 repeats. EGFP-positive mutant fractions were quantitated by flow cytometry, mutation rates were calculated, and the mutant spectrum was analyzed by cycle sequencing. Results: EGFP fluorescence pattern changed with the microsatellite's nucleotide sequence and cell type and clonal variations were observed in mononucleotide repeats. Replication errors (as calculated in HCT116) at A10 repeats were 5 to 10-fold higher than in G10, G16 were 30-fold higher than G10, and (CA) 26 were 10-fold higher than (CA)13. The mutation rates in hMLH1-proficient HCT116+chr3 were 30 to 230-fold lower than in HCT116. Mismatch repair was more efficient in G16 than in A10 clones leading to a higher stability of poly-G tracts. Mutation spectra revealed predominantly 1-unit-deletions in A10, (CA)13, and G10 and 2-unit-deletions or 1-unit-insertion in (CA)26. Discussion: These findings indicate that both replication fidelity and mismatch repair are affected by the microsatellite's nucleotide composition.

The nucleotide composition of microsatellites impacts both replication fidelity and mismatch repair in human colorectal cells

Microsatellite instability is a key mechanism of colon carcinogenesis. We have previously studied mutations within a (CA)13 microsatellite using an enhanced green fluorescent protein (EGFP)-based reporter assay that allows the distinction of replication errors and mismatch repair (MMR) activity. Here we utilize this assay to compare mutations of mono- and dinucleotide repeats in human colorectal cells. HCT116 and HCT116+chr3 cells were stably transfected with EGFP-based plasmids harboring A10, G10, G16, (CA)13 and (CA)26 repeats. EGFP-positive mutant fractions were quantitated by flow cytometry, mutation rates were calculated and the mutant spectrum was analyzed by cycle sequencing. EGFP fluorescence pattern changed with the microsatellite's nucleotide sequence and cell type and clonal variations were observed in mononucleotide repeats. Replication errors (as calculated in HCT116) at A10 repeats were 5–10-fold higher than in G10, G16 were 30-fold higher than G10 and (CA)26 were 10-fold higher than (CA)13. The mutation rates in hMLH1-proficient HCT116+chr3 were 30–230-fold lower than in HCT116. MMR was more efficient in G16 than in A10 clones leading to a higher stability of poly-G tracts. Mutation spectra revealed predominantly 1-unit deletions in A10, (CA)13 and G10 and 2-unit deletions or 1-unit insertion in (CA)26. These findings indicate that both replication fidelity and MMR are affected by the microsatellite's nucleotide composition.

Analysis of cpSSR in triticale plants obtained byin vitroandrogenesis

In vitro androgenesis allows haploid and double haploid homozygous plants to be obtained. However, in cereals, non-viable albino plants are often produced. This limits the use of this technique both in basic studies and in plant genetic improvement programs. We have analyzed eight microsatellites of the chloroplast genome (cpSSR) in a collection of 46 double haploid green lines derived from the intervarietal hybrid ‘Torote’ × ‘Presto’, and 49 haploid albino plants all obtained from the double haploid line ‘ATOPE-41’, in order to know the stability of this sequences after the androgenetic process. The green plants showed no variations at the analyzed loci, while two of the albino plants differed at two loci: WCt2 and WCt9. These variations notably affected the microsatellite nucleotide sequences as well as the sequences surrounding them. The changes observed consisted of insertions, deletions and nucleotide substitutions, with transition and transversion types equally common. The latter albino plants showed several alleles at the polymorphic loci, indicating the existence of heteroplasmy.

A study on relative abundance, composition and length variation of microsatellites in 18 underutilized crop species

In the present work, genomic DNA libraries for 18 underutilized crop species (including 11 dicot and 7 monocot species) were enriched for several di- and tri- nucleotide microsatellites by using an optimized procedure. About 500–960 clones from each library were sequenced and all the sequences were characterized to have a comparative look on relative abundance, composition and length variations of perfect microsatellites among different crops. Sequence analysis revealed contrasting differences in the abundance of di- and tri-nucleotide microsatellites with the predominance of tri-nucleotide microsatellites in 11 crops and that of di-nucleotides in other 8 crops. Among di-nucleotide microsatellites, AG/GA class was the most abundant in all the crops except for four crops in which AC/CA class was predominant. Among tri-nucleotide repeats, AGC/GCA/CAG class was the most abundant in eight crops followed by AGG/GGA/GAG class in four crops, whereas in the remaining six crops, the most abundant class was highly variable. The longest di- and tri-nucleotide repeats were observed in Chinese cabbage and mungbean with the average lengths of 70 and 57 base pairs, respectively. These observations revealed species-specific in the distributions of microsatellite repeat motifs. The present study provides highly valuable information that can be useful in targeted development of specific microsatellites markers for genetic analysis of these underutilized crops.

Existence of microsatellites in expressed sequence tags of common carp (Cyprinus carpio L.) available in GenBank dbEST database

Common carp expressed sequence tags (ESTs) were analyzed for the existence of microsatellites, or simple sequence repeats (SSRs). In the NCBI dbEST database, a total of 10612 sequences were registered before December 31, 2004. A complete search of 2–6 nucleotide microsatellites resulted in the identification of 513 SSR-containing ESTs, accounting for 4.8% of the total. Cluster analysis indicated that 73 sequences of SSR-containing ESTs fell into 27 groups and the remaining 440 ESTs were indenpendent. A total of 467 unique SSR-containing ESTs were identified. These EST-SSRs contained a variety of simple sequence types, and di- and tri-nucleotide repeats were the most abundant, accounting for 42.1% and 27.9% of the whole, respectively. Of the dinucleotide repeats, CA/TG was the most abundant, followed by GA/TC. BLASTx search showed that 38.1% of the SSR loci could be associated with genes or proteins of known or unknown function. BLASTx searches of SSR-containing ESTs also showed high frequencies (98/179) of hits on zebrafish sequences.