Instability of repetitive DNA sequences causes numerous hereditary disorders in humans, the majority of which are associated with trinucleotide repeat expansions. Here we describe a unique system to study instability of triplet repeats in a yeast experimental settings. Using fluctuation assay and the novel program FluCalc we are able to accurately estimate the rates of large-scale expansions, as well as repeat-mediated mutagenesis and gross chromosomal rearrangements for different repeat sequences.
Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.
The Peterhof genetic collection of Saccharomyces cerevisiae strains (PGC) is a large laboratory stock that has accumulated several thousands of strains for over than half a century. It originated independently of other common laboratory stocks from a distillery lineage (race XII). Several PGC strains have been extensively used in certain fields of yeast research but their genomes have not been thoroughly explored yet. Here we employed whole genome sequencing to characterize five selected PGC strains including one of the closest to the progenitor, 15V-P4, and several strains that have been used to study translation termination and prions in yeast (25-25-2V-P3982, 1B-D1606, 74-D694, and 6P-33G-D373). The genetic distance between the PGC progenitor and S288C is comparable to that between two geographically isolated populations. The PGC seems to be closer to two bakery strains than to S288C-related laboratory stocks or European wine strains. In genomes of the PGC strains, we found several loci which are absent from the S288C genome; 15V-P4 harbors a rare combination of the gene cluster characteristic for wine strains and the RTM1 cluster. We closely examined known and previously uncharacterized gene variants of particular strains and were able to establish the molecular basis for known phenotypes including phenylalanine auxotrophy, clumping behavior and galactose utilization. Finally, we made sequencing data and results of the analysis available for the yeast community. Our data widen the knowledge about genetic variation between Saccharomyces cerevisiae strains and can form the basis for planning future work in PGC-related strains and with PGC-derived alleles.
In humans, telomeric repeats (TTAGGG)n are known to be present at internal chromosomal sites. These interstitial telomeric sequences (ITSs) are an important source of genomic instability, including repeat length polymorphism, but the molecular mechanisms responsible for this instability remain to be understood. Here, we studied the mechanisms responsible for expansions of human telomeric (Htel) repeats that were artificially inserted inside a yeast chromosome. We found that Htel repeats in an interstitial chromosome position are prone to expansions. The propensity of Htel repeats to expand depends on the presence of a complex of two yeast proteins: Tbf1 and Vid22. These two proteins are physically bound to an interstitial Htel repeat, and together they slow replication fork progression through it. We propose that slow progression of the replication fork through the protein complex formed by the Tbf1 and Vid22 partners at the Htel repeat cause DNA strand slippage, ultimately resulting in repeat expansions.
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