SUPPLEMENTARY MATERIAL
Strategy for engineering the translocations into S.cerevisiae
In order to generate the reciprocal chromosomal translocation, the FY23 (Kanr, Ura+) strain
was transformed with the plasmid Yep351-cre-cyh, which contains the Cre recombinase
gene, with the Candida maltosa cycloheximide-resistance gene, and the S. cerevisiae LEU2
gene as selectable markers1.
Transformants were selected on SD Leu- minimal medium
plates, and growing the cells in YPGal medium induced the Cre recombinase. The selectable
markers, at the two sites, were excised and, in a proportion of the survivors, centromere-distal
fragments were exchanged between chromosomes VI and VII to generate a reciprocal
chromosomal translocation (VItVII and VIItVI). Survivors were screened for those bearing
the required translocation by diagnostic PCR, using oligonucleotides annealing to regions
flanking either side of the putative translocation breakpoint on chromosome VI or VII (see
Fig.1 in paper). The change in the karyotype was verified by CHEF analysis and the strain,
bearing the required translocation, was designated Sct1.
Sct1 was then manipulated to generate a second translocation between the right arm of
chromosome VI (which comprises the genetic material of chromosome VII, because of the
translocation introduced in Sct1) and the left arm of chromosome XVI, in order to render it
colinear with the second S. mikatae strain, IFO1815. For this construct, the translocation
breakpoints were located between ORFs YGR188c and YGR189c, and YPL103c and
YPL116w2.
In the case of the last breakpoint, the translocation falls in a rather large
chromosomal interval (ca 30 kb) and no genomic sequence is available for 1815 so (for
technical reasons) we chose the largest intergenic region, between ORFs YPL108w and
YPL109c, to insert the loxP cassette. This generated a new strain, Sct1/2, which is colinear
to S. mikatae IFO1815.
During the screen for Sct1/2, colonies carrying the second
translocation (t2) but which had lost the original translocation (t1) from strain Sct1 - due to a
back-translocation between chromosomes VI and VII - were also isolated.
1. Delneri, D. et al. Exploring redundancy in the yeast genome: an improved strategy for
the use of the cre-loxP system. Gene 252, 127-135 (2000).
2. Fischer, G. et al. Chromosomal evolution in Saccharomyces. Nature 405, 451-454
(2000).
Table A: Oligonucleotides used for the construction of the cassettes used for creating the
translocation between chromosome VI and VII, and between chromosome VII and XVI.
Name
Sequencea
Template
YFR06-07F
5’-atgattactagtgcttaaaccttgttattcatgcctcagctgaagcttcgtacgc-3’
PUG6
YFR06-07R
5’-aacctaacaaggttacttcatttttcccgaagagcaatacgcataggccactagtggatc-3’
PUG6
YGR21-22F
5’-actgtaataaagagtaaaaacaacaacaaagacaatcgagctgaagcttcgtacgc-3’
PUGKlURA3
YGR21-22R
5’-gtgtttaaacaaatttcggaagcaatggagcataggccactagtggatc-3’
PUGKlURA3
Ck6F
5’-cgtcgtccacctatttgag-3’
Genomic DNA
Ck6R
5’-atccaatagctgaagaacgtc-3’
Genomic DNA
KanMX-R
5’-caatcgatagattgtcgcac-3’
Genomic DNA
Ck7F
5’-acatcagagacttgggcatc-3’
Genomic DNA
Ck7R
5’-agctgtgctcgatgttgtg-3’
Genomic DNA
URA-R
5’-ctcatcagtcgaacgaacgt-3’
Genomic DNA
YGR188-189F
5’- ccgcactttatattttaacgtttgatggttttctatcttggcagcgcagctgaagcttcgtacg-3’
PUG6
YGR188-189R
5’- cttttattattaagggtagccttcatacaaaggaaatttatgtcatcgcataggccactagtggatc-3’
PUG6
YPL116-103F
5’-cgctgcttctataacacttgttttgacatgacgataagtgtagtcagctgaagcttcgtacgc-3’
PUG-KlURA3
YPL116-103R
5’-ataaattgattctaagtacaggaagtatcaagggggggggttgacgcataggccactagtggatc-3’
PUG-KlURA3
Ck7bF
5’-gttggcacaagttatggagtc-3’
Genomic DNA
Ck7bR
5’-cgtagctttactagccttgc-3’
Genomic DNA
Ck16F
5’-ttattcccctcatatattcgg-3’
Genomic DNA
Ck16R
5’-ttattatgcaagttgtgtaggc-3’
Genomic DNA
a
the underlined bases are the sequences complementary to the templates.
Tanslocation t1: the breakpoint on chromosome VI was located between ORF YFR006 and YFR007 (where a
tRNA TF(GAA)F and an LTR were present; another tRNA TG (GCC) F1 and LTR were also present between
YFR008w and YFR009w); the breakpoint on chromosome VII was located, for technical reasons, between ORF
YGR021w and YGR022w (the only tRNA TD(GUC)G1 was present in between ORF YGR022c/YGR023w and
YGR024c). Following the recent release of the sequence of the S. mikatae 1816 (http://genomewww4.stanford.edu/cgi-bin/FUNGI/FungiMap), it is now confirmed that the exact breakpoints are between
ORF YFR008w and YFR009w for chromosome VI and between ORF YGR022c/YGR023w and YGR024c for
chromosome VII.
Translocation t2: the breakpoint on chromosome VII was mapped precisely between ORF YGR188c and
YGR189c (where a tRNA TK(CUU)G3 and an LTR were present); the breakpoint on chromosome XVI was
placed, again for technical reasons, between ORF YPL108wand YPL109c (only one tRNA TM(CAU)P was
present in the breakpoint interval between ORF YPL111w and YPL112c). No sequence is available for S.
mikatae 1815.
Table B: Oligonucleotides used for the generation of species-specific chromosomal probes.
Chromosomes
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
XVI
Primers specific for S. cerevisiae
Primers specific for S. mikatae
1CF: 5'-GTTGGCTAGTTTCGCATTC-3'
1MF: 5'-TGACTAGCCGTTGGATGTAC-3'
1CR: 5'-CCTTCATTGTTCTTGTCTGATG-3'
1MR: 5'-CAACGACAGATTTCGAGTACC-3'
2CF: 5'-TGCGGTAGCATTTCCATC-3'
2MF: 5'-CTAATTAAATCATTAGTGGGGC-3'
2CR: 5'-GGTTAGTGGCCAGATTGAGA-3'
2MF: 5'-GATGGCAATAGATTACCTATGC-3'
3CF: 5'-ACCTTTCCCTGATGTACGTC-3'
3MF: 5'-TGATATACGGTACAAGAAAGGG-3'
3CR: 5'-CTTCACATCAAACTTGTGCC-3'
3MR: 5'-AAGCTCAAACAAATCCAATG-3'
4CF: 5'-GACAAGCAACACAGTGTTTTAG-3'
4MF: 5'-GCAAATAATTCTTATGGCCC-3'
4CR: 5'-AGCTACTGTGACGACCAGG-3'
4MR: 5'-TGGAGGAACAACATTACAAAAC-3'
5CF: 5'-ACGACATTCCATCTCATCG-3'
5MF: 5'-CTTCTCATAAATAGGCTTGGC-3'
5CR: 5'-GAGAGCGTGAGAAAATACTGC-3'
5MR: 5'-GTTTTACAAAGAAAGCGTGC-3'
6CF: 5'-TTAGCAACTCTATGTACAACCG-3'
6MF: 5'-TGAAATAACTGACTGTCGTTTG-3'
6CR: 5'-TGATAAGTTTGATTGCGTCC-3'
6MR: 5'-AGGAACATGCCATTCAAAG-3'
7CF: 5'-GAATCTCTTGGTAGACTCGACC-3'
7MF: 5'-GCAAAAATCTATAATACTGCTCG-3'
7CR: 5'-GAAATGAACCTGCCAGAAG-3'
7MR: 5'-CTACGGATACTGGTGAAATCG-3'
8CF: 5'-ATAGAATAGTCCCATGGCG-3'
8MF: 5'-TTGTCCTTTTGTCAGTATGTTG-3'
8CR: 5'-CATGGGAGTCAGTTCTTGTTC-3'
8MR: 5'-TTTATCATTTATCTCGTAGGACC-3'
9CF: 5'-ATAATGGCAATTGTGGAATG-3'
9MF: 5'-CTAGAACATCCTCCGGAATC-3'
9CR: 5'-TTACCAGGCGTACTATTTGG-3'
9MR: 5'-GGGTAGACTCCCTAAGTGTTG-3'
10CF: 5'-CGAATCAAGAACCTTGGTG-3'
10MF: 5'-TCCATGGAGCTTAATAGCG-3'
10CR: 5'-AAACGTCGCAGATATGGAC-3'
10MR: 5'-AACTTTGAAGCATCCTTTGAC-3'
11CF: 5'-GCACCATCCTTTAACTCCAC-3'
11MF: 5'-AGATTGAAGTTCGGATACGTG-3'
11CR: 5'-GAAAAAGATGCACTCTGTCG-3'
11MR: 5'-TGATCAGCTAGCACATATTGC-3'
12CF: 5'-TGAGTCGTCATCACCATACG-3'
12MF: 5'-TTTCACAGTCGTCTCGATTG-3'
12CR: 5'-AGATTTTGTCCAAAGTTAGCAG-3'
12MR: 5'-CAATTCGGGTTTCCATAAC-3'
13CF: 5'-GTCATCCTGACATTGCTTTC-3'
13MF: 5'-CACAGTCATAGGTGAACTGAGG-3'
13CR: 5'-ACGATGAGAGAGGAGAAACG-3'
13MR: 5'-TTCGGTAAAAACATCCTGG-3'
14CF: 5'-GGTGAAGAAAAGTTTGTAAAATG-3'
14MF: 5'-AAGAGTGCAATGTTACGGG-3'
14CR: 5'-TCGACCTCGATCTACTTCG-3'
14MR: 5'-TAAGTCATGGCAGGTCGG-3'
15CF: 5'-TGTTAAACTACAATTTCGGATTC-3'
15MF: 5'-GCTCATTTCTTTACTTGCTTG-3'
15CR: 5'-CACTGCCATTTTCATGACC-3'
15MR: 5'-ACGTAGAGCTCAAGTAGACGAC-3'
16CF: 5'-GTCACACCAGGCTATGAGG-3'
16MF: 5'-GTCGCCATATAAATGAGATAGC-3'
16CR: 5'-TCCACCTGTTACCTTCTTGAT-3'
16MR: 5'-ACATACAGAGAAGAATACCTTGC-3'