Text S1
Friedreich's Ataxia (GAA)n•(TTC)n Repeats Strongly Stimulate Mitotic Crossovers
in Saccharomyces cerevisae.
Wei Tang1, Margaret Dominska1, Patricia W. Greenwell1, Zachary Harvanek1, Kirill S.
Lobachev2, Hyun-Min Kim2,3, Vidhya Narayanan2, Sergei M. Mirkin4, and Thomas D.
Petes1*
1Department
of Molecular Genetics and Microbiology, Duke University, Durham, NC
27710, USA. 2School of Biology and Institute for Bioengineering and Bioscience,
Georgia Institute of Technology, Atlanta, GA 30332, USA. 3Present address:
Department of Genetics, Harvard Medical School, Boston, MA 02115. 4Department of
Biology, Tufts University, Medford, MA 02155, USA.
*Corresponding author
Department of Molecular Genetics and Microbiology
Duke University Medical Center
Durham, NC 27710, USA
Strain constructions
Lee et al. [1] previously described the construction of two haploid strains derived
from different genetic backgrounds: PSL2 derived from W303a [2] and PSL5 derived
from YJM789 [3]. Both PSL2 and PSL5 have the wild-type RAD5 gene. These strains
have diverged to the extent of about six single-nucleotide polymorphisms (SNPs) per kb
[3]. Diploids formed by crossing these strains, therefore, are heterozygous for many
markers that can be used to map mitotic recombination events. Genotypes of haploids
used in strain constructions are in Table S1, oligonucleotides used in constructions are
in Table S2, and diploid genotypes are in Table S3.
A precursor strain to PSL2 was the haploid MAB1 ([4]; genotype: MATcan1-100
leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 V9229::HYG V261553::HIS3 RAD5). The
LYS2 gene on chromosome II was removed from MAB1 by the delitto perfetto
procedure [5]. First, the primers LYS2-DEL-F and LYS2-DEL-R were used to amplify a
KANMX-URA3-cassette contained in plasmid pCORE [5]. The resulting fragment was
transformed into MAB1, generating strain HMK151. We subsequently transformed
HMK151 with the partially-complementary oligonucleotides DEL-CORE-F and DELCORE-R, selecting for a 5-FOAR derivative that was also geneticin-sensitive, resulting in
the strain HMK160. HMK160 was transformed with a PCR fragment generated by
amplifying DNA from the plasmid pFL39LYS2 with oligonucleotides LYS2-INS-F and
LYS2-INS-R; the plasmid pFL39LYS2 contains a wild-type LYS2 gene inserted as an
EcoRI-HindIII fragment in the vector pFL39 [6]. The resulting Lys+ strain, HK9, had a
4.5 kb LYS2 insertion between bases 115902 and 115903 of chromosome V (the
GEA2-URA3 intergenic region). HK9 was then transformed with a DNA fragment
generated by BsrGI digestion of pFL39LYS2::CORE; this plasmid is a derivative of
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pFL39LYS2 in which the 3.2 kb KANMX-URA3 cassette is inserted in the BamHI site
within the LYS2 gene. In the resulting transformant (HK10), the 3.2 kb KANMX-URA3
cassette is integrated into the LYS2 gene on chromosome V approximately 3.2 kb from
the beginning of the LYS2 coding sequence.
The next step in the construction was to replace the KANMX-URA3 cassette with
(GAA)n(TTC)n tracts of different sizes and orientations. HK10 was transformed with
PCR fragments containing the tracts, selecting 5-FOAR derivatives and screening for
those that were also geneticin-sensitive. These fragments were generated by
amplification of genomic DNA from previously constructed yeast strains containing
(GAA)n(TTC)n tracts of various sizes [7] using the primers LYS2-TRACT-F and LYS2TRACT-R. The resulting transformed strains were HK11-(GAA)20, HK17-(GAA)230,
HK20-(TTC)20, and HK26-(TTC)230. The tracts in these strains are located about 1.2 kb
centromere-distal to URA3 on chromosome V. All transformants were checked to be
sure that the tracts were of the correct size by PCR using the primers AAG-F and AAGR.
We constructed URA3 derivatives of HK11-(GAA)20, HK17-(GAA)230, HK20-(TTC)20,
and HK26-(TTC)230 (MD500, MD501, MD502, and MD503, respectively) by
transformation of the haploids with a PCR fragment resulting from amplification of
genomic MAB10 DNA [4] with the primers wtURA3F and wtURA3R. As described
below, genomic DNA from strains MD500-MD503 was used to construct the strains
WXT10-WXT13, and WXT30-WXT33.
For strains MD500-503, the (GAA)n(TTC)n tracts are embedded within the 4.2 kb
LYS2 gene. We constructed derivatives of these strains in which the tracts were
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inserted into a truncated lys2 gene about 1.1 kb in size located between GEA2 and
URA3 at the same position as the longer insertion. To construct WXT10, we amplified
genomic DNA from MD502 with the primers AAG-BIG1-F and AAG-BIG1-R. The
resulting fragment, which contains the (GAA)n(TTC)n tract and the closely-linked URA3
gene, was transformed into PSL5, selecting Ura+ transformants. A similar procedure
with the same primers was used to construct WX11-WXT13. For these strains, PSL5
was transformed with a PCR fragment in which the sources of genomic DNA used for
the amplifications were MD503 for WXT11, MD500 for WXT12, and MD501 for WXT13.
The WXT30-WXT33 strains were derived from PSL2 in two steps. First, using the
same primers that were employed in constructing WXT10-13, we generated PCR
fragments that contained the (GAA)n(TTC)n tracts and URA3; these fragments were
used to transform PSL2 to Ura+. The sources of genomic DNA used in these
constructions were: MD502 for WXT20, MD503 for WXT21, MD500 for WXT22, and
MD501 for WXT23. We then constructed ura3 derivatives of WXT20, 21, 22, and 23
(WXT30, 31, 32, and 33, respectively) by transformation of WXT20-23 with a PCRgenerated fragment with a mutant ura3 gene; this fragment was the result of
amplification of PSL2 genomic DNA with the primers URA3-F and URA3-R. Uratransformants were selected with medium that contained 5-fluoro-orotate. The haploids
WXT30-33 were crossed to the haploids WXT10-13 in various combinations to generate
the diploids WXTMD40-46 (Table S3).
The haploid strains MD510 and MD512 are isogenic derivatives of PSL2 with
insertions of (GAA)230 and (TTC)230 near URA3. The context of the tract insertion is
identical to that in strains MD500-MD503. MD510 was a spore of the diploid MD506
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(MAB4 x MD501); MAB4 has been previously described [4]. MD512 was a spore of the
diploid MD508 (MAB4 x MD503). MD510 and MD512 are isogenic except for the
orientation of the trinucleotide tract (Table S1).
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References
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Chromosome fragility at GAA tracts in yeast depends on repeat orientation and
requires mismatch repair. Embo J 27: 2896-2906.
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