Diploid strains of the yeast Saccharomyces cerevisiae homozygous for a recessive chromosome loss mutation (chl) exhibit a high degree of mitotic instability.
CHARACTERIZATION OF A MUTATION IN YEAST CAUSING NONRANDOM CHROMOSOME LOSS DURING MITOSIS PALOMA LIRAS*, JOHN McCUSKER, STEPHEN MASCIOLIt
AND
JAMES E. HABER
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02154 Manuscript received January 24, 1977 Revised copy received December 5 , 1977 ABSTRACT
Diploid strains of the yeast Saccharomyces cerevisiae homozygous for a recessive chromosome loss mutation (chl) exhibit a high degree of mitotic instability. Cells become monosomic for chromosome I I I a t a frequency of approximately one percent of all cell divisions. Chromosome loss a t this high frequency is also found for chromosome I , and at lesser frequencies for chromosomes V I 1 1 and X V I . In contrast, little or no chromosome loss is found for six other linkage groups tested ( I I , V , V I , VIZ, X I and X V I I ) . The eh1 mutation also induces a ten-fold increase in both intergenic and intragenic mitotic recombination on all ten linkage groups tested. The chl mutation does not cause an increase in spontaneous mutations, nor are mutant strains sensitive to UV or Y irradiation. The effects of chl during meiosis are observed primarily i n reduced spore viability. A decrease in chromosome 111 linkage relationships is also found.
ORMAL diploid strains of Saccharomyces cerevisiae heterozygous ( a / a ) at Nthe mating-type locus do not mate with either a or strains. Recently, we described a diploid exhibiting bisexual mating properties (HABER 1974) . Genetic analysis of that strain suggested that bisexual mating was a consequence of a genetically directed, nonrandom chromosome loss in which a small percent of the cells in any clone have become 2n-1 aneuploids for the chromosome carrying the mating-type locus. Thus, in addition to euploid nonmating ( a / a ) diploids, some cells in the clone become 2n-1 and carry only the a allele. They are able to mate with a strains. Others become 2n-1 a and are able to mate with a strains. The bisexual mating phenotype arises from the mosaic nature of each colony (Figure 1). Because this mutant diploid failed to sporulate, a complete genetic description was diffcult, requiring analysis of tetraploid strains (HABER 1974). That analysis suggested that the formation of 2n-1 aneuploids of chromosome ZZZ occurred at a frequency of about one percent of all cell divisions. The loss of chromosome ZZZ seemed to occur at a much greater frequency than that of other linkage groups tested. That study concluded that the formation of aneuploid strains of chromo(Y
* Current address: Departmento Microbiologia, Facultad Ciencias, Universidad Salamanca, Salamanca, Spain.
t Current address: Boston University School of Medicine, Boston, Massachusetts02118. Genetics 88: 651-671 April, 1978.
652
P. LIRAS
et al.
CHROMOSOME LOSS IN BISEXUAL STRAIN
I
1 ”
l a Threonine Requiring
-\ 98 %
”
leu2
leu2
Non-Mating Wild Type
2n-I a
Leucine Requiring
FIGURE 1.-Effect of the frequent loss of chromosome ZZZ in a diploid strain. Most cell divisions result i n the formation of nonmating diploid cells. However, in bisexual colonies (HABER 1974) a significant number of aneuploid cells, monosomic for chromosome ZZZ, arise during mitotic cell divisions. All recessive traits and the mating type of the homologue that remains could then be expressed. Consequently, the colony contains roughly equal numbers of aneuploid cells able to mate with a strains and aneuploid cells able to mate with CY strains. The bisexual mating phenotype thus arises from the mosaic nature of the colony.
some ZZZ is controlled by a single recessive mutation. The mutation did not appear to be linked to the mating-type locus or in fact to chromosome ZZZ at all. We have now been able to analyze this mutation in greater detail. By several successive outcrossings, we obtained a sporulating, bisexual diploid strain, so that further genetic analysis could be carried out more thoroughly. This analysis has shown that the nonrandom loss of chromosome ZZZ is indeed controlled by a single recessive, centromere-linked gene, which is not on chromosome 111. This chromosome loss mutation, designated chl, has been mapped to chromosome XVZ, proximal to radl. In addition to promoting the mitotic loss of chromosome ZZZ, the chl mutation induces chromosome loss of chromosome Z and, to a lesser extent, of chromosomes VIZ1 and XVZ, but not of six other linkage groups tested. I n addition, the mutation causes a greatly increased frequency of mitotic recombination in all ten linkage groups that have been tested. The mutation also results in reduced spore viability and reduced recombination frequencies among viable tetrads following mciosis.
653
M I T O T I C C H R O M O S O M E LOSS MATERIALS A N D METHODS
Strains: The parent haploid strains for most of the diploids constructed in this study were obtained from the Yeast Stock Center, Donner Laboratory, Berkeley, California. Strains exhibiting bisexual mating behavior were derived from the bisexual diploid B1 described previously (HABER 1974). The genotype of those strains discussed in detail are presented i n Table 1. In the course of our work we found that the radl allele in several strains of the Yeast Stock Center does not appear to be linked to tyr7 as had been reported by RESNICK (1968). However, TABLE 1
Diploid strains used in the analysis of mitotic chromosome loss PL34 SM5239 PL2232
+ +
a ura3 ade6 chl __ hisl lys5 aro2 met13 _ _ trp5 ~-~ h 2 a chl his5 chl -lys2 - _a_ chl lys2a trp5
his4 __
+
~
+ + + + + + + ~
~
~~
+ lys2 his4+ a chl + his4 cry1 ~ chl + _ + ~ + a~ thr4 _ trpl _ trp5 ural _ _ eh1 _ _ _ _ _ lys2 his4 leu2a + trpl + + chl cysl adel + _ - + a chl +_ _+- _lys2 leu2a chl adel_ gall tyrl his7 _+ _ + _ _ _lys2 ~ a~ + _ cdc4 _ _ ural ~ ~ade2 _ + _ _ + + +_ _+ _ +_ his4 leu2a trpl + + + chl + _+ ~+ + + ~trp5 arg4 _ ~ his4 _ leu2 _ a _ _ thrl _ chl ~ ~ _ _ gall lys2 tyrl his7 + + ura3 + + + + + _a _rna2_ ural ade2 + adel gall lys2 his7 _ _ _ _tyrl +_ _+_lys2 +_ _+_ leu2a + _+_ ~+ _chl_ his4 _ _ - _l e_u_l a_ + chl his4 + a ura3 + gall_ +~ a _ ade5 lys5 aro2 met13 cyh2 trp5 leu1 + + _ ira3 chl + lys2a +_ +_ _+_ _+ _-++_ _+ + _+_ _4-_ _a _t v_l _+_ _ _+_ _+_ _chl - 4_ _ _+ ___ adel gall lys2 tyrl his7 his4 leu2a + cdc4 ural ade2 + leu2 tyr7 _ _ a_ + - _ ura3 _ _ hisl _ ~ + _ _+ _ ~+ ~ _+_ _+_ _ _+_ _ - _ + eh1 leu2a trpl + + arg9 his6 ilv3 pet8 pet“x” met14 + rad‘Lx” + + + + leu2a chl_ _ _ _+ _ _trpl _ _ tyr7 ___ adel lys2 his4 2eu2a thr4 + + + adel _ lys2 _ +_ leu2 _ cdclOa _ _+ _ _ ~ _ (Y
JH1420 SM5497 PL1 PL2
(Y
PL24 PL4876 PL2219
SM107 A75H160 A89H158 A102H130
+
lys2 his4
A197H157
+ _-
J 10’1
leu2a -adel __
+ +
+ +
(Y
lys2a _ _ radl _ _ _ _ ~ adel lys2a tyr7 chl
adel leu2 01
+
chl
_
_
654
P. LIRAS et
al.
strain JG1 (a radf-1) obtained from the Yeast Stock Center is linked to iyr7, as shown below. The rad alleles in strains X3144-11A and X3104-8C also complemented the rad1 allele in strain JG1. Genetic analysis: Cells were grown on YEPD agar plates (1% yeast extract, 2% bactopeptone, 2?(, dextrose) at 30" for two days before replica plating to sporulation plates containing 1% potassium acetate supplemented with 0.04% dextrose, as well as with amino acids, adenine and uracil (SHERMAN, FINKand LUKINS1970). After three days, the cell walls of asci were digested with the enzyme preparation Glusulase (Endo Labs) and microdissected on agar slabs as described by .MORTIMER and HAWTIIORNE (1969). After germination and growth, the haploid colonies were picked onto master plates and scored for nutritional requirements by replica plating to nutritional omission plates. Further genotypic characterization was accomplished by complcmentntion analysis by crossing the meiotic segregants with tester haploid strains. Analysis of the mak (maintenance of killer) phenotype was performed as described by WICKNER(1974). Testing for mating type and for the presence of the gene controlling bisexual behavior is described below. Mating-type tests: Colonies to be tested for mating type were streaked in a matrix pattern with eight rowc of four colonies across on a YEPD plate. After growth, this master plate was replica plated to another YEPD plate which was then crossed stamped with streaks of a and a
FIGURE 2.-A mating test for the presence of the chromosome loss mutant (chl) in meiotic segregants of a diploid strain heterozygous for chl. Each meiotic segregant was crossed with a strain of opposite mating type carrying chl to produce diploids either heterozygous or homozygous for chl. These diploids were then cross-streaked with rows of both a and a tester strains carrying complementary nutritional markers. The mating plate was then replica plated to minimal medium to allow successful complementing matings to grow. The colonies derived from two of the four segregants displayed in each row show bisexual mating, while two show no mating.
MITOTIC C H R O M O S O M E LOSS
655
tester strains. The tester strains were chosen to have auxotrophic markers not present in the colonies to be tested. Mating in the intersections between the colonies and tester strain were allowed to continue a t 30" overnight. The plate was then stamped to minimal medium to allow only complementing diploids formed a t the intersections to grow. An example of this type of testing can be seen in Figure 2. Radiation sensitivity measurements: The sensitivity of cells to UV light was measured by exposing liquid suspensions of cells in petri dishes to UV light of wavelength 254 n m at 6.3 ergsisec for different periods of time. Samples were removed at intervals, diluted and plated on YEPD plates, which were grown in the dark until colonies appeared. The number of colonies apFearing after UV treatment was measured and the results expressed in terms of the percent of viable colony-forming units relative t o the number of colonies formed without UV exposure. The sensitivity OI cells to Y-rays was determined using a CO source (Shepherd Associates). Liquid suspension of cells in sterile tubes were irradiated at a dose rate of 350 radimin on a revolving platform. Tubes were removed at appropriate intervals to be spread on YEPD plates to determine the number of surviving colony-forming units. RESULTS
Isolation of a bisexual diploid able to sporulate The original bisexual diploid designated B1 (HABER 1974) was unable to sporulate. When this strain was crossed with a/a or ,/a diploids to yield tetraploid strains trisomic for chromosome 111, bisexual s-gregants could be obtained. However, these segregants, too, failed to sporulate. By two subsequent outcrossings of bisexual segregants to an a/a diploid (JIOl), we obtained a number of bisexual diploid segregants, which ranged in sporulation efficiency from less than 1% to approximately 15%. One of these, designated B11/101-7B, sporulated with about 13% efficiency. The four-spored asci that were dissected showed 45% viability. The segregants were tested f o r mating type and showed a 1:l ratio of a or a mating types. These apparently haploid segregants were then used to analyze the genetic control of chromosome loss that had been inferred from previous analyses. Demonstration of a single recessive gene controlling chromosome loss Meiotic segregants of B11/101-7B were crossed with other segregants of the same strain or with haploid strains from our laboratory collection. Diploids were constructed so that they always carried at least one homozygous nutritional marker in order to facilitate examination of their mating behavior by complementation testing. All 11 diploids constructed by crossing two different meiotic segregants of €311/101-7B showed bisexual mating behavior; on the other hand, 15 diploids constructed by crossing one meiotic segregant of B11/101-7B with haploid strains not derived from bisexual parent yielded diploids that were completely nonmating, as is normal for a/, diploids. The properties of these diploids indicated that bisexual mating behavior was indeed controlled by a recessive mutation not generally present in normal laboratory strains. Further, as all 11 diploids constructFd by a random pairwise crossing of meiotic segregants of B11/101-7B were bisexual, the parent bisexual diploid must have been homozygous for the gene or genes v2sponsible for this mating phenotype.
656
P. LIRAS
et al.
Demonstration of a single recessive gene controlling bisexual mating behavior The haploid segregants of B11/101-7B were then used to analyze the nature of genetic control of chromosome loss in bisexual mating behavior. One meiotic segregant, B11/101-7B-IA (a his4 thr4 bisexual) was crossed with strain A40 ( a his4 trp5 ural) to yield a diploid auxotrophic for histidine and heterozygous wild type for all other specified markers. This diploid (designated 7BIA/A40) sporulated with an efficiency of more than 65 %, and the asci dissected had more than 90% spore viability. Each spore of each tetrad was scored for all nutritional markers, as well as €or mating type. The segregants were then crossed with either B11/101-7B-6A ( a his4 leu2 bisexual) or B11/101-7B-7A ( a his4 leu2 bisexual), depending on mating type, to construct a new set of diploids homozygous or heterozygous for bisexual mating behavior, depending on the segregation of this trait in the diploid 7BIA/A40. The newly constructed diploids were purified on minimal medium plus histidine, and the purified diploids were then crossstreaked with a and a ade5 mating-type testers. After growing the cross-streaks on YEPD medium, the strains were replica plated to minimal medium to test for mating behavior of the diploids. An example of the pattern of segregation of bisexual mating behavior is shown in Figure 2. Two of each four segregants in any tetrad, after backcrossing to a strain containing the chromosome loss trait, exhibit bisexual mating, whereas the other two segregants do not. Of the first 227 tetrads analyzed from this and subsequent tetrad analyses of strains heterozygous for the gene controlling chromosome loss and bisexual mating behavior, 209 segregated 2:2 for this trait. We conclude that bisexual mating behavior, as caused by chromosome loss of chromosome 111, is controlled by a single recessive gene. This single recessive mutation is designated chl (chromosome loss) .* Mapping of the chl mutation Despite its pronounced effect on the mitotic stability of chromosome 111
(HABER 1974), the chromosome loss mutation does not appear to be linked to any markers on that chromosome (Table 2A), as an equal number of parental ditypes (PD) and nonparental ditypes (NPD) is found in the tetrad analysis of chl and each marker. The chl mutation does, however, seem to be closely linked to another centromere, as the percentage of tetratype asci with either leu2 or cdclO (31% and 15% respectively) ,both of which are quite close to the chromosome ZZZ centromere, is considerably less than the 67% expected if chl were not centromere linked. Further analysis showed that the chronlosome loss mutation is closely linked to the centromere of chromosome XVZ. Tetrad analysis for a diploid heterozygous f o r both chl and tyr7 (located about 27 cM from the centromere of chromosome X V Z ) is shown in Table 2B. There is clear evidence of linkage between chl and tyr7 from the very large ratio of parental ditypes t o nonparental ditypes (79/1). From these data, chl is approximately 28 CMfrom tyr7. Given the known gene-centromere distances for the other markers tested in the cross, we could * In our initial deacrbtion of this mutation (HABER 1974) It was provisionally deslgnated her (hermaphrodite). A more accurate dealgnation has now been chosen
65 7
MITOTIC CHROMOSOME LOSS
TABLE 2 Tetrad analysis of chromosome loss mutation (chl) and centromere-linked genetic markers ~
EKpenment*
Gene pairi
Ascus types PD : NPD : T
Percent tetratype
A
chl-leu2 chl-cdcl0 chl-mat chl-his4
112 28 80 84
119 23 96 86
1M 9 188 123
31 15 52 42
B
chl-adel chl-trpl chl-tyr7 adel-trpl
33 69 79 28
34 67 1 28
5 19 85 1
6 12 52 2
C
chl-rad1 chl-t yr7 radl-t yr7 adel-rad1 adel-chl adel -tyr7
38 21 16 24 26 17
0 0 4 24 20 12
12 26 47 25 4
24 55 70 34 8 60
4.0
* Tetrad analysis shown in Experiment A was performed with diploids AlOQH30.Experiment B utilized diploids A75H160 and A89H58. Experiment C employed diploid A197H157. I. The chromosomal location of each of the known centromere-linked markers is given in order in parentheses. also determine the distance of chl from the centromere (WHITEHOUSE 1950). Based on the data for pairwise segregations of trpl, adel and chl, the chromosome loss mutation appears to lie about 8cM from its centromere. The chl mutation was located more precisely on chromosome XVZ by using radl, the other marker known to map on this linkage group (RESNICK 1968). Based on the data from 75 tetrads heterozygous f o r chl, tyr7 and radl, we conclude that chl lies on the same arm as radl, about 7 CMfrom the centromere and about 12 cM from radl (Table 2C). Effect of chl on chromosome I11 during mitosis A preliminary examination of the effect of a homozygous chl mutation on mitotic cells indicated that the mutation promoted the loss of chromosome ZZZ at a frequency of approximately 1% of all cell divisions, a much greater frequency than that observed for the loss of other chromosomes (HABER1974). This analysis has been extended to determine the spectrum of action of the chl mutation on many linkage groups. Several diploid strains were constructed in which one or more chromosomes were marked with auxotrophic markers on both arms of the same homologue. Such diploids, either homozygous or heterozygous for chl, were grown on YEPD plates, suspended in water, diluted and spread on YEPD agar plates to a density of approximately 200 colony-forming units per plate. After the colonies had grown up, the plates were replica plated to minimal medium supplemented only with those amino acids that the original diploids required for growth. Colonies that had become auxotrophic for additional nutri-
658
P. LIRAS
et al.
tional requirements were then selected and analyzed further to identify the loci which had become hemizygous or homozygous for the requirement. We first examined the extvmtand nature of chromosome ZZZ loss in the presence of chl, using diploids carrying recessive markers on both homologues. The data in Table 3 confirm previous findings that en-1 aneuploid strains for chromoper cell division. In these some ZZZ are detected at a frequency of about experiments, 134 of 10,378 colonies appear to be 2n-1 (a frequency of approxiTABLE 3 EfJect of chl on chromosome I11 loss and mitotic recombination* Genotype of auxotrophic colonies
Case
No. of colonies detected in strain H30104 H3540 H3527
Ploidy
cryl
a
thr4
2n-lt
34/5265
17/51 13
0/7944
his4
+
a
thrl
2n-I
0/5265
2/5113
0/7944
his4 ___
cryl
a
2n- 1
1/5265
0/5113
0/7944
2n-l$
69/5265
9/5113
0/7944
his4 0 --
-leu2 -
leu2
o-
-
+ + +
a
+ +
a
thr4
2n-I
0/5265
2/5113
0/7944
thrl
2n
4/5265
2/5113
0/7944
2n
0/5265
O/5113
2/794+
2n
1/5265
0/5113
1/7944
2n
2/5265
0/5113
0/7944
his4 cryl " -~ leu2 cryl
+
his4
+ cryl -+ -/+ - cryl
a
a
-/-
a
thr4
a
+
O
-0
his4 his4
+ his4
leu2
+
_____
+
leu2
-0
-0
a thr4
f
a
thr4
cryl
a
thr4
cryl
a
thr4
* Diploid strains H3540 and H30104; both heterozygous for markers on chromosome ZZZ: his
+
+
-po
cryf a thr4 chl -0
o------
+
+
-
leu2 a chlo was grown at 25" on YEPD and spread for single colonies. Plates were replica plated to minimal medium at 25", YEPD plates containing cryptopleurine at 25", and YEPD plates a t 34" to select auxotrophic colonies and cryptopleurine resistant colonies. Auxotrophic colonies were then scored f o r specific nutritional requirements. Colonies still exhibiting bisexual mating behavior were scored as 2n diploid a/a.Single colonies from these strains were examined to detect the presence of recessive heterozygous markers. Colonies with a single mating type and showing recessive phenotypes for at least one marker on each arm of chromosome 111 were assumed to be 2n-l aneuploids for chromosome ZZZ, as demonstrated in Table 4 and in previous analyses (H~BER 1974). An equivalent selection was carried out for a diploid heterozygous for chl, H3527. -j- Four h i s cryr a t h r colonies were mated with an a/a leu2/leu2 diploid and analyzed to show that the auxotrophic colonies were 2n-1 for chromosome ZZZ (see Table 4A). t: Three leu- a colonies were mated with an cy/a adel/adel diploid and analyzed to show that these colonies were 2n-1 for chromosome ZZZ (see Table 4B).
MITOTIC CHROMOSOME LOSS
659
mately 1.3X10-2). These colonies could be shown to be 2n-1 aneuploids. Four colonies that had become a! mating and both histidine and threonine requiring were crossed with an a/a Ieu2/leu2 diploid and the complementingmatings purified and sporulated. The pattern of segregation of mating types was consistent with an a!/a/a trisomic cell that would have been formed if the original a! mating parent was 2n-1 for chromosome ZZZ (Table 4A). Whereas an a/a/a!/a! tetraploid may generate tetrads in which all four meiotic segregants are a/a! , nonmating diploids, such cells cannot yield tetrads containing 2 nonmating (a/.) segregants and 2 a mating-type segregants (SEIAFFER et al. 1971). Conversely, an a/a/m trisomic cell cannot yield four nonmating segregants but can give rise to t-etrads such as 2 nonmating:2 a mating. All of the four putative 2n-1 colonies analyzed by this method gave results consistent with the segregation of a/a/a!, not of ./././a!. Further, in these tetrads, there was generally a 2+: 2- segregation for leucine, as would nearly always be expected for a strain +/-/-, but only about 25% of the time for a +/+/-/- tetraploid. Again, this result confirms that the a! colonies auxotrophic for histidine and threonine were 2n-1 aneuploids for chromosome ZZZ and, thus. hemizygous for the LEU2+ allele. In a similar fashion, three colonies that were a leu- (case 4, Table 3) were shown to be 2n-1 monosomic for chromosome 111 by crossing them with an a / m adeZ/adel diploid and analyzing the tetrads. The results in Table 4B confirm that these a leu- colonies were indeed 2n-1 aneuploids. Some tetrads in these analyses are apparently derived from tetraploids a/a/a!/a for chromosome ZZZ. These tetrads indicate that strains monosomic for chromosome 111 are not stable and revert to 2n euploids by duplication of the remaining 1970). Nevertheless, the results clearly homologue (BRUENNand MORTIMER demonstrate that a 2n-1 colonies were induced by the chl mutation. Sometimes chromosome loss appears in conjunction with a recombinational event. Six of 108 colonies expressing recessive markers on both sides of the centromere (cases 2, 3 and 5 of Table 3) do not exhibit a parental configuration of markers in the homologue remaining after chromosome loss. There are also three cases in which one or more markers had become homozygous in the absence of chromosome loss, presumably by mitotic recombination. In contrast, no chromosome loss was detected in chZ/+ heterozygous cells.
Effect of chl on other linkage groups The effect of chl in promoting chromosome loss of other linkage groups is detailed in Tables 5 and 6. Besides chromosome I l l , three other linkage groups showed a significant increase in chromosome loss in chl/chl strains. The appearance of auxotrophs in strains heterozygous for markers on chromosome Z occurs at a frequency even greater than that for chromosome ZZZ (Table 5A). The very high frequencies of adel auxotrophs seen in two of the four diploids tested represent cases in which the red pigment associated with adel facilitated the identification of colonies in which only a sector was adenine requiring. For those
660
P. LIRAS
et al.
TABLE 4
Demonstration that chl diploids become monosomic for chromosome I11 A. Analysis of his- cryr a t h r colonies Auxotrophic Colony tested'
1
2
Mating type Segregation Number
Leucine requirement Segregation Number
2a:2a:On+ 2a : Oa : 2n 2a : la : In Oa : Oa : 4n l a : l a : 2n
2 5 2 18
2a:h:On 2a : Oa :2n 2a : la : In l a : la : 2n
3 7 2 1'I
2+ : 2I+ : 3-
12 1
2a : 2a : On 2a : Oor : 2n 2a : la : I n
2
e+:%-
4 5
3f : 1-
10 1 I/
2a : 2a : On 2a : Oa : 2n 2a : 18a: In 3a : 18a: On Oa : Oa : 4n la:la:2n l a : 1.a: 3n
2 2
e+
: 2-
11
3f
: 1-
211
4
4f
: 0-
5 11
2+ :2If : 34+ : 0-
9 1 1 II
1s
2 3s
4s 1s
B. Analysis of leu- a colonies Auxotrophic Colony t e s t e a
5
6
7
Mating type Segregation Number
2a Oa la la
: 2a : On : 2a : 2n : 2a : I n :3,a:On
2a : 2a : On Oa:h:2n l a :2oL : In la : lor :2n 2a:h:OOn Oa:2a:2n la:2a:ln la:lor:2n
6 7
5 2 2
5 8 29 2 4
3 1'1
* Four his- cryr a t h r colonies presumed to be 2n-1 monosomic for chromosome ZZZ were mated with a diploid a/a ZeuZ/leuZ and the resulting tetraploid was then sporulated and dissected. 1- The symbol n designates a nonmating or bisexual segregant. $ Three leu- a colonies presumed to be 211-1 monosomic for chromosome ZZZ were mated with a diploid a/a adel/adel and the resulting tetraploid was then sporulated and dissected. Segregants not compatible with an a/a/a trisomic (4n--1) tetraploid. 11 Segregants not compatible with a ZeuZ/leu2/+ trisomic (411-1) tetraploid. 7 Segregants not compatible with an a/cy/a trisomic.
s
66 1
MITOTIC CHROMOSOME LOSS
TABLE 5 Effect of chl on chromosomes I, VI11 and X V I A. Chromosome Z Number colonies tested
Number of auxotrophs
-
5897
38 adel cysl 3 adel 6 cysl
chl
5591
Genotype
cysl
adel
chl
+ +
--
cysl ___
-0
chl
adel
-
+ + + + adel cdcl5 + + + -0
PO--
+
+ + +
adel
cdd5
+
adel
+
adel
-0
chl
-
113 adel cdcl5
__
2633
chl -
8028
3 adel
chi chi
6295
134 cdcl5 598 adel
eh1
9247
1 cdcl5 2 adel
chl
+
+
--
cdcl5
+ +
--
chl
0
6210
173 adel
chl
B. Chromosome VIZ1 Genotype
mak7
arg4
eh1
-
+ "-7eh1
Number colonies tested
Number of auxotrophs
6953
32 mak7 arg4 38 arg4
7275
8058
22 arg4
0
C. Chromosome X V I Genotype
radl
+ radl + radl + radl +
-o---
tyr7
-
chl chl
+ tyr7 chl chl + tyr7 chl + chl tyr7 chl + +
Number colonies tested
Number of auxotrophs
5160
7 radl lyr7 2 radl
9653
6 radl tyr7 7 tyr7
6823
1 radl t.y-7
7623
1 tyr7
662
P. LIRAS
et al.
TABLE 6 Effect of chl on chromosomes 11, V, VI, VII, XI and XVII A. Chromosome Z l Npber colonies tested
Genotype
ilsl gall lys2 tyrl his7 chl --to+ +f chl
7422
ilsl
8058
+
gall
-----o
lys2 tyrl
his7
+ + + + +
chl -
+
Number of auxotrophs
7 gall lys2 tyrl his7 . . 3 lys2 tyrl his7 11 gall 12 ilsl 1 lys2 tyrl his7
1 tyrl
1 gall
B. Chromosome V Npber colomes tested
Genotype
+ arg9 + ura3 + hisl + arg9 + ura3 + hisl 0-
chl -
5253
2 ura3
-
8824
1 arg9 0
chl eh1
+
0-
Number of auxotrophs
C. Chromasome VI Npber colomes tested
Genotype
cdc4 -_
cdcl4 his2
chl -
+ + +
7275
Number of auxotrophs
12 cdcl4 his2
5 cdc4
chl
D. Chromosome VZT Genotype
++ -
Number - . .. colonies tested
lys5 aro2 met13 trp5
+
-I-+
ade6
eh1
6210
chl
lys5 aro2 met13 trp5
__
adeb
+ + + + + -0
lys5 aro2 met13 trp5
__
ade6
chl
__
chl
-
+ + + + + + -0
11294
chl
5290
Number
of auxotrophs
2 lys5 aro2 met13 trp5 a h 6
3 lys5 aro2 met13 trp5 5 adeb 1 lys5 met13 aro2 trp5 adeb 2 lys5 met13 aro2 trp5 2 ZYS5 2 iys5 met13 1 met13 5 ade6 0
E. Chromosome X I Genotype ._
ural --- met14
chl -
+ met14 + chl chl + + + F. Chromosome XVZI ural
-o-
Genotyp+
lys10
met2
chl
+ + chl lys10 met2 chl --_ + + + -0
-0
__
Number colonies tested
5253
Number of auxotroDhs
3 urd Imet14
8824
0
Number colomes tested
Number of auxotrophs
9946
1 met2 5 lys10
5850
0
MITOTIC CHROMOSOME LOSS
663
TABLE 7 Effect of chl on intragenic mitotic recombination A. Prototroph formation in ade2-l/ade2-2 diploids* chl genotype
Prototrophs/colony forming units
9.6 +. 3.0 X leE 1.3 -t 1.0 x l o r 6
chl/chl
chi/+ B. Prototroph formation in adeZ-l/adeZ-1 diploidst chl genotype
Prototrophs/colony forming units
chl/chl
5.9 2 2.7 x 10-7 4.5 2 4.1x l o r 7
chi/+
* Diploid strains heteroallelic for ade2 (ade2-l/ade2-2) and either heterozygous o r homozygous for chl were spread from fresh colonies grown on YEPD plates onto adenine drop-out plates. After 2 to 3 days, the number of adenine independent prototrophic colonies were scored. The total number of colony forming units plated was determined by spreading serial dilutions of the original cell suspension on YEPD plates. The results are based an an average of five experiments. -f Diploid strains homaallelic for ade2 (ade2-l/ade2-l) and either heterozygous o r homozygous for chl were tested for adenine independent prototrophic colony formation, as described in part A. The results are based on an average of five experiments.
cases in which it was possible to detect simultaneous auxotrophy on both sides
,it has not been possible to show une) quivocally that these strains are 2n-1 monosomic for chromosome 1. The viability
of the centromere
strains
of tetrads from four independently isolated ade- cys- colonies was very poor, averaging only 17%. Hence, the fact that all 35 tetrads dissected showed only two or fewer viable spores cannot be viewed as a proof that the colonies were 2n-1. However, when other diploids not containing cysl were used, the adel colonies that appeared also gave only two viable spores per tetrad in all 5 cases tested. These results indicate that chZ/chZ strains lose chromosome Z at a frequency of at least 1% of cell divisions. Chromosome loss was also found for linkage groups VZZZ and XVZ, although at lower frequencies than for chromosomes ZZZ and 1. I n the case of chromosome VZZZ (Table 5B), approximately 0.3% of colonies tested became simultaneously arg4 and mak7. None of these colonies were shown to be 2n-1 for chromosome VZZZ, because these strains did not sporulate (even though the parent diploid sporulated well). Similarly, a small number of chromosome XVZ heterozygotes expressed recessive markers on both sides of the centromere (radl tyr7). Here the frequency of such putative 2n-1 strains was only 0.15% (Table 5C). Again, none of these strains could be shown to be 2n-1 tetrad analysis, as none of these strains sporulated. The fact these strains could not sporulate even though the parent strain and strains homozygous for either radl or tyr7 can sporulate (data not shown) may be an indication that these strains are indeed 2n-1 aneuploids. Six additional linkage groups have been examined for chromosome loss using auxotrophic markers on both sides of a particular centromere. For chromosomes ZZ, V , VIZ and XVZZ there were few if any cases of actual chromosome loss
664
P. LIRAS
et al.
(Table 6). On the other hand, there were a significant number of instances in which one or more markers on one arm of a chromosome became homozygous. I n these cases, colonies exhibited recessive auxotrophic requirements for one or more markers on one arm of a chromosome, while recessive markers on the other arm remained heterozygous wild type. Dissections of tetrads from these colonies yielded four viable spores and a 2:2 segregation f o r the unexpressed recessive markers. These colonies most likely arose by a mitotic recombination event in which markers on one arm of a chromosome became homozygous without affecting heterozygous markers on the opposite arm. Instances such as cases 2 and 5 in Table 3 appear to be gene conversion of a single locus rather than the coincident homozygosis of all markers distal to a particular crossing over event. As before, the number of spontaneous recombination events was much greater when chl was homozygous than when heterozygous (cf., Table 3 ) . These results suggest that while chromosome loss is found for only a few chromosomes, an increased level of spontaneous mitotic recombination occurs on perhaps all linkage groups. E ~ e coft chl on intragenic recombination
The effect of the chromosome loss mutation on mitotic recombination was also studied in terms of its effect on the level of intragenic recombination. Diploid strains heteroallelic for ade2 and either heterozygous or homozygous for chl were grown from single colonies in YEPD liquid culture and then spread after appropriate dilutions on both adenine dropout plates and complete medium plates. The number of adenine prototrophs per colony-forming unit was recorded in each case (Table 7 A ) . The level of prototroph formation in a diploid homozygous for chl is seven-fold greater than for the heterozygous strain. This difference in prototroph formation is due to recombination rather than to a difference in the rate of back mutation to wild-type. When diploid strains homoallelic for ade2-l and either heterozygous o r homozygous for chl were examined for prototroph formation, nearly identical rates were found (Table 7B). If one subtracts frequency of homoallelic back mutation from the frequency of prototrophs in the heteroallelic diploids, then the rate of prototroph formation attributable to recombination is 9.0 x for the chl/chl strain and 0.8 x for the chl/+ strain. Thus, after correction, the difference in intragenic recombination between strains homozygous and heterozygous for chl is more than ten-fold. These results f o r intragenic recombination support those shown for intergenic recombination in Tables 5 and 6, namely, that the chromosome loss mutation appears generally to increase mitotic recombination events about ten-fold on all linkage groups examined. Effects of chromosome loss mutation on meiosis
Diploid strains homozygous for chl are able to sporulate as efficiently as strains that are heterozygous for the mutation. The viability of the spores is, however, markedly different. While the viability of spores from diploids heterozygous for this mutant usually exceeds 75%: spore viability in tetrads from strains
665
MITOTIC CHROMOSOME LOSS
TABLE 8
Viability of spores in tetrads of diploid strains homozygous or heterozygous for chl Diploid strain
4
A. chl/chl diploids P L 34 SM 5239 PL 2232 JH 1420 SM 5497
1 2 64 23 9
__
Total
99
Number of viable spores per tetrad' 3 2 1
0
39
Percent spore viability
27.4 49.2$ 82.6 57.6 71.6$
8 5 17 30 3
33 15 18 48 8
56 10 7 17 2
63
122
92
49
54.2s
51 103 18 12 40
11 6 0 0 7 14
0 0 0 0 9
37
34 44 5 3 18 20
76.4 73.5 92.8 83.7 74.8 86.l$
261
124
42
--t 0 10
--t ~--~
B. chl/+ diploids PL PL PL PL PL
1 2 4876 24 2219 SM 107
65 44 74 27 58
143 ~
Total
41 1
_ _ _ _ _ _ _
--t
9
80.2s
* The distribution of tetrads in which 4, 3, 2, 1 or no spores were viable is shown for some of the diploid strains used in this work. 1- The number of tetrads with no viable spores was not recorded. $Viability overestimated, as tetrads with no viable spores were not included. 0 Viability overestimated, as some tetrads with no viable spores were not included. homozygous for chl is generally much lower. Several examples of spore viability in different tetrad analyses are shown in Table 8. In tetrads containing at least one viable spore, 49% of asci dissected from diploids homozygous for chl have 4 viable segregants. Such a lack of viability may be due to nondisjunction or chromosome loss during meiosis, leading to the formation of n-1 spores, which are not viable. However, our attempts to rescue aneuploid spores by using a macromanipulator to place spores adjacent to other haploids (to construct 2n-1 diploids) were not successful. I n tetrads in which only three or two spores germinate, it also seems possible that any reciprocal nondisjunction of chromosome ZZZ might lead to the formation of n+l disomic spores. Had spores disomic for chromosome ZZZ of a/a genotype been formed these spores would be detected as bisexual segregants, as we have unpublished found that a/.. disomic strains carrying chl are bisexual (HABER, observations). No such segregants have been found among the viable spores, but this observation does not rule out the possibility of forming n S 1 spores in the absence of genetic exchange between homologous, yielding only a/a or ./cy disomic haploids that would not have been distinguished from normal a or cy segregants. Among those tetrads where all four spores were viable, the recombination distances for markers on chromosome ZZZ were approximately one half the distance measured when diploids strains heterozygous for chl were used (Figure 3 ) .
666
chl/+
P. LIKAS
et al.
linkage map:
i
I
his4
leu2
"
21.6 (251)
_ chl/chl _
~
I thr4
-1
ala 22.1 (65)
32.9
(2512
linkage map: 1 -
I
his4
I
leu2 8.6 (75)
I
a/a 22.6 (156)
thr4 14.2 (165)
FIGURE3.-A comparison of apparent genetic linkage of markers on chromosome ZZZ determined by analysis i n diploid strains either heterozygous or homozygous for chl. The number of tetrads for which each distance was calculated is given below each interval.
Th.3map distances for chZ/+ diploids show general agreement with the published (1973). map of MORTIMER and HAWTHORNE Radiation Sensitivity of chl
It was possible that a strain altered in spontaneous mitotic recombination might also be altered in radiation sensitivity, as in the case of rad51 or rad52 (GAMEand MORTIMER 1974) or rad18 (Bonuiv and ROMAN1976). Strains carrying the ch2 mutation are not radiation sensitive. From a diploid strain heterozygous for the chZ mutation, all four spores from a single tetrad were grown, tested f o r genotype and also for radiation sensitivity by measuring the fraction of cells surviving UV treatment for increasing lengths of time. The two chl segregants were approximately as sensitiv-2to radiation as their non-chl sisters. For example, after an exposure of 500 ergs, the two chl colonies showed 31.7% and 26.0% survival, while the two chZ+ strains showed 27.5% and 28.5% survival. Similarly, when haploid and diploid strains were tested for sensitivity to gamma irradiation, we found no significant differences in the sensitivity of chZ and nonchl strains. Haploid chl strains are also not sensitive to the mutagen MMS (J. THORNER, personal communication). DISCUSSION
Diploid cells of Saccharomyces cereu 'siae are usually genetically very stable. We have described the properties of a single recessive mutation (chl), which has two different consequences on mitotic cells. First, the mutation causes the appearance of a bisexual mating genotyp? in a clone of a/.! diploid cells. The bisexual phenotype has been shown to be a consequence of a significant frequency of loss of one or the other homologue of chromosome 111 carrying the mating-type locus. Aneuploid cells 2n-1 for chromosome ZZZ are generated at a frequency of approximately 1% in all cell divisions. An equivalent mitotic chro-
MITOTIC CHROMOSOME LOSS
667
mosome loss is also found for chromosome Z, and lower frequencies of loss are found for linkage groups VZZZ and XVZ. The effects of chl on chromosome loss are not general, as there is little or no loss of chromosomes ZZ, V, VZ, VIZ, XZ or XVZZ in surveys o€ between approximately 5,000 and 12,000 colonies. The absence of 2n-I aneuploids for these linkage groups cannot be attributed to the possible lethality of cells monosomic for these chromosomes, as other studies have shown that yeast can tolerate significant aneuploidy (PARRY and Cox 1970; BRUENNand MORTIMER 1970). In particular, stable monosomes for chromosome VZ have been constructed (BRUENN and MORTIMER 1970) even though no such monosomes were produced in chl/chl diploids. Thus, it seems that the chl mutation induces mitotic chromosome loss in an extremely nonrandom fashion, primarily affecting chromosomes ZZZ and Z. The second mitotic phenotype of chl is an increased frequency of spontaneous mitotic recombination in all the linkage groups that have been tested. The increase in mitotic recombination is at least ten-fold greater than is found for strains heterozygous for chl (Tables 3, 5, 6 and 7). The frequency of mitotic crossing over in chl/chl diploids may, in some cases, be several orders of magnitude greater than that observed for c h l / f heterozygotes or wild-type cells. There is also an increase in intragenic recombination or gene conversion events in diploids homozygous for chl. The observed increases cannot be attributed to a difference in the incidence of spontaneous mutations, as the appearance of prototrophs in diploids homoallelic for ade2-l and the appearance of canavanine resistant haploids were not different in chl or wild-type strains. Although the relation between these two aspects of the action of chl is not yet understood, it appears that chromosome loss may also be accompanied by some recombinational event in the same linkage group. This can be seen in the two different experiments involving the loss of chromosome ZZZ shown in Table 3. Cells were judged to have undergone chromosome loss when they became simultaneously hemizygous for markers on both sides of the centromere. In 5 of 134 cases, however, the remaining homologue was not of the parental type. For example, in case 3 of Table 3, a strain that had become histidine requiring, cryptopleurine resistant and (Y mating type did not require threonine for growth, as would hare been expected for a simple chromosome loss event (and which was observed in 34 other instances). We interpret these cases to repPG -sent a recombination event that occurred in conjunction with the chromosome loss event. A similar set of observations showing a correlation between chromosome loss and recombination was presented by CAMPBELL, LUSNAK and FOGEL (1974). In their study, haploid strains disomic (n f 1) a/. for chromosome ZZZ revert to haploid conditions at a frequency of about lo4. In approximately 10% of the cases when chromosome loss occurred to produce an either a or OL haploid strain, a recombination event involving gene conversion at either leu2 or his4 was found. Further, CAMPBELL and FOGEL (1977) showed that among gene convertants of leu2 in such disomic strains, approximately 10% exhibited chromosome loss. How-
668
P. LIRAS
et al.
ever, a causal relationship between chromosome loss and recombination in that chromosome has not been established. Possible correlations between the mitotic eflects of chl Why does chl cause a general increase in mitotic recombination, but a nonrandom pattern of chromosome loss? We have considered two models. A. It is possible that chl acts “directly” on a few linkage groups and exerts indirect effects on mitotic recombination in other linkage groups. A somewhat analogous situation has been studied in Drosophila, in which a number of different Minute mutations cause increased somatic crossing over in the chromosome on which they are located, and in some other chromosome arms as well (STERN1936; KAPLAN1953). Similarly, some autosomal inversion heterozygotes also increase X-chromosome mosaicism ( ROEN 1964). The effect could also represent the response of cells to a significant inbalance in metabolism caused by chl, analogous to the spontaneous n 1 chromosome X I and HENRY duplication arising in strains requiring fatty acids ( CULBERTSON 1973) in yeast. B. Alternatively, it is possible that chl has a single direct effect whose consequences are seen more dramatically on chromosomes 111 and Z than on other linkage groups. We suspect that the primary effect of chl is to cause a generalized increase in somatic crossing over. The losses of particular linkage groups would then arise because of particular features of those chromosomes that make them unstable after mitotic recombination. For example, it is well known that mitotic and meiotic chromosome loss is promoted by recombination between inversion heterozygote chromosomes (BECKER1969; MERRIAM, NOTHIGER and GARCIA-BELLIDO 1972). Meiotic chromosome loss is also well documented in cases of crossing over between a ring and a rod chromosome (MORGAN 1933). The loss of chromosome I may stem from the presence of reiterated copies of rDNA. At least 100 rDNA cistrons appear to lie on chromosome I (@YEN 1973), although the detailed arrangement of these genes is not known. If some of these homologous sequences are inverted or if they are found on both sides of the centromere, mitotic recombination events between rDNA cistrons may result in either large deletions or chromosome loss. Similarly, the loss of chromosome IZZ may be promoted by repeated blocks of genetic information at different places on the chromosome or by the presence of inversions in a/, diploids. Experiments concerning the arrangement of matingtype information on chromosome ZZZ have led to the suggestion that there are three blocks of mating-type information widely separated on that linkage group (HICKS,STRATHERN and HERSKOWITZ 1977). Chromosome loss may then occur because of recombination between homologous mating-type sequences at different places in the chromosome or at the same site in different orientations. The loss of chromosome ZZZ cannot, however, be explained simply as an inversion of sequences at the mating-type locus, as a / a strains show chromosome loss as frequently as a / a strains (unpublished observations).
+
669
MITOTIC CHROMOSOME LOSS
We have found preliminary evidence that eh1 does promote recombination at or near different blocs of mating-type information. An unusual, large deletion of a portion of the right arm of the chromosome ZZZ results in the conversion of mating type from OL to a (HAWTHORNE 1963). This deletion appears to extend from the mating-type locus to the gene HMa, which is believed to be a silent copy of a mating-type information (STRATHERN 1977). We have found that chl causes a ten-fold increase in the appearance of such deletions (HABER, ROSENKRANTZ and MCCUSKER, manuscript in preparation). Thus, chromosome loss may arise as a consequence of increased mitotic crossing over in those linkage groups that have two or more sets of homologous sequences. Whatever the mechanism of chromosome loss, it does not result in reciprocal chromosome loss. Chromosome loss events in chromosome Z could be detected by the appearance of red colonies that are adel. When sectored colonies, half red and half white were analyzed, the white, adenine independent side of the colony was still a 2n diploid, heterozygous for adel (MCCUSKER, WILSON and HABER, manuscript in preparation). E ~ e c oft chl on meiosis Diploids homozygous for chl yield spores with reduced viability. I n the absence of a method to “rescue” inviable sporcs, we can surmise only that some segregants are n-1 nullosomics unable to grow. Compared to chl/+ diploids, the reduction in viability is clearly significant, but there is a considerable overlap in the degree of viability found in particular crosses (Table 8). W e cannot conclude whether the lack of viability can be solely accounted for by chromosome loss of chromosomes ZZZ and 1. No evidence was found for n 1 a / a (bisexual) segregants from diploids homozygous for chl. Thus, we can say that there does not appear to be a measurable frequency of reciprocal nondisjunction leading to n - 1 and n 1 spores. However, only n 1 segregants in which crossing over occurred between the centromere and the mating-type locus (about 25 cM) would have generated n 1 a / a spores. Spores n 1 and a/a or a/a would not have been detected by mating phenotype testing. A striking observation about the effects of chl on meiosis is the finding that meiotic recombination is reduced about two-fold on chromosome ZZZ. Preliminary evidence from another linkage group (chromosome VZZ) also shows a decrease in most map distances as compared to published map intervals. These data suggest that there may be a selective recovery of tetrads in which no crossover has occurred in certain regions of one or more chromosomes. However, more detailed studies of the effects of chl during meiosis are required.
+
+
+
+
+
MARKROSENKRANTZ helped in some of the recombination experiments. We are grateful for the discussion with JEFF HALL,AMARKLARand SEYMOUR FOGEL. This work was supported by the National Science Foundation Grants GB 43730 and PCM 76-11749. The comments of R. E. ESPOSITO have been extremely helpful in revising this paper.
670
P. LIRAS
et al.
LITERATURE CITED
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HABER, J. E., 1974 Bisexual mating behavior in a diploid of S. cereuisiae: Evidence for genetically controlled non-random chromosome loss during vegetative growth. Genetics 78: 843-858.
HAWTHORNE, D. C., 1963 A deletion in yeast and its bearing on the structure of the mating type locus. Genetics 48: 1727-1729.
HICKS,J., J. STRATHERN and I. HERSKOWITZ, 1977 The casette model of mating type interconversion. In: DNA insertion elements, plasmids and episomes. Edited by A. BUKHARI, J. SHAPIROand S. ADHYA.Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (in press).
W. D., 1953 Influence of Minutes on somatic crossing over in Drosophila melanogaster. KAPLAN, Genetics 38: 630-651. J. R., R. NOTHIGER,A. GARCIA-BELLIDO, 1972 Are dicentric anaphase bridges formed MERRIAM, by somatic recombination in X chromosome inversion heterozygotes of D. melanogaster? Molec. Gen. Genet. 115: 294-301. MORGAN, L. V., 1933 A closed X-chromosome i n D.melanogaster. Genetics 18: 250-283. 1969 Yeast genetics. In: The Yeasts, Vol. 1. Edited MORTIMER, R. K. and D. C. HAWTHORNE, by A. H. ROSEand J. S. HARRISON. Academic Press, New York. -, 1973 Genetic mapping in Saccharomyces. IV. Mapping of temperature-sensitive genes and use of disomic strains in localizing genes. Genetics 74: 33-54. OYEN,T. B., 1973 Chromosome I as a possible site for some rRNA cistrons in Saccharomyces cereuisiae. FEBS Lett. 30: 53-56.
PARRY, E. M. and B. S. Cox, 1970 Tolerance of aneuploidy i n yeast. Genet. Res. 16: 333-340. RESNICK,M. A., 1968 Genetic control of radiation sensitivity i n S. cereuisiae. Genetics 71: 548. ROEN,A., 1964 Interchromosomal effects on somatic recombination. Genetics 50: 649-658. SHAFFER,B., I. BREARLEY, R. LITTLEWOOD and G. R. FINK,1971 A stable aneuploid of Saccharomyces cereuisiae. Genetics 67 : 483-495.
F., G. R. FINKand H. B. LUKINS,1970 Methods in Ymst Genetics. Cold Spring SHERMAN, Harbor, New York.
M I T O T I C C H R O M O S O M E LOSS
671
STERN,C., 1936 Somatic crossing over and segregation in D. melanogaster. Genetics 21:
625-730. STRATHERN, J., 1977 Regulation of cell type in Saccharomyces cerevisiae. Ph.D. Thesis. Univ. of Oregon.
H. L. H., 1950 Mapping chromosome centromeres by the analysis of unordered WHITEHOUSE, tetrads. Nature 165: 893. WICKNER, R. B.,1974 Chromosomal and nonchromosomal mutations affecting the “killer character” of Saccharomyces cerevisiae. Genetics 76:423-432. Corresponding editor: R. E. ESPOSITO
