Supplementary Material
Strain constructions.
Five haploid spores with the tel1 mec1-21 genotype were derived from sporulation of
two diploids (24 and 56) that were heterozygous for these markers. These strains were
isogenic with W303a (THOMAS and ROTHSTEIN 1989) and were provided by P.
MIECZKOWSKI (Duke University School of Medicine, Durham, NC). The progenitor
W303a strain has the markers: MATa leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1100 rad5-535). There were multiple steps in the constructions of diploids 24 and 56.
First, the haploid JMY309-1b (MATalpha mec1-21; MIECZKOWSKI et al. 2003) was
crossed to RCY58 (MATa rif1::KANMX rif2::HIS3 TELVR::URA3; MIECZKOWSKI et al.
2003). A spore derived from the resulting diploid with the genotype MATalpha
rif1::KANMX mec1-21) was crossed to RCY56 (MATa tel1::URA3 rif2::HIS3
TELVR::URA3, CRAVEN and PETES 1999) to yield the diploid 56. The first step in the
construction of diploid 24 was also a cross of JMY309-1b and RCY58. A spore of the
resulting diploid with the genotype MATalpha mec1-21 rif2::HIS3 was crossed to RCY24
(MATa tel1::ura3 rif1::KANMX; CRAVEN and PETES 1999), yielding the diploid 24.
Three of the tel1 mec1-21 strains (MV24-15, MV24-17 and MV24-18) were spore
derivatives from strain 24 and two (MV56-3, and MV56-5) were spore derivatives from
strain 56. Two single-colony isolates were derived from each of these strains (MV2415-1, MV24-15-2, MV24-17-1, MV24-17-2, MV24-18-1, MV24-18-2, MV56-3-1, MV56-32, MV56-5-1, and MV56-5-2) and were subsequently sub-cultured 10 times, and
examined by microarray analysis. The summary of the analysis of the MV24 and MV56
spores is in Supp. Table 1.
Another pilot study was done with spores derived from RCY278, a diploid of the
W303 genetic background that was heterozygous for the tel1 and mec1-21 mutations
(CRAVEN and PETES 2001). We examined two spore cultures of each of the four
possible genotypes by microarrays; these strains were not sub-cultured. All mec1-21
and tel1 mec1-21 haploid strains had two copies of VIII, whereas the wild-type and tel1
strains had only one copy.
These pilot studies indicated that acquisition of an extra chromosome VIII occurred
very quickly in strains with the tel1 mec1-21 genotype. To generate diploid strains in
which the mec1-21 haploid spores would be haploid before sub-cloning, we constructed
two isogenic diploids MV58 and MV59. These strains were generated by mating the
haploid MV40-3b (tel1::KANMX arg4::HYG CAN1) with RCY308-7b (MATalpha mec121 RAD5; CRAVEN et al. 2002); microarray analysis showed that MV40-3b was euploid
and RCY308-7b was disomic for chromosome VIII. We sporulated these strains and
dissected tetrads. We identified tetrads in which the ARG4 marker segregated two to
two since, in such tetrads, the Arg- spores should receive only a single copy of
chromosome VIII (as discussed in the text). We selected three Arg- spores each of the
three mutant genotypes (tel1, mec1-21, and tel1 mec1-21). We struck each of these
strains on rich growth medium and from each of the original spores, we isolated seven
colonies. Each of these colonies was then sub-cloned separately ten times. Thus, we
examined 21 strains of each genotype.
Because of the very high rate of de novo chromosome aberrations in mec1-21
haploid strains, we constructed a doubly-heterozygous tel1 mec1-21 diploid (MV70) in
which the mec1-21 allele was not introduced by a cross. The construction required
several steps; all strains are isogenic with W303a except for the markers noted. First,
as described in Materials and Methods of the paper, we constructed a plasmid (pMD92)
containing the mec1-21 allele on an integrating plasmid with the wild-type URA3 gene
(YIp5). We then crossed SPY40-FR, a strain with a tel1::ura3 mutation, (2) to
HLK1042-1c (CAN1 hom3-10 RAD5) to generate the diploid MD316. This diploid was
transformed with BsrGI-treated pMD92, selecting for a Ura+ transformant (MD317). We
selected a 5-fluoro-orotate (5-FOA)-resistant derivative of MD317 (MD318) and
confirmed by PCR that this strain had a substitution of mec1-21 in one of two
homologues. We found that MD318, although derived from a strain (MD317) that was
heterozygous for the tel1::ura3 allele, had lost this mutant allele, possibly as a
consequence of a mitotic crossover stimulated by the 5-FOA treatment. Consequently,
we introduced a different null allele of TEL1 (tel1::KANMX) by transforming MD318 with
a PCR fragment containing the mutant allele. This fragment was generated by PCR
amplification of genomic DNA of the strain MV40-3b (MATa tel1::kanMX CAN1
arg4::HYG) with PCR primers: TEL1-Downstr-For and TEL1-Upstr-Rev. This diploid
(MV70), when examined by CGH microarrays, was euploid and had no chromosome
rearrangements resulting in deletions or duplications.
We sporulated MV70 and selected two spores each of the four possible genotypes:
wild-type (MV70-1d, MV70-2c), mec1-21 (MV70-6d, MV70-7b), tel1 (MV70-1c, MV706c), and tel1 mec1-21 (MV70-5a, MV70-11d). Without sub-culturing, we analyzed
cultures of all of the strains by CGH microarrays. No aneuploidy, duplications or
deletions were detectable in any of the wild-type or tel1 strains. All of the mec1-21 and
tel1 mec1-21 strains were disomic for chromosome VIII, and one of the tel1 mec1-21
strains (MV70-11d) was also disomic for chromosome III. These results demonstrate
that strains with the mec1-21 allele become disomic for chromosome VIII very rapidly.
To determine whether extra copies of DNA2 (a gene encoding a
helicase/endonuclease located on chromosome VIII suppressed the tendency of tel1
mec1-21 strains to become aneuploid for chromosome VIII, we transformed MV70 with
the plasmid pRS316-DNA2, a CEN-containing plasmid with the wild-type DNA2 and
URA3 genes (LEE et al. 2000). The resulting diploid transformant (MV71) was
sporulated and tetrads were dissected. Eight plasmid-containing spores, two each of
the four genotypes were characterized. The strain names and genotypes were: MV7111c and MV71-13a (wild-type), MV71-4b and MV71-7c (mec1-21), MV71-1c and MV712a (tel1), and MV71-14a and MV71-17d (tel1 mec1-21).
Analysis of chromosome alterations in tel1 mec1-21 strains derived from
spores of MV58 and MV59.
As described above, we generated haploid strains from sporulation of the diploids
MV58 and MV59 and sub-cultured 21 derivatives of each of three genotypes, tel1,
mec1-21, and tel1 mec1-21. Following the tenth sub-culturing, we picked a single
colony from each of the 21 strains for further analysis. DNA isolated from each of these
strains was analyzed by DNA microarrays, CHEF gel electrophoresis, and other
methods described below. The sequences of oligonucleotides used to generate
hybridization probes or to analyze chromosome rearrangements are in Supp. Table 2.
Before examining the 21 sub-cultured tel1 mec1-21 strains, we examined DNA isolated
from the three progenitor haploids (MV58-20a, MV59-16c, MV59-6a) used to generate
the 21 sub-cultured strains. MV58-20a and MV59-6a are MATalpha, and MV59-16c is
MATa. All strains were disomic for chromosome VIII, but had no other changes.
Many of the chromosome rearrangements described below involve recombination
between Ty elements or solo delta elements (the LTR associated with Ty). Some of
these elements have not been annotated in SGD. Since the strain W303a (THOMAS and
ROTHSTEIN 1989) is not identical to S288c (the sequenced strain in SGD), this lack of
annotation might reflect strain differences. It should be noted, however, that not all of
the Ty elements present in S288c are annotated in SGD (LEMOINE et al. 2005). As
described below, many of the chromosome rearrangements that we observed involve
Ty or delta elements on chromosome III. W303a has the same two pairs of Ty
elements on the right arm noted previously in LEMOINE et al. (2005). Two of these
elements are in tandem in a region termed “FS1” and two are in inverted orientation at
“FS2” (5). In addition, there is a Watson-oriented Ty1 element located centromeredistal to a Ty2 element on the left arm of III (UMEZU et al. 2002); this region of
chromosome III has been termed the “Left-Arm Hot Spot” (LAHS), since it appears to be
a hotspot for the insertion of Ty elements (W ARMINGTON et al. 1987). The position of
insertion of this element is an unannotated solo delta at SGD coordinates 82,70083,036. As noted below, there are other Ty1 and Ty2 elements that are present in the
W303 genome that are not present or not annotated in SGD. The information
concerning the location of these elements was provided by ABRAM GABRIEL (Rutgers
University). The unannotated Ty elements that are relevant to our analysis (SGD
coordinates in parentheses) are: Ty1 elements on VII (700589 and 734219), a Ty1 on X
(355473), and a Ty2 on XII (168498). Information about the Ty element on
chromosome XII was also provided by DAVID CARTER (Sanger Center).
MV58-20a#1: By DNA microarray analysis, this strain was disomic for chromosomes
III and VIII and had no other obvious rearrangements. By CHEF gel analysis, the strain
had no chromosome III of the normal size (about 340 kb). Southern analysis with a
HIS4 probe (a gene located on IIIl, generated by PCR amplification of yeast genomic
DNA with primers his4-51 and his4-31) showed that there were two chromosomes that
hybridized to HIS4, one about 90 kb larger than the wild-type chromosome, and one
about 90 kb smaller. The larger, but not the smaller, chromosome hybridized to THR4
(a gene located on IIIR, generated by PCR amplification of yeast genomic DNA with
primers thr4-5 and thr4-3). The simplest explanation of these results is that there was
an unequal crossover between two copies of III in a disomic strain, involving MAT and
HMR. Since these two repeats are about 90 kb kb apart, this event would result in two
monocentric chromosomes, one about 90 kb larger and the other 90 kb smaller than the
wild-type chromosome. Since THR4 is located between MAT and HMR, the smaller
chromosome would lack THR4. We confirmed this rearrangement by PCR analysis
using the primers BUD5-5Prime-Rev and ARS318-Rev to detect the MAT-HMR fusion.
We found the 1.9 kb PCR fragment expected for this fusion. We confirmed the HMR to
MAT fusion by PCR using the primers ARS317-Upstream-For and TAF2-3Prime-For.
The expected fragment size (about 2.8 kb was observed).
MV58-20a#2: The microarray showed disomy for VIII, and the two copies of VIII
were of the normal size; chromosome VIII was examined using CUP1 as a hybridization
probe (probe generated by PCR using primers cup1-amp3 and cup1-amp5). The
microarray indicated two copies of III from the telomere of IIIL to the MAT locus, one
copy from MAT locus to HMR, and two copies from HMR to the telomere of IIIR. This
result suggests that there are two copies of III, one normal and one with the Hawthorne
deletion (deletion between MAT and HMR). The expected chromosome sizes, about
340 kb and 240 kb were seen by CHEF and Southern analysis. As expected, genes
from the left arm of III (CHA1 [generated by a PCR reaction using primers c/s III 15838
and c/s III 16857] and HIS4), hybridized to both chromosomes, but THR4, which is
located between MAT and HMR, hybridized only to the 340 kb chromosome. We
confirmed the MAT to HMR fusion by a PCR reaction using the BUD5-5Prime-Rev and
ARS318-Rev primers described above. The microarray indicated Y’ amplification.
MV58-20a#3: The microarray showed disomy for chromosome VIII and CHEF gel
electrophoresis showed that the chromosomes were the normal size. There was an
interstitial duplication on chromosome VII with the breakpoints at approximately
YGRWdelta19 and YGRWdelta21. According to A. GABRIEL (personal communication),
there are two Ty elements very near the locations of YGRWdelta19 and YGRWdelta21.
The duplication is likely to reflect unequal crossing over between these two Ty
elements. Below, we will indicate these Ty elements as YGRWdelta19/Ty and
YGRWdelta21/Ty. On the CHEF gel, there is a novel chromosome of the expected
size, assuming that the duplication occurred within VII. By microarray analysis, there
were three copies of the chromosome III sequences located between the left arm hot
spot (LAHS) and the IIIL telomere; the LAHS is a region with two Watson-oriented Ty
elements, YCLWTy2-1 and an unannotated Ty1 element. There were two copies of
chromosome III sequences between LAHS and FS1 (a pair of Watson-oriented Ty
elements located on the right arm of III; LEMOINE et al. 2005). There was one copy of
the chromosome III sequences located between FS1 and the right telomere of III. The
CHEF gel showed that there were two chromosomes that hybridized to the HIS4 probe
(located centromere distal to LAHS), one of 340 kb (wild-type chromosome III) and one
of 240 kb. The signal of hybridization for the HIS4 probe was stronger for the 240 kb
chromosome. THR4 hybridized only to the normal chromosome. These results suggest
that there were originally two chromosome IIIs in MV58-20a#3. One broke in FS1 and
repaired the break using a Crick-oriented delta (such as YCLCdelta1) in LAHS as a
template for a BIR event. This mechanism would generate a short (about 240 kb)
isochromosome with two copies of sequences between LAHS and the left telomere and
a deletion of the sequences between FS1 and the right telomere.
MV58-20a#4: The microarray indicated disomy for chromosome VIII. The
microarray also showed that there are three copies of the region between the left
telomere of III and LAHS, two copies of III from LAHS to YCLCdelta6 on the right arm of
III, and one copy of III from YCLCdelta6 to the IIIR telomere. There were two
chromosomes that hybridized to CHA1 (gene on the left arm of III), one at the size of
the wild-type chromosome and one at about 210 kb; the hybridization to the CHA1
probe is more intense for the smaller chromosome. The larger, but not the smaller
chromosome, also hybridized to THR4 located on the right arm of III. All of these
results suggest the strain was initially disomic for III. One of the chromosomes had a
double-strand break at YCLCdelta6 that was repaired by a BIR event utilizing the LAHS
sequences as a starting point. Using the PCR primers SUF2/Delta6-For and Ty13Prime-Alt-For, we observed a PCR fragment of about 1.4 kb. This size is consistent
with a recombination event involving YCLCdelta6 and one of the 3’ delta elements of
the Watson-oriented Ty elements of LAHS.
MV58-20a#5: The microarray analysis showed that the strain was disomic for
chromosome VIII and the CHEF gels demonstrated that the chromosome VIIIs were of
the normal size. By microarray analysis, we found that there were three copies of the
sequences between the LAHS and the left telomere of III, two copies of the sequences
between LAHS and FS2 (LEMOINE et al. 2005), and one copy of the sequences between
FS2 and the right telomere of III. On the CHEF gel, there were two chromosomes that
hybridized with the HIS4 probe (located on the left arm of III), one at 340 kb and one at
260 kb; the 260 kb band hybridized more strongly. The 340 kb, but not the 260 kb,
chromosome also hybridized to the THR4 probe (located on the right arm of III). This
pattern indicates that there are two chromosome IIIs, one normal chromosome and an
isochromosome in which a DSB at FS2 was repaired using the sequences in LAHS as a
template. Microarrays also indicate Y’ amplification.
The position of MscI sites results in an MscI fragment that hybridizes to the KCC4
probe (generated by amplification of genomic DNA with primers KCC4-1 and RKCC4-1)
of about 23 kb in the wild-type strain. FS2 has two Ty elements with a Crick
centromere-proximal Ty and a Watson centromere-distal Ty (5). There are also two
Watson Ty elements at LAHS, the centromere-distal element is a Ty1 and the
centromere-proximal element is a Ty2. If the Crick Ty of FS2 had a BIR involving the
Watson Ty1 of LAHS, we should get a 14 kb band. If the Crick Ty of FS2 had a BIR
with the Watson Ty2, we expect a 22 kb band. The Southern showed that MV58-20#5
had a band of hybridization with KCC4 that was about 22 kb, indicating that BIR was
between the Crick Ty at FS2 and the Ty1 element of LAHS.
MV58-20a#6: The microarray indicated no chromosome VIII disomy. There was an
approximately 100 kb interstitial duplication on chromosome IV between two paired Ty
elements (YDRWTy2-2/YDRCTy1-2 and YDRWTy2-3/YDRCTy1-3). From the CHEF
gel, it was clear that chromosome IV in this strain was larger by the size consistent with
the duplication reflecting unequal crossing over between the paired elements. There
were four copies of sequences between the left telomere of III to LAHS, two copies from
LAHS to YCLCdelta6, and one copy between YCLCdelta6 and the right telomere of III.
By CHEF gel analysis, there were three bands that hybridize to the CHA1 and HIS4
probes on the left arm of III, one of the usual size (340 kb), one of 220 kb and one of
640 kb. The THR4 probe hybridized only to the 340 kb fragment. There was no
“normal” band for chromosome VIII at 560 kb, but there was a band at 640 kb (the same
band that hybridized to the HIS4 and CHA1 probes) that hybridized to the chromosome
VIII-specific CUP1 probe. Based on the microarray analysis and the size of the III-VIII
translocation, it is likely that this translocation reflects a BIR event in which a DSB in
YHRCTy1-1 was repaired using either the Watson Ty1 or the Watson Ty2 elements of
LAHS; these orientations are correct for the translocation.
To confirm this conclusion, we treated DNA from W303 and MV58-20a#6 with NcoI,
separated the resulting fragments by gel electrophoresis, and did Southern analysis
with two different hybridization probes. One probe (LAHS-D) was generated by PCR
amplification of genomic DNA with the primers (LAHSD-F and LAHSD-R); this probe is
located within the KCC4 gene located immediately centromere-distal to the Ty1W of the
LAHS. The second probe (VIIIP) was generated by PCR amplification of genomic DNA
with the primers VIIIP-F and VIIIP-R, and contained sequences from the region of VIII
immediately centromere-proximal to YHRCTy1-1. Since the region of VIII near
YHRCTy1-1 is very similar to a telomere-adjacent region of chromosome I, the probe
also hybridized to a restriction fragment from chromosome I. When the NcoI digests
were hybridized to the LAHS-D probe, we found a band of about 32 kb in both W303
and MV58-20a#6 as expected from information in SGD. The MV58-20a#6 strain had an
additional band of 13.4 kb, the size expected from a recombination event between
YHRCTy1-1 and the Ty1W of the LAHS. When we did Southern analysis of the NcoI
digests using the VIIIP probe, W303 had a single hybridizing fragment of 10.6 kb and
MV58-20a#6 had two hybridizing fragments, one of 10.6 kb and one of 13.4 kb. The
observation that the 13.4 kb fragment hybridized to both the LAHS-D and VIIIP probes
strongly indicates that the MV58-20a#6 strain had a translocation between YHRCTy1-1
and the Ty1W of the LAHS.
The 220 kb chromosome is the size expected for a DSB at YCLCdelta6 that was
repaired using the LAHS in a BIR event. The structure of the rearrangement in the 220
kb chromosome was confirmed by Southern and PCR analysis. In a MscI-EcoRI digest
hybridized to the YCR006C probe (located centromere-proximal to YCLCdelta6,
generated by PCR amplification of yeast genomic DNA with primers YCR006C-F and
YCR006C-R), one should observe a 4.2 kb band in W303. A fusion between the 3’
delta of the Ty element in LAHS with YCLCdelta6 would produce a 5 kb band. The
MV58-20a#6 strain has two bands of the expected sizes. Digests of the DNA were also
done with the BlpI, NcoI, and NheI digests. With the YCR006C probe, one expects
fragments of 7.5 kb (BlpI), 6.5 kb (NcoI), and 23.5 kb (NheI). A fusion of YCLCdelta6
with the Ty1 of LAHS would result in additional fragments of: 18.5 kb (BlpI), 12 kb
(NcoI), and 18 kb (NheI). Fragments of these sizes were seen. However, in the NcoI
digest, there was an additional fragment in the MV58-20a#6 strain of unknown origin.
The fusion was also confirmed by PCR analysis. With the primers SUF2/Delta6-For
(located centromere-proximal to YCLCdelta6) and Ty1-3Prime-Alt-For, PCR
amplification of MV58-20a#6 DNA resulted in a fragment of about 850 bp, as expected
for a fusion between YCLCdelta6 and a Ty element. Similarly, PCR amplification of
MV58-20a#6 DNA with primers YCR006C/SUF2-For and Ty1-3Prime-Alt-For resulted in
a fragment of about 1.4 kb, as expected. In summary, MV58-20a#6 has three
chromosomes with sequences derived from the left arm of III. One is a normal
chromosome, one is an isochromosome in which a DSB within YCLCdelta6 was
repaired using the LAHS to initiate a BIR event, and one is a III-VIII translocation in
which a DSB within YHRCTy1-1 on chromosome VIII was repaired by a BIR event
utilizing the LAHS as an initiation site.
MV58-20a#7: This strain had several chromosome rearrangements. The strain
was disomic for chromosome VIII. By Southern analysis using the CUP1 (VIII-specific)
probe, there were two chromosomes with VIII sequences, one at the normal size (about
565 kb) and one about 600 kb. A large (355 kb) region on chromosome X was
duplicated, from YJLCdelta4 to the left telomere. The copy number of sequences on III
was unusual. From HML to the left telomere, sequences were less than one copy;
since a nearly identical copy of these sequences is also near the telomere of
chromosome XI, the interpretation of this result is not completely clear. From HML to
the LAHS, there appeared to be one copy of the sequences. From LAHS to FS1, there
were two copies, and from FS1 to the MAT locus, there appeared to be three copies.
Finally, between the MAT locus and the right telomere, there appeared to be a single
copy of the sequences. There are at least three chromosomes that had sequences
from III in this strain. The HIS4 hybridization probe labeled chromosomes of about 300
and 1000 kb. The BUD5 hybridization probe (located immediately centromere-proximal
to the MAT locus; generated by PCR amplification of genomic DNA with primers BUD5For and BUD5-5Prime-Rev) hybridized to chromosomes of about 600 and 1000 kb. In
addition, the BUD5 probe strongly hybridized to the well of the CHEF gel, suggesting
the possibility of a circular chromosome containing BUD5 sequences. The results of the
CHEF gels were not completely concordant with the microarray analysis. For example,
if there are only a single copy of the region of chromosome III with the HIS4 sequences
(the region between HML and the LAHS), it is not clear how there can be two different
chromosomes that hybridized to the HIS4 probe. One possibility is that the MV5820a#7 strain has a mixture of cells with different chromosome rearrangements.
We further analyzed the 600 kb novel chromosome that hybridized to the BUD5, but
not the HIS4, probe. This chromosome was purified from a CHEF gel and hybridized to
a microarray. It contained all of the sequences of III from the right telomere to the LAHS
and all of the sequences from the left telomere of X to YJLCdelta4. At the position of
YJLCdelta4, according to ABRAM GABRIEL (personal communication), there is a Ty1
element (coordinate 355473). Thus, the translocation is likely to reflect a DSB in the
Ty1 element at 355473 on X, and repair of the DSB by a BIR event utilizing the Ty1
element at the LAHS.
MV59-6a#1: The microarray analysis indicated disomy for chromosome VIII and the
CHEF gels (CUP1 probe) showed that these chromosomes were of normal size.
Chromosome III had a complex pattern of gene dosages. There were three copies of
sequences between LAHS and left telomere of III, two copies of sequences between
LAHS and YCLCdelta6, one copy of sequences between YCLCdelta6 and HMR, and
the sequences from HMR to the right telomere were deleted. By CHEF gel, there is no
normal III but a single 220 kb chromosome that hybridizes to both CHA1 and HIS4. The
BUD5 hybridization probe, located just centromere-proximal to MAT, did not hybridize to
any chromosome on the gel, but hybridized strongly to the well of the gel.
These results suggest the following scenario. The strain was originally disomic for
two normal IIIs. One of these chromosomes had a DSB at YCLCdelta6, followed by a
BIR at LAHS, generating an isochromosome (losing DNA distal to YCLCdelta6 and
duplicating DNA on the left arm distal to LAHS). Using the PCR primers SUF2/Delta6For and Ty1-3Prime-Alt-For, we observed a PCR fragment of about 1.4 kb. This size is
consistent with a recombination event involving YCLCdelta6 and one of the 3’ delta
elements of the Watson-oriented Ty elements of LAHS.
It is likely that the second chromosome III had a recombination between HMR and
HML recombination, circularizing III, and resulting in loss of sequences distal to HMR
and HML. The HMR/HML fusion was confirmed using the primers located centromereproximal to HML (YCL065W-Upstr-Rev) and centromere-proximal to HMR (ARS317Upstream-For). A fragment of the expected size (2.6 kb) was observed. Finally, the
microarrays indicated amplification of Y’ elements.
MV59-6a#2: By microarray analysis, this strain was disomic for chromosome VIII,
and CHEF gel analysis (CUP1) probe showed that these two copies were of normal
size. By microarray analysis, there were two copies of III, except in region between
MAT and HMR where there was only a single copy. By CHEF gel analysis, two
chromosomes hybridized to CHA1 and HIS4, one at normal size of III (about 340 kb)
and one at about 240 kb. The 240 kb band did not hybridize to the THR4 probe,
although the 340 kb band did. These results suggest that the strain had one normal III
and one with the Hawthorne deletion, a deletion resulting from recombination between
MAT and HMR.
The fusion between MAT and HMR was confirmed by PCR with the primers BUD55Prime-Rev (sequences centromere-proximal to MAT) and ARS318-Rev (sequences
centromere-distal to HMR). The expected PCR product about 2 kb in size was
observed. The MAT-HMR fusion was also confirmed by Southern analysis using AflIItreated DNA. Using the hybridization probes RBK1 (located centromere-proximal to
MAT, generated by PCR amplification of genomic DNA with primers RBK1-For and
RBK1-Rev) or GIT1 (located centromere-distal to HMR, generated by PCR amplification
of genomic DNA with primers GIT1-For and GIT1-Rev), we found the expected
fragments in the parental strain of about 12 kb (MAT) and 7 kb (HMR). As expected the
MV59-6a#2 strain had a recombinant fragment of about 18 kb in size and that
hybridized to both probes. Microarray analysis also indicated Y’ amplification.
MV59-6a#3: Microarrays, CHEF, PCR, and Southern analysis were identical with
those observed for MV59-6a#2. Therefore, this strain was also disomic for VIII, had one
normal III, and one III with the Hawthorne deletion.
MV59-6a#4: From microarray analysis, this strain was disomic for chromosome VIII
and has no other chromosome rearrangements. By CHEF gel analysis, chromosome III
(analyzed with the HIS4 and THR4 probes) and chromosome VIII (analyzed with the
CUP1 probe) were of normal size. Microarray analysis also indicated amplification of Y’
elements.
MV59-6a#5: By microarray analysis, the strain had two copies of chromosome VIII.
By CHEF gel analysis, however, the chromosome VIII-specific CUP1 probe hybridized
to a chromosome of about 640 kb; the normal size of VIII is about 560 kb. By
microarray analysis, there were three to four copies of III sequences between LAHS and
the left telomere, two copies of the sequences between LAHS and FS1, and one copy
of sequences between FS1 and the right telomere. There were two chromosomes that
hybridized to the HIS4 and CHA1 (probes from the left arm of III), one of 640 kb and
one of 440 kb. The THR4 and BUD5 probes hybridized to the 440 kb chromosome, but
not the 640 kb chromosome. We isolated the 440 kb chromosome, labeled it, and
hybridized to a microarray. It contained only chromosome III sequences.
The size and hybridization patterns observed with the 640 kb chromosomes indicate
that it was formed by recombination between LAHS (Watson-oriented Ty elements) and
the Crick-oriented Ty1 element (YHRCTy1-1) located near the right telomere of VIII.
The predicted size for such a translocation is 545 kb + 85 kb or 630 kb. This
translocation was confirmed by Southern analysis of NcoI-treated DNA, as was done for
MV58-20a#6. The III-VIII translocations in MV58-20a#6 and MV59-6a#5 appear to be
identical.
The pattern of hybridization and size of the 440 kb chromosome indicated that this
chromosome had a duplication of chromosome III sequences of about 100 kb without
any deletion. Since there were two copies of the sequences between LAHS and FS1,
and since the 640 kb translocation did not have this region, it is likely that the 440 kb
chromosome had a duplication of the region between LAHS and FS1. Such a
duplication could arise as a consequence of an unequal crossover between the Watson
Ty elements of LAHS and the Watson Ty elements at FS1. The distance between
these repeats is about 60 kb. Depending on which Ty element was involved in the
exchange, the amount of DNA added by the unequal crossover is between 66 and 78
kb. Thus, the predicted size of the chromosome with the duplication is between 406
and 418 kb, about the size observed. In addition, the size of the chromosome with the
duplication could be increased by Y’ amplification, since the microarray analysis
indicated that Y’ elements were amplified in this derivative. It should be noted,
however, that the chromosome generated by this mechanism would be expected to be
dicentric.
MV59-6a#6: By the microarray and CHEF gel analysis, chromosome III was disomic
and the two copies of III were of normal size. Chromosome VIII was not disomic. By
CHEF gel analysis, chromosome V was about 60 kb larger than the normal
chromosome. We have not identified the source of this extra DNA.
MV59-6a#7: By microarray and CHEF gel analysis, chromosome VIII was not
disomic and was of normal size. By microarray analysis, the region between MAT and
HMR was duplicated. By CHEF gel analysis, there was no chromosome III of normal
size, but a 440 kb chromosome that hybridized to both HIS4 (located on the left arm of
III) and THR4 (located on the right arm of III). The size of this chromosome and the
microarray analysis indicated that this chromosome has a duplication between MAT and
HMR as a consequence of an unequal crossover between these two regions. The
distance between these repeats was about 95 kb.
The HMR-MAT fusion was confirmed by PCR using one primer located centromereproximal to HMR (ARS317-Upstream-For) and one located centromere-distal to MAT
(TAF2-3Prime-For). A PCR fragment of the size predicted of the fusion (about 2.8 kb)
was observed in MV59-6a#7, but not in the parental strain.
MV59-16c#1: Microarray analysis showed that the strain was disomic for VIII. The
sequences from LAHS to the left telomere of III were duplicated. By CHEF gel analysis,
there are two chromosomes that hybridized to the HIS4 and CHA1 probes (genes on
the left arm of III), one of the normal size and one at about 250 kb. The THR4 probe
(right arm probe) hybridized only to the normal chromosome. In addition, there were
two copies of the left arm of chromosome XII, the centromere of XII, and a portion of the
right arm. The duplicated region was about 170 kb. There is a Crick sigma element
(YLRCsigma1) located about 168 kb from the left telomere of XII and an unannotated
partial Watson delta element located about 200 bp centromere-distal to the sigma
element. By PCR analysis using the primers TEN1-5 prime-Rev and Ty1-3Prime-AltFor, we found a PCR fragment of about 1.5 kb in MV59-16c#1 as well as in W303a.
This result suggests the presence of a Ty1 or Ty2 element in the Crick orientation on XII
at about position 168,660. ABRAM GABRIEL (unpublished information) has evidence for a
Ty2 element at position 168498 in W303a and DAVID CARTER (unpublished information),
from sequence analysis of W303a, has evidence of a Ty element in the Crick orientation
at position 168671. These data, taken together, argue that the III-XII translocation is
likely to reflect a DSB in a Ty2 element on XII that was repaired by a BIR event
involving either the Ty2 or Ty1 elements at the LAHS on III.
To confirm the III-XII translocation, we did Southern analysis of NcoI digests of DNA
derived from W303 and MV59-16c#2. The probe for chromosome III sequences was
LAHS-D used in the analysis of MV58-20a#6 (described above). The chromosome XII
probe (XIIP) was generated by PCR amplification of genomic DNA with the primers
XIIP-F and XIIP-R. This probe is located immediately centromere-proximal to the Ty2
element on XII. The W303 strain had restriction fragments of about 32 kb and 17 kb
when hybridized to the LAHS-D and XIIP probes, respectively. In addition to these
fragments, in MV59-16c#2, there was a novel fragment of 23 kb that hybridized to both
probes. This fragment is the size expected as a consequence of recombination
between the Ty2 element on XII and the Ty elements in the LAHS.
This strain also had a duplication of a 35 kb region of VII located between
YGRWdelta19/Ty and YGRWdelta21/Ty similar to that observed in MV58-20a#3. In
addition, by microarray analysis, this strain had Y’ amplification.
MV59-16c#2: By microarray analysis, this strain was disomic for VIII. By CHEF gel
analysis using the CUP1 probe, there were two copies of VIII, one of normal size (565
kb) and one of about 640 kb. There was a terminal duplication of about 120 kb on
chromosome XV; the breakpoint of the duplication is near YOLWTy1-1. A BIR event on
chromosome VIII at YHRCTy1-1 that was repaired using YOLWTy1-1 as a initiation site
would generate a chromosome of 650 kb. Thus, it is likely that the 640 kb chromosome
is a VIII-XV translocation. The strain also had a duplication of the region of III distal to
LAHS and a duplication of a 170 kb region on XII. The breakpoints determined by
microarray analysis, as well as the analysis by CHEF gels, showed that this strain has
the same III-XII translocation found in MV59-16c#1. We further confirmed this
translocation by the same Southern analysis described above for MV59-16c#2. The
microarrays indicated no evident Y’ amplification.
MV59-16c#3: The microarray analysis indicated disomy for chromosome VIII. The
CHEF gel, when hybridized to the CUP1 probe, had the same pattern as observed in
MV59-16c#2: one chromosome VIII of normal size and one of about 640 kb. In
addition, we observed the same terminal duplication of XV. Thus, this strain is likely to
contain the same VIII-XV translocation observed in MV59-16c#2, with breakpoints at
YHRCTy1-1 and YOLWTy1-1.
By microarray analysis, there were three copies of III sequences located between
LAHS and the left telomere, two copies of sequences between LAHS and YCLCdelta6,
and one copy of sequences between YCLCdelta6 and the right telomere. The strain
had two chromosomes that hybridized to the HIS4 and CHA1 chromosome III probes,
one of the normal size and one of about 220 kb. The larger, but not the smaller
chromosome, also hybridized to THR4 located on the right arm of III. All of these
results suggest the strain was disomic for III. One of the chromosomes had a doublestrand break at YCLCdelta6 that was repaired by a BIR event utilizing the LAHS
sequences as a starting point. This strain also had an interstitial duplication on
chromosome VII with the breakpoints at approximately YGRWdelta19/Ty and
YGRWdelta21/Ty similar to that observed in MV58-20a#3 and MV59-16c#1. In
addition, by microarray analysis, this strain has Y’ amplification.
The fusion was also confirmed by PCR analysis using the primers. With the primers
SUF2/Delta6-For (located centromere-proximal to YCLCdelta6) and U1 LAHS-F
(located centromere-distal to the Ty1 element of the LAHS), PCR amplification of MV5916c#3 DNA resulted in a fragment of about 860 bp, as expected for a fusion between
YCLCdelta6 and the 5’ delta element of the Watson Ty1 element at the LAHS.
Similarly, PCR amplification of the DNA sample with primers SUF2/Delta6-For and U2
LAHS-F (located about 200 bp centromere-proximal to U1 LAHS-F) resulted in a
fragment of 670 bp. These results demonstrate that the chromosome alteration that
generated the 220 kb isochromosome was a homologous recombination between
YCLCdelta6 and the 5’ delta element of the Ty1 located at the LAHS.
MV59-16c#4: From the microarray analysis, the strain was disomic for I and VIII. In
addition, as in MV59-15c#3, there were three copies of III sequences located between
LAHS and the left telomere, two copies of sequences between LAHS and YCLCdelta6,
and one copy of sequences between YCLCdelta6 and the right telomere. CHEF
analysis, using the same probes as for MV59-16c#3, demonstrated the same
chromosome composition: a wild-type III and a second small (220 kb) derivative that
reflected a BIR event between YCLCdelta6 and LAHS. The two copies of VIII were of
normal size. Microarrays also indicated Y’ amplification.
We confirmed that the 220 kb rearrangement reflects homologous recombination
between YCLCdelta6 and the 5’ delta element of the Watson Ty1 at the LAHS using the
same primers as were used for MV59-16c#3.
MV59-16c#5: In this strain, microarray analysis showed no gene dosage changes
on III. On VIII, about 280 kb of the left portion of VIII, including the centromere,
appeared to be duplicated, with the rest of the chromosome being present in a single
copy; the transition between the duplication and single-copy sequences, however, was
unclear. Only a single chromosome of 600 kb (about 50 kb bigger than the normal VIII)
hybridized to the CUP1 probe. In addition, chromosome III hybridized to the probe as a
smeary fragment that varied in size from 340 kb to about 390 kb. The nature of the
chromosome rearrangement involving VIII is unclear. The size heterogeneity of III
probably reflects Y’ amplification, since the microarray indicated a very high level of Y’
amplification in this strain.
MV59-16c#6: Microarray analysis showed disomy for VIII. Both III and VIII were
about 50 kb larger than normal, possibly as a consequence of a high level of Y’
amplification, which was evident by microarray analysis in this derivative.
MV59-16c#7: This strain was disomic for chromosome VIII of normal size and had
only a single normal III. There was an interstitial duplication on chromosome VII with
the breakpoints at approximately YGRWdelta19/Ty and YGRWdelta21/Ty. The strain
had no obvious amplification of Y’ elements.
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