Copyright 0 1984 by the Genetics Society of America
H E A L I N G OF BROKEN LINEAR D I C E N T R I C
CHROMOSOMES I N YEAST
JAMES E. HABER'
AND
PATRICIA C. THORBURN
Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University,
Waltham, Massachusetts 02254
Manuscript received June 14, 1983
Revised copy accepted October 20, 1983
ABSTRACT
In yeast, meiotic recombination between a linear chromosome III and a
haploid-viable circular chromosome will yield a dicentric, tandemly duplicated
chromosome. Spores containing apparently intact dicentric chromosomes were
recovered from tetrads with three viable spores. The spore containing the
dicentric inherited URA3 (part of the recombinant DNA used to join regions
near the ends of the chromosome into a circle) as well as HML, HMR and
MAL2 (located near the two ends of a linear but deleted from the circle). The
Ura+ Mal+ colonies were highly variegated, giving rise to as many as seven
distinctly different stable ("healed") derivatives, some of which were Ura+ Mal+,
others Ura+ Mal- and others Ura- Mal'. The colonies were also sectored for
five markers (HIS4, LEUZ, CRYI, MAT and THR4) initially heterozygous in the
tandemly duplicated dicentric chromosome.-Southern
blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic breakage of the dicentric chromosome, followed by one of several different healing
events. The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome. A smaller proportion of events resulted
in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere. In two instances we recovered chromosome
111partially duplicated with a novel right end. We have also found one derivative that had also experienced rearrangement of repeated DNA sequences
found adjacent to yeast telomeres.
ROKEN chromosomes, lacking a telomere, are mitotically unstable in both
B
maize (MCCLINTOCK1941) and the yeast Saccharomyces cerevisiae (MCand HABER1981 HABER,THORBURN
CUSKERand HABER1981 WEIFFENBACH
;
;
and ROGERS 1984). In both organisms, broken chromosomes can become
"healed" (that is, mitotically stable). In maize, such healing events often involved chromosomal rearrangements that in essence allowed a broken chromosome to acquire a new telomere by translocation involving another, nonhomologous chromosome. In other cases, MCCLINTOCK(1 94 1) observed terminally deficient chromosomes in which the origin of the telomeric region
could not be determined.
I
To whom correspondence should be addressed.
Genetics 106207-226 February 1984
208
J. E. HABER AND P. C. THORBURN
In yeast, in which cytology is not good enough to examine individual chromosomes for rearrangements, the mechanisms of chromosome healing have
only been addressed genetically. Our previous studies, based on the healing of
chromosome breaks generated specifically at the yeast MAT locus, indicated
that two major types of healing events occurred (MCCUSKER
and HABER1981;
WEIFFENBACH
and HABER1981). The majority of events involved homologous
recombination events between a broken chromosome end and its homologous
sequence on an intact homologous chromosome. Less frequent were those
resulting in terminally deficient chromosomes. In these instances, it was not
known whether the healed chromosome acquired a telomere by a translocation
event with another, nonhomologous chromosome or whether a bona fide new
telomere was created at the broken chromosome end.
An alternative approach to studying broken yeast chromosomes has been to
examine chromosomes that had become broken at apparently random sites.
We have followed the breaking and healing of linear, tandemly duplicated
dicentric chromosomes that were formed by recombination between a linear
chromosome ZZZ and a circular derivative of that chromosome (HABER, THORBURN and ROGERS1984). Both parental chromosomes were haploid-viable and
uniquely marked: the circular derivative with URA? and pBR322 sequences
not normally found on chromosome ZZZ, and the linear homologue by the distal
markers MAL2, HML and HMR that were deleted in forming the circle.
Meiotic recombination between the two homologues yielded a dicentric structure carrying both URA3 and MAL2 (Figure 1). Colonies derived from a haploid spore containing part or all of the dicentric chromosome ZZZ were identified by finding meiotic tetrads containing only three viable spores, one of
which carried both URA? and MAL2. The other two spores contained either
a parental circular (Ura+) or linear (Mal+) homologue, respectively (HABER,
THORBURN
and ROGERS1984). The colonies containing both URA? and MAL2
were highly variegated with the mitotic progeny derived from a single haploid
spore expressing a variety of different combinations of dominant and recessive
chromosome ZZZ markers.
In this paper we have characterized the different types of rearranged chromosomes derived from spores containing tandemly duplicated linear dicentric
chromosomes. Unlike previous studies in either maize or yeast, there were no
intact, normal homologues with which a broken chromosome end could interact; however, as shown in Figure 2, broken ends created in a tandemly duplicated dicentric could interact with homologous sequences on the opposite end
of the dicentric chromosome. In addition, it was likely that a cell actually
contained two different broken chromosomes ZZZ, as we have shown (HABER,
THORBURN
and ROGERS1984) that dicentrics generally did not break during
meiotic chromosome segregation; all of the regions present on the dicentric
were inherited into a haploid spore. In these cases, the initial chromosome
break would occur in a mitotic cell, after chromosome replication, so that a
cell might contain two different chromosome fragments, including all of the
regions carried by the original dicentric (Figure 3). Thus, one might envision
two major types of outcomes: the re-formation of circular or linear chromo-
BROKEN YEAST CHROMOSOMES
209
LL2
+
thr4
To
I
URA3
H M L HIS4 leu2
MATO
thr4
HMR MAL2
MATw T H R 4 URA3 his4 LEU2
FIGURE1.-Generation of a dicentric linear chromosome during meiosis. A haploid-viable circular chromosome III was constructed essentially by joining the two silent copies of mating type
and ROGERS
information (HML and HMR) via a MAT-URA3-pBR322 plasmid (HABER,THORBURN
1984). The HMR/MATa-URA3-pBR322-MAT/HMLa
region is designated URA3. The Ura+ ring
chromosome III lacks MAL2 and other sequences distal to HML and HMR, whereas a normal linear
chromosome III carries MAL2 and lacks any homology with URA3 and pBR322. Meiotic recombination between a ring and a rod chromatid yields a tandemly duplicated linear dicentric chromosome III. Thus, a meiosis with one exchange along chromosome yields a tetrad containing one
linear, one circular and one dicentric chromosome. Genetic evidence (HABER,THORBURN
and
ROGERS1984) indicates that, at least half the time, such a dicentric does not break and is inherited
intact into a single spore. The fourth spore, lacking a chromosome III, would be inviable.
somes by recombination between the broken end and a homologous region on
the same chromosome (or on another chromosome fragment), or the generation of partially duplicated chromosomes containing a novel chromosome end.
MATERIALS AND METHODS
Strains: Diploid PT139 (Table IA), containing both a linear (MAL?) and circular (URA3) chromosome IZI was sporulated and dissected. Tetrads containing three viable spores (one Ura+, one
210
J. E. HABER AND P. C. THORBURN
MATa T H R 4 U R A 3
HML
HIS4
leu2
HMR
/
MAL2
A.
B.
H M L his4
leu2
HML his4
leu2
MAT&
thr4
HMR M A L 2
MATH
thr4
URA3
C.
--
HIS4
FIGURE2.-Types of healing events involving dicentric chromosomes inherited into mitotic
cells. Broken chromosome ends are highly recombinogenic (MCCUSKER
and HABER1982) and can
recombine with homologous sequences within the tandemly duplicated dicentric chromosome. In
a mitotic cell that inherited an intact dicentric chromosome, subsequent chromosome replication
and centromere segregation could lead to a cell containing fragments of two broken homologues.
Recombination can either lead to the re-formation of circular (A) or linear (B) derivatives that are
mitotically stable. In addition, however, more novel healing events can occur (C) in which a broken
chromosome (which carries all of the genetically essential segments of the chromosome) becomes
mitotically stable.
Mal+ and one Ura+ Mal+) were obtained (HABER,THORBURN
and ROGERS1984). The Ura+ Mal+
colonies were genetically variegated, as evidenced both by sectoring for various recessive markers
on chromosome III and from subclone analysis. A variety of genetically distinct derivatives from
several different Ura+ Mal+ colonies were collected; their phenotypes are listed in Table 1B. (The
chromosome III genotypes of these strains are the subject of this paper.)
Genetic analysis: Standard yeast genetic methods, described by SHERMAN,
FINKand LAWRENCE
BROKEN YEAST CHROMOSOMES
c
211
\
/
I
FIGURE3.-A spore inheriting an unbroken dicentric chromosome would replicate the chromosome prior to mitosis. Mitosis could either lead to the creation of two daughter cells each of
which contained an unbroken dicentric chromosome or each of which received two different broken
chromosomes, depending on the alignment of centromeres, The two broken chromosomes would
be expected to have different breakpoints. Thus, a colony of cells derived from a single spore
containing a dicentric chromosome would likely give rise to a colony in which several entirely
independent chromosome breaks had occurred.
(1979) were used. Additional methods are described in the accompanying paper (HABER,ROGERS
and THORBURN
1984).
Analysis of DNA: DNA from various strains was isolated as described previously (HABER,ROGERS
and MCCUSKER
1980). DNA was digested with restriction endonucleases (New England Biolabs),
and fragments were separated and transferred to nitrocellulose filters according to the method of
SOUTHERN
(1975). The nitrocellulose filters were probed with s2P-labeled probes. The plasmid
pJH3 is a pBR322 plasmid containing the EoRI-Hind111MATa region, which is homologous not
only to MATa and MATa but also to two other loci on chromosome 111, HML and HMR, on
distinct restriction fragments (HABER,ROGERSand MCCUSKER
1980). This probe is also homologous to the HMR/MATa-URA3-pBR322-MAT/HMLa
construction creating the ring chromosome
III. Some Southern blots were probed with a 0.7-kb PvulI-Sac1 fragment from plasmid pSZ220,
which contains a telomere-adjacent sequence (TAS) apparently found near the ends of various
yeast chromosomes (SZOSTAK
and BLACKBURN
1982). The purified fragment was generously provided by T. CLAWS
and J. SZOSTAK.
In addition, '*P-labeled plasmid pB161 that hybridizes to most
T Y 1 elements in the yeast genome was used to probe Southern blots to look for rearrangements
of TYl elements. Plasmid pB161, containing the BglII fragment of TYl was subcloned from
plasmid ,5513 (CAMERON,
LOH and DAVIS1979) and was generously provided by R. T. SUROSKY,
B.-K. TYEand G. R. FINK.
RESULTS
Aizalysis of stable derivatives from Urn+M a l + colonies: We have used both genetic
and molecular approaches to examine the structure of healed chromosomes ZZZ
212
J. E. HABER AND P. C. THORBURN
TABLE I
Phenotypes of healed derzuatiues from three spores containing a linear dzcentrzc
chromosome 111
A. Diploid PTI?Y, used to generate dicentric spores, had the genotype:
circle III"
[HIS4 leu2 CRY1 MATa THR4 HMRIMATa-URA?-MATIHMLa]
linear IiI HMLa his4 LEU2 cry1 MATa thr4
HMRa
MAL2
ura?
adel
_
__
_ -lys2
-ura? adel
+
+
leu1
can1
+
B. Derivatives of segregants PT1?9-7?AA,PTl39-72A, and PT139-76B had the following chromosome 111
phenotypes:
PT139-73A
PT139-73A
PTl39-73A
PT139-73A
PT139-73A
PT139-73A
PT139-72A
PT139-72A
PT139-72A
PT139-72A
PTl39-72A
PT139-72A
PT139-72A
PT139-76B
PTl39-76B
PT139-76B
PT139-76B
PT139-76B
PTl39-76B
#9
#32
#27
#2
#1
#6
#5
#21
#33
#8
#46
#6
#4
#4
#66
#31
#45
#1
#48
Ura3- Ma12+ His4'
Ura3- Ma12' His4Ura3+ Ma12- His4Ura3+ Ma12+ His4+
Ura3+ Ma12- His4'
Ura3- Ma12+ His4Ura3+ Ma12+ His4+
Ura3' Ma12' His4+
Ura3+ Ma12- His4'
Ura3- Ma12+ His4+
Ura3+ Ma12+ His4'
Ura3- Ma12' His4+
Ura3- Ma12+ His4+
Ura3' Ma12' His4+
Ura3- Ma12+ His4+
Ura3+ Ma12+ His4+
Ura3- Ma12+ His4+
Ura3- Ma12' His4+
Ura3+ M a K His4+
Leu2' Thr4+ weak a mating
Leu2+ Thr4+ weak a mating
Leu2+ Thr4+ nonmating
Leu2+ Thr4+ a mating
Leu2' Thr4+ a mating
LEU2+ Thr4- a mating
leu2- Thr4+ nonmating
Leu2+ Thr4' weak a mating
Leu2- Thr4+ weak a and a mating
Leu2- Thr4+ a mating
Leu2- Thr4' a mating
Leu2+ Thr4- a mating
Leu2- Thr4- a mating
Leu2- Thr4+ a mating
Leu2- Thr4+ a mating
Leu2- Thr4+ a mating
Leu2- Thr4- a mating
Leu2- Thr4+ a mating
Leu2- Thr4+ a mating
"The circular chromosome I I i was constructed by joining the distal markers HMR and HML via
a recombinant DNA plasmid containing MAT-URA?-pBR322 [see Figure 2 of accompanying article
and ROGERS1984)) This ring chromosome therefore lacks MAL2.
(HABER,THORBURN
in the wide variety of stable derivatives from Ura+ Mal+ colonies. We could
assess the kinds of healing events that occurred in different instances by examining Southern blots of BamHI-digested DNA from different subclones
probed with a 32P-labeled plasmid containing MATa. As we showed in the
and ROGERS 1984) the MAT probe
accompanying paper (HABER, THORBURN
is homologous to six different regions along the length of a dicentric chromosome ZZZ HML (near the left end); two MAT regions; H M R (near the right
end) and two BamHI fragments from the (HMR/MAT-URA3-pBR322-MAT/
HML) fusion that was originally part of the circular chromosome (4 Figure 1
and Figure 4A, lanes 1 and 2). Thus, from the pattern of bands homologous
to a MAT probe we could find examples of stable derivatives that were characteristic of either the linear or circular parent chromosomes. In addition, this
probe also allows the detection of rearranged chromosomes. For instance, in
example C illustrated in Figure 2 , the healed chromosome would be expected
213
BROKEN YEAST CHROMOSOMES
B
A
1
2
3
4
5
6
7
1
2
3
4
5
6
c
FIGURE4.-Examples
of different healing events arising from a single duplicated chromosome
III. DNA from genetically distinct subclones from two different Ura+ Mal+ spores were digested
with EamH1, and the Southern blot was probed with "P-labeled plasmid pJH3 containing the yeast
MAT locus. A, Derivatives of strain PTl39-73A are shown, in comparison with the patterns of
the two parent haploids (lanes 1 and 2) that gave rise to the diploid PT139. The lanes represent:
(1) PT49-32D haploid containing a circular chromosome III; (2) PT69-23A haploid containing a
linear chromosome III. T h e top band in each lane contains the MAT locus. T h e two other bands
(U and P) in lane 1 contain the HMR/MAT-URA3 and pBR322-MATIHML portions of the structure
creating the circle. T h e middle band in lane 2 carries HMR, whereas the bottom band carries
HML. (3) PT139-73A no. 9 Ura- Mal+; (4) PT139-73A no. 32 Ura- Mal-; (5) PTl39-73A no. 27
Ura' Mal-; (6) PTl39-73A no. 2 Ura* Mal*; (7) PTl39-73A no. 1 Ura+ Mal-. The complete
phenotypes of these derivatives are given in Table 1. B, Derivatives of strain PT139-76B. Lanes
include: (1) PT139-76B no. 4 Ura+ Mal+; (2) PTl39-76B no. 66 Ura- Mal+; (3) PT139-768 no.
31 Ura+ Mal'; (4) PTl39-76B no. 45 Ura- Mal+; ( 5 ) PT139-76B no. 1 Ura- Mal+; (6) PT13976B no. 48 Ura+ Mal-. Some derivatives have the pattern characteristic of a circular chromosome
III; some appear to contain a linear chromosome III. Other derivatives have all of the bands found
in both a ring and a rod chromosome. Finally, a small number exhibit novel patterns suggesting
other chromosomal structures (e.g., A, lane 5; and B, lanes 2 and 6).
to include restriction fragments containing MAT, HML, the two fragments
construction, but not HMR.
from the HMR/MAT-URA3-pBR322-MAT/HML
Southern blots of BumHIdigested DNA from five different derivatives s u b
cloned from one Ura+ Mal+ segregant, PT139-73A,are shown in Figure 4A
(lanes 3-7), whereas similar blots from six derivatives of another segregant,
PT139-76B,are presented in Figure 3B. Similar analyses of derivatives from
Ura+ Mal+ segregants PT139-29A,PT139-72A,and PT.139-98Bwere also
carried out (data not shown). An inspection of these patterns revealed that
most of the Ura+ Mal- derivatives were apparently circular chromosomes, with
the same restriction bands as the parent haploid (e.g., Figure 4B, lane 6).
Similarly, nearly all of the Ura- Mal+ derivatives appeared to contain only
those bands characteristic of a linear chromosome, with both HML- and HMRcontaining restriction fragments (e.g., Figure 4A, lanes 3 and 4; Figure 4B,
lanes 4 and 5). These results suggested that, in many cases, the healing of the
dicentric chromosome involved the re-formation of an intact linear or circular
chromosome (Figure 3). Subsequent genetic analysis, involving tetrad analysis
after crossing these stable derivatives with marked linear chromosomes (data
214
J. E. HABER AND P. C. THORBURN
not shown), confirmed that most of the Ura- Mal+ derivatives behaved as linear
chromosomes (giving good spore viability), whereas most of the Ura' Malcells appeared to carry circles (being mitotically unstable in diploids and giving
poor spore viability).
Diploid derivatives arising from haploid spores: In the course of this analysis we
discovered that some of the Ura+ Mal- and Ura- Mal+ derivatives were able
to sporulate. These appeared to be instances in which diploid cell had been
produced, quite possibly by the mating of genetically different derivatives of
the initially haploid spore containing the dicentric. For example, the apparently
linear Ura- Mal+ derivative PTl39-29A-25 was nonmating and sporulated,
yielding more than 80% tetrads with four viable spores. The segregants were
homozygous for his4, LEU2 and thr4 but heterozygous for cryl and MAT. Thus,
this diploid contained two genetically distinct linear chromosomes ZZZ.
The Ura+ Mal- derivatives PTl39-29A-23 and 72A-33 both contained only
BamHI restriction fragments characteristic of circular chromosomes but were
also able to sporulate. Spore viabilities were poor, with fewer than 30% of the
tetrads yielding four viable spores. The recovery of four viable spores in some
tetrads again indicated that the diploid contained two (circular) chromosomes
ZZZ. Derivative 29A-23 apparently contained two circular chromosomes that
were genetically distinct, as these diploids were heterozygous for MAT and
cryl. Diploid 72A-33 was homozygous for MATa and apparently was able to
sporulate because of the presence of the very weakly expressed HMRIMATa
sequence adjacent to URA3.
Thus, at least some of the time, several different healing events must have
occurred within a Ura+ Mal+ colony, generating cells of opposite mating type
that could then mate to form diploids either with two different linear chromosomes, or with two circular chromosomes, or (as we will discuss later) with
one linear and one circular chromosome.
Among the Southern blots of Ura' Mal- and Ura- Mal+ derivatives shown
in Figure 3, there were several that did not contain the BamHI patterns expected of either a circular or a linear chromosome ZZZ. Two such Ura+ Malderivatives are seen in Figure 4A, lanes 5 and 7; an unusual Ura- Mal+ derivative is shown in Figure 4B, lane 2. These three cases, which represent examples of more novel healing events, will be discussed in detail later.
Diploid and disomic haploid derivatives containing both a ring and rod chromosome:
A third large group of derivatives were still Ura+ and Mal+. As judged from
inspection of several hundred subclones, these cells were essentially mitotically
stable (unlike the original highly variegated Ura+ Mal+ colonies germinated
from spores containing the dicentric chromosome). When we examined Southern blots of these derivatives, we found that these cells usually contained the
restriction fragments characteristic of the URA3-pBR322 plasmid joining H M L
and H M R as well as bands containing intact HML and H M R sequences (Figure
4A, lane 6; Figure 4B, lanes 1 and 3). Such structures would be expected in
strains carrying both a circular and linear chromosome but could be found in
other structures as well.
One subclone that we have examined in detail (PTl39-72A-21) was weakly
BROKEN YEAST CHROMOSOMES
215
A
1
2
3
4
5
U
HML
B
1
2
3
4
5
MAT
HMR
P
U
FIGURE5.-Meiotic segregation of MAT-homologous &"I
fragments in two different diploid
derivatives from spore PT139-72A.A. Diploid PT139-72A-21contained all of the &"I
fragments homologous to the MAT-containing probe pJH3 expected of a dicentric linear chromosome
III (lane 1). D N A was isolated from all four meiotic segregants of one tetrad. The pattern of
segregation is consistent with the presence of a normal linear chromosome in two segregants (lanes
4 and 5 ) and a circular chromosome in the other two spore colonies (lanes 2 and 3). B, Diploid
PT139-72A-5also contains all of the BumHI fragments in an intact dicentric chromosome (lane
1). However, when D N A from all four segregants from one tetrad were, analyzed, it became
evident that two carried a normal linear chromosome III (lanes 2 and 3), whereas the other two
had a novel pattern (lanes 4 and 5). The novel, healed chromosome III contains MAT, HML. plus
region. but does not conthe U and P fragments from the HMR/MAT-lJRA3-pBR322-MAT/HML
tain HMRa.
5B, lane 1). T h e weakly a-mating phenotype was characteristic of a disomic or diploid strain that carried MATa on a linear chromosome
and MATa on a circle. This nonmating strain would become a mating when
the circular chromosome was lost at a low frequency (about lo-' per cell
and ROGERS1984). Indeed, further tests showed
division) (HABER,THORBURN
a mating (Figure
216
J. E. HABER AND P. C. THORBURN
that the cells within this colony that actually mated simultaneously became
Ura- and Thr-. Thus, subclone 72A-21 appeared to carry a Ura' Thr+ M A T a
circular chromosome and a Mal+ MATa linear chromosome (but with a different arrangement of markers on the two homologues than had been present in
the original parent diploid). Derivative 72A-2 1 was apparently diploid rather
than a disomic haploid; the strain sporulated well and gave rise to viable spores.
Spore viability was characteristic of a ringlrod diploid, with fewer than 25%
of the tetrads containing four viable spores. When DNA was isolated from one
tetrad with four viable spores, a Southern blot confirmed that the two Mal+
spores contained the pattern expected of a linear chromosome, whereas the
two Ura+ segregants contained restriction fragments characteristic of a circle
(Figure 5A, lanes 2-5). This colony was heterozygous for leu2, cryl, MAT and
thr4 but homozygous for HZS4, on chromosome ZZk it was also homozygous
for the three markers on other linkage groups. Thus, this derivative apparently
arose from a series of events in which a dicentric chromosome was broken,
and various fragments were recovered by several healing events in two different cells, which then mated to form a diploid.
A similar analysis has been carried out on another weakly mating Ura+ Mal+
derivative whose Southern blot showed the presence of all the bands expected
in a ring/rod diploid (PT139-29A-70). The results were similar to those described earlier for PT139-72A-2 l . Thus, these diploid derivatives containing
both a ring and a rod chromosome ZZZ had been generated from an initially
haploid spore carrying a dicentric chromosome.
Novel healing of a broken chromosome at the right arm of chromosome 111: One
Ura+ Mal+ derivative (PT 139-72A-5) was completely nonmating, suggesting
that there was no mitotically unstable circular chromosome, even though the
Southern blot (Figure 5B, lane 1 ) contained all of the bands characteristic of
both a linear and a circular chromosome. This strain proved to be diploid and
could be sporulated and dissected. Spore viability was excellent, again not
characteristic of a ringlrod situation: 17 of 20 tetrads had four viable spores.
In each case, MAL2 and URA3 segregated to opposite segregants, indicating
that they were in strict repulsion. Further genetic analysis indicated that the
two chromosomes ZZZ were both stable, linear chromosomes, one of which
carried URA3 near the end of the right arm, closely linked to the position of
MAL2 on the other homologue. T o confirm this interpretation we also analyzed the Southern blot of DNA from four segregants of one tetrad. As shown
in Figure 5B, the two Mal+ segregants (lanes 2 and 3) contained BamHI fragments corresponding to M A T a , HMLa and HMRa, whereas the two Ura+
segregants (lanes 4 and 5) did not contain HMRa but did include M A T a ,
HMLa, plus the two bands (HMRIMATa-URA3 and pBR322-MATIHMLa) characteristic of the plasmid insertion of the ring chromosome.
Further evidence to support this contention has been the finding that the
healed Ura+ chromosome ZZZ is actually duplicated for HZS4. Haploid meiotic
segregants of PT139-72A-5 containing a His+ Ura+ chromosome with the
novel right end were crossed to a his4 LEU2 ura3 strain. Tetrad analysis
showed that there were two HZS4 alleles segregating independently. One HIS4
BROKEN YEAST CHROMOSOMES
217
allele was tightly linked to URA3, whereas the other segregated independently
(but linked to leu2).
These data suggest that a dicentric chromosome had broken somewhere
between HIS4 and the centromere of the left arm of chromosome ZZZ (as
illustrated in Figure 2 , example C) and subsequently became healed by the
formation of a new telomere. Thus, the Ura+ segregants did not contain HMRa
but had the structure:
HMLa HIS4 leu2
M A T a THR4 (HMRIMATa URA3 MATIHMLa) HIS4.
We have also recovered a second, independent derivative with a similar novel
healing event on the right end of the chromosome. The Ura+ Mal- derivative
PTl39-73A-27 (Figure 4A, lane 5 ) contained four BamHI restriction fragments
homologous to a MAT probe: H M L , M A T , and the two plasmid insertion fragments, pBR322-HMLlMATa and URA3-HMRIMATa. There was no H M R fragment, suggesting that the normal telomeric sequences beyond H M R were not
present. In this case we do not know whether the region including his4 had
been duplicated.
Formation 0 f a Ura- circular chromosome 111: We have also recovered one UraMal+ derivative that did not appear to be simply a linear chromosome. With
strain PT139-76B-66, the Southern blot of this strain (Figure 4 B , lane 2)
showed BamHI restriction fragments containing M A T , H M L and H M R and at
a new restriction fragment, different from those characteristic of the plasmid
insertion. Further analysis of the Southern blot showed that this new restriction
fragment contained an HMRlHMLa fusion, identical with that found in a Uraderivative of the original circular chromosome [Figure 2 of accompanying
article (HABER,ROGERS and THORBURN
1984)]. Genetic analysis has shown
that this strain is a haploid, but disomic, with one linear and one Ura- circular
chromosome ZZZ. The circular chromosome apparently carried T H R 4 , whereas
the linear carried thr4. When this strain was subcloned, approximately 5 % of
the subclones became Thr-; subsequent Southern blot analysis has shown that
these Thr- subclones no longer contained the HMRIHMLa band. Furthermore, both homologues carry M A T a , so that when this strain (PT139-76B-66)
was crossed with a normal rod-containing haploid, we found tetrads consistent
with MATaIMATaIMATa segregation (data not shown). T h e formation of the
Ura- circular chromosome could have arisen in two ways: it may have resulted
from a rare intrachromosomal recombination event between homologous matconstrucing-type sequences in the HMR/MATa-URA3-pBR322-MAT/HMLa
tion (such events were found at a frequency of less than
among unselected
Ura+ haploids); alternatively, it could represent another example of healing a
broken chromosome by recombination with an homologous sequence. In the
latter case, the broken end of the chromosome may have arisen within the
MATIHMLa and the broken end then recombined with its homologous sequence at HMR.
A novel healing event involving the HML/MATa-URA3-pBR322-MAT/HMLa
construction: From the same colony (PTl39-73A) that gave rise to the mono-
218
J. E. HABER AND P. C. THORBURN
centric partially duplicated chromosome that we described before (PTl39-73A27), we have also recovered an entirely different chromosomal rearrangement.
When subclone PT139-73A-1 was examined by Southern blots of BumHI fragments, we found a novel band homologous to the MAT probe (Figure 4A, lane
7). This strain had bands characteristic of a circular derivative, plus one additional band lying between the two fragments derived from the HMRIMATURA3-pBR322-MAT/HML construction (arrow). This strain was then crossed
to a normal linear (MAL2) parent (PT148-69C), and the diploid was sporulated
and dissected. Spore viability was poor, as expected from a ring/rod diploid,
but eight complete tetrads were recovered.
Tetrad analysis revealed several unusual features. First, there were two c o p
ies of URA3, MATa and THR4 segregating in the cross, as evidenced by tetrads
with, for example, either 2 Ura+:2 Ura-, or 3 Ura+:l Ura-, or 4 Ura' segregants. Second, approximately half of the MAL2 segregants (presumably inheriting a linear chromosome) were Ura+. Some of these were weakly a mating,
and further investigation showed that the cells that actually mated also lost
URA3. All of these phenomena suggested that the original strain carried two
Ura+ chromosomes.
Southern blot analysis of DNA from meiotic segregants has shown that one
of the URA3 genes cosegregated with the unique BumHI restriction fragment.
Southern blots from three tetrads are shown in Figure 6. The three tetrads
represent the three types of tetrads that we described earlier: in the first type
FIGURE6.-Meiotic segregation of a novel MAT-homologous BamHl tragmcnt In the healed
derivative PTl39-73A-1. This strain was crossed with strain PT148-69C carrying a novel chromosome Ill. DNA from three complete tetrads was isolated and cleaved with EumH1, and the
Southern blot was probed with the pBR322-MAT. plasmid pJH3. The diploid apparently contained
both a linear chromosome I l l (lanes 3A and 3D, for example) and a circular chromosome (lanes
1A and lD, for instance), which segregated 2:2. In addition. another partial chromosome I11
appears to segregate independently of either the linear or the circular chromosomes Ill. The novel
chromosome contains MATa and two other MAT-homologous bands, one of which is apparently
identical with the HMR/MATa-lJJZA3 EamHI fragment. The other band, distinctly smaller than
the pBR322-MAT/HMLa EamHl fragment in a circular chromosome (cg.. lanes lB, 1C).
BROKEN YEAST CHROMOSOMES
219
(lanes lA, B, C and D) there were two Ura' Mal- and two Ura+ Mal+ segregants; the second (lanes 2A, B, C, D) contained a tetratype ascus, with one
Ura+ Mal+, one Ura- Mal+ and one Ura+ Mal- spore; the third had two Ura'
Mal- and two Ura+ Mal- segregants. The Southern blot showed that all Mal+
segregants contained the BamHI fragments characteristic of a linear chromosome ZZZ (i.e., HMLa and HMRa). In addition, those Mal+ segregants that were
also Ura+ carried two additional BamHI fragments homologous to the probe.
T h e Ura+ Mal- segregants all had the bands characteristic of a circular derivative, but again, half of these segregants contained the novel fragment. Thus
the "extra" URA3 sequence was associated with the segregation of two BamHI
fragments, one of which was the same size as the HMRIMAT-URA3-containing
fragment found in the circular parent. The second novel fragment was now
8.7 kb instead of the 9.0-kb pBR322-MATIHML BamHI fragment found in
the original circle. It was also important to note that the segregation of the
novel fragment was not linked to the segregation of either HML or HMR (or
MAL2).
Among the eight complete tetrads that we have analyzed we have not found
an instance in which a viable segregant contained only the BamHI fragments
associated with the altered extra URA3 gene. This suggests that the novel
URA3-associated chromosome is either deficient for some of the regions on
chromosome ZZZ essential for haploid viability or, alternatively, may not be able
to pair with or compete with the two intact chromosome ZZZ homologues during
meiosis.
All of these data suggested that the original strain PT139-73A-1 was a
disomic haploid and contained both a circular (Ura+) chromosome and a second, apparently partial Ura+ chromosome. This new chromosome (which must,
at least, carry MATa, THR4 and the altered HMLIMATa-URA3-pBR322-MATI
HMRa region) may not contain all of the regions of chromosome ZZZ essential
for viability but does segregate as if on a centromere-containing chromosome.
Evidence presented later in the paper also suggests that this chromosome may
be a healed linear rather than circular structure.
Rearrangements of TAS: From the data we have described it was not at all
clear what was the structure of one of the healed chromosomes in PT13973A-1. This Ura+ Mal- chromosome apparently lacked normal HML or HMR
sequences (and was also Mal-) and might, therefore, be a circular derivative,
although with some rearrangement of the MATIHMLa sequences. Alternatively, this novel chromosome might also be linear, with novel telomeres at
one or both ends. To investigate this further we have used a TAS that has
been described recently by SZOSTAK
and BLACKBURN
(1982). The cloned yeast
telomere that they described contained a Sad-PvuI fragment that is homologous to a large number of KpnI restriction fragments in yeast (presumably
near the ends of other chromosomes). Further evidence that nearly all of the
copies of this TAS are near yeast telomeres comes from o u r observation that
these sequences are preferentially digested by the endonuclease Ba13 1 (J. E.
HABER,P. S. LIU and P. C. THORBURN,
unpublished results), as are telomeric
sequences in both Tetrahymena (YAO 1981) and trypanosomes (WILLIAMS,
220
J.
E. HABER AND P. C. THORBURN
YOUNG and MAJIWA 1982; DELANCEand BORST 1982). We have used this
sequence as a probe to look for chromosomal rearrangements associated with
the healing of broken chromosomes.
Three different stable derivatives of PT139-73A represented an ideal set of
strains for this purpose. All three derivatives were derived from the same
haploid spore containing the same dicentric chromosome; consequently, all
three derivatives must have begun with the same chromosome ends. This is
an important point, because we have discovered that different yeast strains,
even reasonably closely related, have widely varying patterns of restriction
fragments homologous to the TAS probe (J. E. HABER,P. S. LIU and P. C.
THORBURN,
unpublished results). Such variation can be seen in comparing the
pattern of KpnI fragments homologous to the two parental strains used to
construct PT 139 (Figure 7A). The pattern of restriction fragments in the three
PT139-73A derivatives is different from either parent, as expected from the
segregation into one spore of many independently segregating restriction fragment length polymorphisms. More important, however, is the clear presence
of two novel fragments (arrows) in the DNA from strain PT139-73A-1. These
two novel bands are not found either in PT139-73A-2-1B (a diploid with both
a linear and a circular chromosomes ZZZ) nor in PT139-73A-29, which, nevertheless, does apparently have a novel chromosome end on the right arm of
the healed chromosome.
The fact that strain PT139-73A-1 contained two novel fragments homologous to the telomere-adjacent probe suggested that both new fragments were
adjacent to novel telomeres on the ends of the extra chromosome that also
harbored a rearrangement in the BamHI fragment containing pBR322 and
MAT/Hil4La. An indication that all three novel fragments were contained on
a single chromosome has come from our discovery that the strain occasionally
loses all three regions simultaneously. The Southern blot of one such subclone
of strain 73A-1 showed that it is missing both new bands homologous to TAS
(data not shown). The novel MAT-homologous band on a BamHI digest was
also missing (data not shown).
We have also followed the segregation of the two new telomeric regions
during meiosis. Using DNA from the same three tetrads in which we analyzed
the segregation of the novel BamHI fragment, we have examined the segregation of the two new TAS-homologous bands. One tetrad, corresponding to
tetrad no. 1 in Figure 7, is shown in Figure 7B. It is evident that the two
bands segregate independently of each other, as one segregant has both bands,
one has neither and the others have one band each. Furthermore, when the
segregations are compared with the novel BamHI fragment, we find that neither TAS-homologous band is closely linked to the URA3-pBR322-containing
region; for example, segregant 1B has neither TAS-homologous bands whereas
segregant 1C has both.
We have also examined seven different healed derivatives of PTl39-73A for
changes in TAS-homologous fragments. No such rearrangements were detected.
Exaininatiorz of repeated (TYl) sequences in cells that have undergone healing:
MCCLINTOCK
(194 1) first detected the transposable element Ds in maize that
BROKEN YEAST CHROMOSOMES
A
221
B
A
B
C
D
1 2 3
-W.
F,
I
FIGURE7.-Appearance of two novel KpnI fragments homologous to a TAS in derivative
PT139-73A-1. A, Kpnl fragments homologous to the TAS probe are shown for three genetically
distinct derivatives of one spore PT139-73A. Both derivatives 73A-2-1B (lane 2) and 73A-27 (lane
3) contain only TAShomologous fragments found in the parental diploid (not shown), whereas
derivative 73A-1, (lane 1) contains t w o novel TAShomologous bands (arrows). B, Meiotic segre
gation of TAShomologous bands found in derivative PT139-73A-1. This strain was c r d with
strain PT148-69C. DNA from four segregants of the same tetrad (IA, IB, IC. ID) shown in
Figure 6 was digested with KpnI and hybridized with '*P-labeled TAS probe. T h e segregation of
many polymorphic bands can be seen, often showing 2:2 segregation expected of largely unique
fragments carrying a region homologous to the TAS probe. T h e segregation of the two novel
KpnI fragments is also seen. T h e two fragments segregate independently, as evidenced by the fact
that one segregant (B) has neither fragment, one (D) has both and the other two have one novel
fragment each.
had previously experienced chromosomal breakage and healing. We wondered
whether chromosomal breakage and rearrangements might also stimulate the
movement of the yeast transposable element, T Y l . When Southern blots of
genomic yeast DNA were digested with the endonuclease BamHI and probed
with pB161 containing a BgIII fragment common to most T Y l elements, a p
proximately 30 distinct bands homologous to the probe could be counted. T h e
sizes of some of these fragments varied in different strains (data not shown).
We examined the sizes of TYl-homologous fragments in a series of different
healed derivatives from four different Ura+ Mal+ spores, PTl39-72A, PT139-
222
8
J. E. HABER AND P. C. THORBURN
MAT
9
THR4 U R A 3 HIS4 L E U 2
A.
M A T S T H R 4 U R A 3 HIS4
leu2
A.
H M L HIS4 l e u 2
MAT
H M L HIS4 l e u 2
MATo
lhr4
HMR M A L 2
H M L HIS4 L E U 2
MATo
lhr4
HMR M A L 2
THR4 HMR M A L 2
8.
HMR M A L 2
H M L his4 L E U 2
MATU
H M L his4 L E U 2
MATS
H M L hls4 L E U 2
M A T o T H R 4 HMR M A L 2
HML hlS4 LEU2
MATS T H R 4 URA3
HML h1s4 LEU2
MATS
thr4
0
C.
T H R 4 HMR M A L 2
C
D.
H M L H I S 4 leu2
MATo
H M L HIS4
IOU2
MAT#
H M L HIS4 l e u 2
MATo
lhr4
HMR M A L 2
THR4 URA3
HIS4
D
thr4
HMR M A L 2
E.
thr4
HMR M A L 2
h's4u
URA3
W
E.
URA3
H M L hb14 L E U 2
MAT%
thr4
HMR M A L 2
F
I
e
n
a
H1S4J1/THR4
HIS4
URA3
HR4
URA3
F.
h's4
UT,,,
URA3
_____
MAT& T H R 4 URA3'
- - .-
FIGURES8 AND 9.-A summary of different healing events derived from a single spore containing a dicentric linear chromosome III. Based on the fact that each spore came from a triad in
which the other two parents had entirely parental arrangements of chromosome III markers, we
presume that the dicentric chromosome were heterozygous for h i d , leu2, MAT and thr4. The
exact arrangement of the markers on the dicentric depends on the interval in which crossing over
BROKEN YEAST CHROMOSOMES
223
73A, PT139-76A and PT139-98B. Although there were some differences between the two sets of derivatives (reflecting the Mendelian segregation of
various T Y 1-containing fragments into different haploid spores), there were
no differences among the various subclones derived from a single spore.
DISCUSSION
It is clear that we can recover a wide variety of genetically distinct, stable
derivatives from a single spore containing a dicentric chromosome. An examination of these different mitotic offspring has raised several questions. How
did these different types arise? Can they be accounted for by a single breakage
and immediate healing of a dicentric chromosome during the first mitotic cell
cycle, or is there evidence of a more complex series of events? Is there, for
example, evidence of a breakage-fusion-bridge (BFB) cycle, resulting in a potentially large number of different chromosomal arrangements?
By characterizing the different subclones from one spore colony, we can
attempt to reconstruct the sequence of events leading to the formation of all
of these stable types. We have considered two examples, the derivatives of
segregants PT139-72A and PT139-73A. We have chosen these two cases because they appear to have arisen from meioses in which there was only a single
crossover between one ring and one linear parent (that is, the other two viable
spores-one ring and one rod-had an entirely parental arrangement of chromosome ZZZ markers) [Table 3 in accompanying article (HABER,ROGERSand
THORBURN
1984)]. The exact arrangement of markers along the dicentric was
likely different in the two cases, depending on the position of the exchange
event that produced the dicentric. We assume that two major types of healing
events were possible: recombination between homologous segments elsewhere
on a broken chromosome fragment or the formation of a new telomere at the
broken chromosome end.
In Figure 8 are represented the apparent genotypes of the different types
of stable subclones obtained from these spores, based on both genetic and
Southern blot analysis. From strain PT139-72A (Figure 8) we have recovered
two different normal linear chromosomes, two different circular homologues
and one novel linear chromosomal rearrangement. From strain PTl39-73A
(Figure 9), we found three different normal h e a r s , two different rings plus
two linear chromosomal rearrangements. It is difficult to account for all of
these different arrangements simply on the basis of a single breakage of a
dicentric chromosome and its immediate healing. (The most obvious example
is strain PTl39-29A-23, which contains two Ura+ ring chromosomes, one of
which carries MATa, and the other, MATa.)
occurred between the ring and rod chromosomes. Figure 8, Stable derivatives of spore PT13972A. In addition to differently arranged linear and circular chromosomes, one derivative (D)
carried a healed chromosome that must carry a novel right telomere. Figure 9, Derivatives of
spore PT139-73A. In addition to apparently n o m 1 healed linear and circular chromosomes, one
derivative (C) has a novel healing event on the right end of the chromosome. Another (F) contained
an apparently partial linear chromosome 222 in which the URA3-containing region had been altered.
In addition, the derivative contained two novel TAS-homologous fragments.
224
J.
E.
HABER AND P.
C.
THORBURN
It should be stressed that we have, as yet, no direct evidence that dicentric
chromosomes undergo repeated rounds of a BFB cycle; rather, we know only
that dicentric chromosomes must be broken and that eventually a number of
quite different chromosomal rearrangements are produced. It is difficult to
see how this could occur without some continuing process to generate unstable
chromosomes that could heal in different ways. However, it must be recalled
that the initial Ura+ Mal+ spore most likely contained an unbroken dicentric
chromosome (Figure 3), so that after chromosome replication, segregation of
the two centromeres of the dicentric to the same pole would lead to two
daughter cells still containing the intact dicentric. In subsequent cell divisions,
however, segregation of the dicentric centromeres to opposite poles would
yield broken chromosomes. Under these circumstances, breakage and healing
would then occur totally independently in several different lineages within the
colony; and a wide variety of chromosomal rearrangements could be produced,
even without a true BFB cycle.
It is important to distinguish among the reasons why a dicentric chromosome
might not break in meiosis and mitosis. In meiosis, the two centromeres (from
two homologous chromosomes ZZZ) should obligately segregate to opposite
poles. Therefore, recovery of intact dicentric chromosomes in haploid spores
must represent a true failure to break the dicentric bridge during meiosis. In
contrast, during mitosis, unbroken dicentrics can be inherited, but for a different reason ( i e . , when two centromeres of a replicated dicentric move to the
same mitotic pole). However, when the two centromeres move to opposite
poles, it appears that the dicentric bridge is generally broken, unlike the equivalent situation in meiosis.
Mechanisms of healing broken chromosomes
We have recovered two distinct types of healing events. The majority of
cells contained linear or circular chromosomes that carried different combinations of genetic markers from the original parent chromosomes but were
otherwise indistinguishable from the parental ring and rod chromosomes. One
likely way in which these chromosomes were derived from the dicentric would
be entirely analogous to the most frequent way that chromosome ZZZ, broken
and HABER1981;
specifically at the MAT locus, became healed (MCCUSKER
WEIFFENBACH
and HABER1981). In those instances, the highly recombinogenic
broken chromosome end interacted with homologous sequences in a second,
intact chromosome ZZZ. In the case of a broken dicentric chromosome, we
imagine that a broken end would recombine with homologous sequences on
the same or on a second monocentric fragment produced by chromosome
breakage (Figures 2 and 3). Depending on which broken ends recombined
(and on the position of the break), one could recover either stable circular or
stable linear chromosomes by such recombination events.
A smaller number of healing events have occurred in an apparently different
way. In these cases, we recovered rearranged chromosomes that gave clear
evidence of having been derived from a dicentric. These chromosomes were
linear, but lacked either the normal right end or left end of chromosome ZZZ.
BROKEN YEAST CHROMOSOMES
225
In one instance, PT139-72A-5, the rearrangement was also shown to include
the duplication of a region including HZS4, at opposite ends of the chromosome. These chromosomes are examples of true healing events, in which a
broken chromosome end becomes stable, presumably by the formation or acquisition of a new telomere. We do not know whether the new, stable end
represents the de nouo formation of a telomere at a broken end, as has been
proposed in Tetrahymena (KING and YAO 1982), or was formed by a translocation event with a genetically unmarked distal portion of some other chromosome.
Because S. cerevisiae contains so few repeated DNA sequences, it should be
possible to determine whether terminally deficient healed chromosomes actually result from recombination between repeated sequences, one proximal to
the chromosome breakpoint and the other proximal to the telomere of another
chromosome. Our first investigation, which simply looked at the size of fragments homologous to the T Y 1 element, has not revealed such rearrangements.
One healed derivative contained two new fragments homologous to a TAS.
It is possible that these two new fragments may represent new telomeres associated with a healed chromosome ZZZ. Alternatively, they may reflect rearrangements on other chromosomes in which a broken chromosome ZZZ caused
a more general “genome shock” (MCCLINTOCK
1941 and B. MCCLINTOCK,
personal communication).
We hope to be able, by recovering the new end of the healed chromosome,
to demonstrate how such healing events occur. This should be possible by
taking advantage of recent advances both in the cloning of yeast telomeres
(SZOSTAK
and BLACKBURN
1982) and in the physical separation of entire yeast
et al. 1982). We have, in fact, carried out prelimchromosomes (D. SCHWARTZ
inary chromosome separation experiments in which we have been able to
demonstrate that the partially duplicated healed derivative shown in example
D, Figure 8 is nearly twice as large as a normal chromosome ZZZ. In contrast,
a similar healed derivative (example C, Figure 9) appears to be slightly smaller
C. CANTOR,
P. THORBURN
than a normal-sized chromosome ZZI (D. SCHWARTZ,
and J. HABER,unpublished results).
Recently, MANN and DAVIS(1983) reported that yeast plasmids containing
two cloned yeast centromere (CEN) fragments could not be maintained as
dicentric structures when transformed into yeast. They recovered a variety of
deletions within the plasmid that eliminated one CEN region. In addition,
SCHERER,
MANN and DAVIS(1982) found that either dimer formation in E.
coli (prior to transformation) or sister chromatid exchange events in yeast (after
transformation) also apparently yielded dicentric chromosomes that underwent
MANNand
apparent breakage and healing. The system employed by SCHERER,
DAVIS(1982) only recovered unusual events that also affected the transcription
of HIS3 on the plasmid. Consequently, they could not assess what proportion
of plasmid breakage and healing involved recombination between homologous
sequences on the tandemly duplicated plasmid. The rare events that they recovered were deletions joining apparently nonhomologous regions together to
re-form a circular plasmid. Our results are entirely compatible with the notion
226
J. E. HABER AND P. C. THORBURN
that most healing events involve homologous recombination, but that less frequent, nonspecific joining of broken chromosome ends may also occur at a
low frequency.
We are grateful for several discussions with BARBARA
MCCLINTOCK,
as well as for the comments
of JOHN MCCUSKER
and SUESTEWART.This work was supported by National Science Foundation
grant PCM-8110633.
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Corresponding editor: D. BOTSTEIN