Current Genetics 1, 241-248 (1980)
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© by Springer-Verlag 1980
Mitotic Versus Meiotic Recombination in Saccharomyces cerevisiae Robert E. Malone, John E. Golin* and Michael S. Esposito Department of Biology and Committee on Genetics, The University of Chicago, Chicago, Illinois USA
Summary. As part o f a comparative analysis o f spontaneous mitotic and meiotic recombination we have compared the mitotic and meiotic maps o f the wild type and yeast hybrids homozygous for reml-1, a mitosis-specific hyper-rec mutation (Golin and Esposito, 1977; Golin, 1979). In wild type yeast strains recombination in centromere proximal intervals occurs relatively more frequently in mitosis than in meiosis. In reml-1/reml-1 hybrids the distribution of mitotic exchange events is more similar to the distribution observed in meiosis. Key words: Recombination Hyper-rec mutants Yeast
Introduction We have recently begun a comparative analysis of mitotic and meiotic exchange in wild type, hyper-rec (Golin and Esposito, 1977; Golin, 1979), and hypo-rec diploids (Malone and Esposito, 1980). These studies were prompted in part by the observation that spontaneous mitotic recombination in Saccharomyces cerevisiae occurs almost exclusively at the two-strand stage (Esposito, 1978) rather than at the four-strand stage as originally proposed for Drosophila melanogaster by Stem (1936). The purpose o f the experiments described below was to examine the rates and distribution of mitotic exchange events in the wild type and diploids homozygous for the mitosis-specific hyper-rec mutation reml-1 (Golin and Esposito, 1977).Mitotic recombinationinreml-1/reml-1 diploids occurs at the two-strand stage as in the wild * Present address: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA
Offprint requests to." M. S. Esposito
type (Golin, 1979). Meiotic recombination in reml-1 diploids occurs at the wild type level in all genetic intervals thus far tested (Golin, 1979). We have compared the mitotic maps o f chromosome VII generated by wild type and reml-1/reml-1 with the meiotic map. We find: (1) The mitotic and meiotic maps of the wild type differ with respect to the relative distribution o f exchange events. Mitotic recombination occurs relatively more frequently in regions close to the centromere. (2) The rein1-1 mutation not only increases the rate but also alters the distribution of mitotic exchange events resulting in a mitotic map more closely resembling the meiotic map.
Materials and Methods a) Media. The amounts indicated are those required for preparation of one liter of medium. Yeast Peptone Dextrose (YPD): dextrose 20 g, peptone 20 g and yeast extract 10 g. For solid YPD 15 g agar were added. Synthetic Complete: agar 15 g, dextrose 20 g, yeast nitrogen base without amino acids (Difco) 1.7 g, ammonium sulfate 5 g, adenine sulfate 10 rag, uracil 20 rag, Larginine-HCl 50 rag, L-histidine-HC1 20 rag, L-leucine 100 mg, Lqysine-HC1 50 rag, L-methionine 20 rag, L-phenylalanine 50 rag, L-threonine 100 rag, L-tryptophan 50 rag, L-tyrosine 50 rag, adjusted to pH 5.8 with HC1 or KOH. Cycloheximide: synthetic complete medium containing 2 rag/m! of cycloheximide. Sporulation Medium for Replica Plating: agar 15 g, potassium acetate 20 g, dextrose 1 g, yeast extract 2.5 g, 75 mg/1 of each of the following: adenine sulfate, uracil, L-histidine-HC1, Lqeucine, Llysine-HC1, L-methionine, L-phenylalanine, L-tryptophan, Ltyrosine, and adjusted to pH 7 with acetic acid or KOH.
b) Strains Used. The complete genotypes of all strains used in the experiments discussed in this paper are shown in Table la. Note that the cyh2r allele is recessive to wildtype. Strains JG44, JG43, RM13, and RM15 were dissected and at least 40 tetrads analyzed; the meiotic map distances obtained for chromosome VII are shown in Table lb. The standard values for chromosome VII come from the published data of Mortimer and Hawthorne (1973). / O172-8083/80/0001/0241/$ 01.60
242
R. E. Malone et al,: Mitotic Versus Meiotic Exchange
Table la. Genotypes of strains employed Strain
Genotype
REM1/REM1
Strains:
JG44
a D CAN1 -D CAN1
ura3-1 HOM2HIS1 ura3-313 H O M 3 h i s l
lys2-2 t y r l - 2 his7-1 lys2-1 tyrl-1 HIS7
ade5 m e t l 3 - c cyh2 r trp5-c leul-c ADE6 CLY8 ADE5 m e t 1 3 - d CYH2trp5-d l e u l - 1 2 ADE6 CLY8
ade2-1 ade2-1
JG85
a D CAN1 c~ d CAN1
ura3-1 URA3
HOM3 HIS1 HOM3 HIS1
lys2-2 t y r l - 2 his7-1 lys2-! tyrl-1 his7-2
ade5 met13-c cyh2 r trp5-c leul-c ADE6 CLY8 ADE5 met13-d CYH2trp5-d l e u l - 1 2 ade6 cly8
ade2-1 ade2-1
D can1-100 ura3-1 d CAN1 URA3
HOM3 hisl horn3 HIS1
lys2-1 TYR1 his7-7 LYS2 tyrl-1 HIS7
ADE5 MET13 CYH2TRP5 leu1-12 ADE6 CLY8 ADE5 m e t l 3 - c cyh2 r trp5-c leul-c ade6 CLY8
ade2-1 ade2-I
JG88
a
RM7
a d c~ d
can1-100 ura3-1 HOM3 HIS1 CAN1 ura3-313 HOM3 HIS1
lys2-2 t y r l - 2 his7-1 lys2-1 tyrl-1 his7-2
ADE5 met13-c cyh2 r trp5-c leul-c ADE6 CLY8 ade5 m e t l 3 - d CYH2trp5-d l e u l - 1 2 ADE6 CLY8
ade2-1 ade2-1
RM13
a a
d d
c a n l - 1 0 0 ura3-1 h o m 3 HIS1 CAN1 ura3-313 HOM3 h i s l - 1 9
lys2-1 TYR1 HIS7 LYS2 tyrl-1 HIS7
ade5 MET13 CYH2trp5-d leu1-12 ADE6 CLY8 ADE5 m e t l 3 - c cyh2 r trp5-c leul-c ade6 CLY8
ade2-1 ade2-1
RM15
a a
d d
c a n l - 1 0 0 ura3-313 HOM3 HIS1 CAN1 ura3-1 HOM3 HIS1
lys2-1 tyrl-1 his7-2 lys2-2 t y r l - 2 his7-1
ade5 met13-d CYH2trp5-d l e u l - 1 2 ADE6 CLY8 ADE5 m e t l 3 - c cyh2 r trp5-c leul-c ade6 CLY8
ade2-1 ade2-!
f e r a l - I / r e i n 1 - 1 Strains:
JG43 JG83
a
D CAN1 D CAN1
ura3-1 HOM3 HIS1 ura3-313 HOM3 his1
lys2-2 t y r l - 2 his7-1 lys2-1 tyrl-1 HIS7
ade5 m e t l 3 - c cyh2 r trp5-c leul-c ADE6 CLY8 ADE5 m e t l 3 - d CYH2trp5-d l e u l - 1 2 ADE6 CLY8
ade2-1 ade2-1
a D CAN1 c~ D CAN1
ura3-1 HOM3 HIS1 ura3-313 HOM3 HISt
LYS2 t y r l - 2 HIS7 lys2-1 TYR1 HIS7
ade5 m e t l 3 - c cyh2 r trp5-c leul-c ADE6 CLY8 ADE5 m e t l 3 - d CYH2trp5-d l e u l - 1 2 ADE6 CLY8
ade2-1 ade2-!
Gene symbols are as follows: a, ~ - mating type alleles; ade adenine auxotroph; can - canavanine resistance; D - diploidization (homothallism); his - histidine auxotroph; horn - threonine auxotroph; leu - leucine auxotroph; lys - lysine a u x o t r o p h ; m e t methionine a u x o t r o p h ; r e m - hyper-recombination; trp - trptophan auxotroph; and t y r - tyro sine auxotroph.
Table l b , Meiotic map distances on c h r o m o s o m e VII Strain
Standard RM13 RM15 JG44 JG43
No. of tetrads 100 80 200 40
V
IV
III
II
I
0
0'
1/2% TT ura3-leul
> > > >
86.6 50 50 50 50
22.9 24.4 16.2 18.2 26,6
43.2 42.6 33.8 48.7 35,0
16.6 19.7 20.9 21.7 25,0
2.9 -
31.7 -
40.4 43.4 -
9.7 a 10.0 7.5 8.4 12,2
Intervals on c h r o m o s o m e VII are designated as a d e 5 - ( V ) - m e t l 3 ( I V ) - c y h 2 - ( I I I ) - t r p 5 - ( I I ) - l e u l - ( I ) - c e n t r o m e r e - ( O ) - a d e 6 . Region 0' in RM13 and RM15 is the l e u l - a d e 6 distance. Some intervals could not be measured in strains used in these experiments because appro100 x TT + 6NPD priate markers were not present. Map distances were calculated by the formula map distance in centimorgans = of 2(PD + NPD + TT) Perkins (1949) where PD = parental ditype, NPD = non-parental ditype and TT = tetratype asci respectively. One-half of the percent TT asci for the unlinked centromere markers ura3 and l e u l closely approximates the sum of the gene to centromere distances for these markers. The meiotic map distances of R E M 1 / R E M 1 and r e m l - 1 / r e m l - 1 are similar as previously reported (Golin and Esposito, 1977; Golin, 1979). a
S u m of the gene-centromere distance for l e u l and ura3,
c} Isolation a n d A n a l y s i s o f C y c l o h e x i m i d e R e s i s t a n t (cyh r) Colonies f r o m Culture. All cultures were started from single
colonies diluted to a concentration of 1,000 cells/ml in 5 ml of YPD. The cultures were grown at 30 °C, with aeration, to a concentration of approximately 2 x 107. Samples o f the cultures were plated on complete plates (for total cell n u m b e r ) and on cycloheximide plates for c y h r colonies. Approximately
200 c y h r colonies from each culture were picked to YPD masters to be analyzed further. The position of the recombination event which led to the c y h r colony was located by the following procedure. The YPD master was replicated to sporulation m e d i u m and cycloheximide m e d i u m (to verify the p h e n o t y p e again). After 4 days, the sporulation plate was replicated to media lacking leucine or trypto-
243
R. E. Malone et al.: Mitotic Versus Meiotic Exchange Table 2. Frequency, number, and position of exchange events resulting in eyh r colonies in wild type diploids Strain
Culture number
Cells per 5 ml culture x 10 - 8
Total cyh r frequency x 104
Interval I Number
Intervall II Number
Interval III
eyh r
Frequency x 104
cyh r
Frequency x 104
Number
eyh r
Frequency x 104
JG44
1 2 3 4 5 6
1.0 0.5 2.4 3.0 3.4 1.7
3.04 4.85 3.68 4.37 3.07 4.90
55 90 24 31 18 7
1.98 3.40 1.25 0.79 0.61 0.39
3 9 8 85 15 10
0.12 0.34 0.40 2.18 0.52 0.53
26 39 38 53 57 70
0.94 1.50 1.99 1.36 1.93 3.92
JG85
7 8 9
1.0 0.6 1.0
1.84 1.08 1.19
57 36 22
0.66 0.46 0.38
20 9 9
0.24 0.12 0.15
83 38 37
0.96 0.50 0.64
RM7
10 11
0.8 0.6
0.98 1.98
62 58
0.29 0.51
38 35
0.19 0.32
104 128
0.50 1.15
RM13
12 13 14
0.7 2.0 0.6
1.19 0.83 3.40
143 20 28
0.86 0.08 0.67
15 86 41
0.10 0.37 0.99
41 87 72
0.25 0.37 1.74
RM15
15 16
0.7 0.6
1.88 1.59
35 41
0.43 0.37
31 37
0.39 0.33
84 99
1.05 0.89
JG88
17 18
0.2 0.3
3.06 3.21
50 16
1.00 0.29
12 24
0.24 0.44
91 134
1.82 2.45
The number of eyh r ceils is the actual number of cyh r colonies observed. The frequency refers to the number of cyh r cells per ml divided by the total number of cells per ml.
phan or methionine (dropout media). It was assumed that colonies which had undergone gene conversion events of CYH2 s to cyh2 r were those that remained heteroallelic at leul, trp5, and met13. They therefore generated prototrophic papillae on all dropout media after sporulation. Such colonies could also be due to double crossover events; these, however, should be rare even in rein1-1. Colonies which had undergone crossovers in interval I (between the centromere and leul) were detectable because they had become homozygous for all markers on the left arm of chromosome VII. After sporulation, such colonies could not give rise to prototrophs on met, leu, or trp dropout media because they were homoallelic. Crossovers in region II (between leul and trp5) left leul heteroallelic while both trp5 and met13 were homoallelic. Crossovers in region III (between trp5 and cyh2) left leul and trp5 heteroallelic while met13 (which is distal to cyh2) was homoallelic. In at least two cultures of every strain used, the alleles which were homozygosed by the crossover event were tested and shown to be the ones on the cyh2 r marker chromosome. In those strains where the leul, trpS, or met13 loci were heterozygous instead of heteroallelic (e.g., met13 in RM13), the recombination event could be located because the recessive marker was in coupling with the eyh2 r marker chromosome.
d) Isolation andA nalysis of independent cyh r Colonies. A culture of JG88 was grown to a concentration of 107 ceUs/ml and plated on complete medium in order to obtain single colonies (about 50 colonies/plate). (At the same time the frequency of cyh r colonies was measured and found to be normal. Data not shown.) Five hundred colonies were picked and small "patches" of ceils made on YPD masters. Following 2 days of growth, the YPD masters were replicated to cyclohex~mide plates. After 3 to 4 days, a number of cyh r papillae arose (due to mitotic recombination) in
each patch. A single cyh r papillus was picked from each patch for further study; each cyh r colony thus acquired represents an independent event. The cyh r clones were analyzed as described in Section C.
e) Isolation and Analysis of Non-Selected Chromosome VII. Re. combinants. JG44 and JG43 are homozygous for the ade2.1 mutation which causes the production of a red pigment. The production of this pigment can be blocked by a mutation in the ade5 gene (Roman, 1956). JG44 and JG43 are heterozygous for the ade5 mutation, the most distal marker on the left arm of chromosome VII. Recombination events which render the diploid homozygous for the ade5 mutation block the production of pigment and lead to a white colony. In order to study unselected events, white colonies (which appear at an average frequency of 2.6 x 10 - 3 in JG44 and 2.1 x 10 - 2 in JG43) were picked from complete plates to a YPD master and analyzed for the other markers on chromosome VII as discussed above.
Results The Distribution o f Mitotic Exchange Events in Wild T y p e Diploids. In these e x p e r i m e n t s we m e a s u r e d t h e rates/cell division o f m i t o t i c e x c h a n g e events in genetic intervals on c h r o m o s o m e VII e m p l o y i n g six wild t y p e hybrids. Their c o m p l e t e g e n o t y p e s are given in Table la. We isolated r e c o m b i n a n t c y c l o h e x i m i d e resistant (cyh r) colonies f r o m 18 i n d e p e n d e n t cultures. The interval in w h i c h the e x c h a n g e events o c c u r r e d was d e t e r m i n e d as described in Materials and M e t h o d s . The results are
244
R.E. Malone et al.: Mitotic Versus Meiotic Exchange
Table 3. Rates and distributions of exchange events in wild type diploids Culture number
Interval I
Interval II
Interval III
Events % per cell of division total x 105
Events % per cell of division total x 105
Events % per cell of division total x 105
1.7 3.1 1.0 0.62 0.47 0.32 0.57 0.42 0.33 0.26 0.46 0.77 0.066 0.61 0.39 0.39 1.2 0.33
65 70 34 18 20 8 36 43 32 30 26 72 10 21 23 23 33 9
0.10 0.31 0.32 1.7 0.41 0.44 0.21 0.11 0.13 0.17 0.29 0.09 0.30 0.90 0.35 0.35 0.30 0.50
4 7 11 50 17 11 13 11 13 19 16 8 45 31 21 21 8 14
0.82 1.4 1.6 1.1 1.5 3.3 0.83 0.45 0.56 0.44 1.1 0.22 0.30 1.6 0.95 0.95 2.3 2.8
31 23 54 31 63 80 52 46 54 51 58 21 45 48 56 56 59 77
32
0.32 +0.38
14
1.22 -+ 0.84
53
Geometric Mean 0.52
28
0.29
15
1.11
57
Median Method
25
0.48
19
1.4
56
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Arithmetic Mean 0.73 -+ 0.72
0.64
Rates are calculated from frequencies (Table 2) as described in text. Table 4. Sporulation ability of interval I recombinants Strain
Culture number
No. of recombi- Average nant clones % tested sporulation
JG44
1 2 Parent
25 13 -
61 70 70
JG85
7 8 9 Parent
14 14 14 -
91 86 71 81
RM7
10 11 Parent
16 9 -
86 89 83
RM13
12 Parent
11 -
85 83
RM15
15 16 Parent
12 12 -
68 81 83
summarized in Table 2. From the frequencies of Table 2 we calculated the recombination rates shown in Table 3 for each culture from the equation r = 0.4343f/(logN-logNo) where r = r a t e , f = frequency,N = final total cell number, a n d N o = initial cell fiumber (Drake, 1970). Because recombination events occurring early during exponential growth lead to an overestimation of rates, we calculated both the arithmetic and geometric mean rates. The latter reduces the skewing of the means due to cultures with early "jackpot" events. In addition, we applied the method of the median (Lea and Coulson, 1948) to the distribution of frequencies. This gives rates of 6.4 x 10 - 6 for interval I, 4,8 x 10 -6 for II, and 1.4 x 10 - s for III (see Table 3). It is apparent that the relative distribution of rates (i.e., events) is similar no matter which method is used to calculate them. Approximately 28% of the intergenic recombination events occur between the centromere and leul (I), 16% between leul and trp5 (II) and 56% between trp5 and cyh2 (III).
Cyh r Recombinants in Region I are not due to Loss of Chromosome VII. Given the method by which we assign a recombination event to interval I, II, or III, a monosome arising from loss of chromosome VII would appear to have had a crossover event in interval I. In order to determine whether a substantial fraction of our interval I recombinants were really monosomes for chromosome VII, we sporulated and dissected a representative sample for several of the strains used (Tables 4, 5). The spomlation values and spore viability of interval I recombinants were essentially the same as the parental diploid. In every case the spore viability was significantly different than the expected 50% for a monosome; of 63 different interval I recombinants examined, none had lower than 85% spore viability (Table 5). In addition, when the parental diploid was heterozygous for ade6 (on the opposite side of the centromere), the interval I recombinants remained heterozygous for ade6 with one exception. In one culture of JG85, 7/10 interval I recombinants were homozygous for ADE6 and CL YS. All seven had spore viability greater than 90% so they may have been clonally related double recombinants. If chromosome VII was originally lost, then it must have been restored during growth of the clone. We conclude that chromosome loss does not account for a significant fraction of the interval I recombinants observed.
Selective Growth Advantages Cannot Account for the Observed Distribution of Mitotic Exchange Events. It was possible that the distribution of mitotic crossingover we observed was due to growth rate differences between the different classes of recombinants. A reconstruction experiment was done to test this possibility. Approximately equal numbers of interval I, II, and III
245
R. E. Malone et al.: Mitotic Versus Meiotic Exchange Table 5. Spore viability of interval I recombinants Strain
Culture number
No. inter- Average val I no. clones tetrads/ tested clone
Classes of asci observed a 4 :0
3:1
2: 2
i :3
0: 4
Total asci
% viable spores
Range
JG44
1 2
8 9
10 !0
79 64
5 21
1 2
0 0
0 0
85 87
98 93
93-100 85-98
JG85
2 3 4
9 10 8
5 5 5
31 41 28
8 8 10
5 1 2
0 0 0
0 0 0
44 50 40
90 95 91
85-100 85-100 85-100
RM13
1
9
5
38
5
1
0
0
44
96
95-100
RM15
1 2
5 5
5 5
19 20
5 4
1 1
0 0
0 0
25 25
92 93
90-100 85-100
A number of interval I recombinants from several different cultures were sporulated and dissected. a
Number of asci containing 4 viable (4 : 0), 3 viable (3 : 1), 2 viable (2: 2), 1 viable (1 : 3) and no viable ascospores (0:4).
Table 6. Selective growth advantages of various cyh r recombinant classes Recombinant in interval
I II III
Culture 1 Fraction of total B 0.38 0.25 0.37
Culture 2
h
Relative increase A/B
Fraction of total B
0.41 0.34 0.25
1.08 1.36 0.68
0.23 0.39 0.38
Culture 3
h
Relative increase A/B
Fraction of toal B
A
Relative increase A/B
0.21 0.47 0.32
0.91 1.21 0.85
0.36 0.17 0.46
0.5 0 0.18 0.32
1.38 1.06 0.70
Average relative increase
1.12 1.21 0.74
Selective growth advantages of the various cyh r recombinant types were determined by mixing approximately equal amounts of each at a total concentration of 3 x 103 cells/ml in the presence of 105 cells/ml of the parental type. The mixed culture was then grown to a total cell concentration of 2 x 107 ceUs/ml. Cells were plated before and after growth of the cultures to determine the exact ratio of i : II :III. B = before growth; A = after growth.
Table 7. Independent events in a wild type diploid, JG88 Total number cyh r examined Colonies with events in Interval I Colonies with events in Interval II Colonies with events in Interval III Colonies with gene conversions
298 61 (29%) 21 (10%) 132 (61%) 85
Independent cyh2 r colonies were isolated and analyzed as described in the text. The numbers in parenthesis represent percent of total crossover 6vents
r e c o m b i n a n t s were inoculated at a total c o n c e n t r a t i o n o f 3 x 10 3 cells/ml in the presence o f parental cells at a c o n c e n t r a t i o n o f 10 s cells/ml. Such r e c o n s t r u c t e d cultures were t h e n grown to a total c o n c e n t r a t i o n o f app r o x i m a t e l y 2 x 10 7 cells/ml after which the relative ratios o f interval I, II, and III were determined. A comparison o f the ratios o f the various types o f r e c o m b i n a n t s
before and after g r o w t h shows that interval II recombinants have a slight advantage over interval I recombinants which in turn have a m o d e r a t e growth advantage over interval III recombinants (Table 6). These relatively small selective advantages are n o t sufficient to explain the large differences we observe b e t w e e n the m i t o t i c and m e i o t i c maps. A Mitotic Map Obtained f r o m Independent cyh r Reeombinants. Because the r e c o m b i n a t i o n events o b t a i n e d f r o m culture m a y be clonally related, we analyzed 298 i n d e p e n d e n t cyh r colonies f r o m J G 8 8 (see Materials and Methods for details). E a c h cyh r c o l o n y m a y be treated as representative o f an i n d e p e n d e n t r e c o m b i n a t i o n event and the distribution o f such events is shown in Table 7. A p p r o x i m a t e l y 29% o f the crossovers leading to cyh r clones o c c u r r e d in interval I, 10% in interval II, and 61% in interval III. This is similar to the average distribution o b t a i n e d f r o m the 18 cultures e x a m i n e d .
246
R.E. Malone et al.: Mitotic Versus Meiotic Exchange
Table 8. Frequency, number, and position of exchange events resulting in cyh r colonies in rein1-1 diploids Strain
Culture number
Cells per 5 ml culture x 10 -8
Total cyh r frequency x 102
Interval I Number cyh r
Interval II
Interval III
Frequency x 102
Number cyh r
Frequency x 102
Number cyh r
Frequency x 102
JG43
1 2 3 4 5
0.44 1.1 0.55 0.39 1.3
9.41 2.33 0.92 0.54 1.01
29 29 12 130 17
0.16 0.15 0.08 0.14 0.06
32 43 35 94 29
0.53 0.22 0.22 0.10 0.11
505 382 102 295 81
8.42 1.96 0.64 0.31 0.30
JG83
6 7
0.84 0.69
0.77 1.13
17 16
0.06 0.14
24 16
0.08 0.14
187 98
0.62 0.85
The number of cyh r cells is the actual number of cyh r colonies observed. The frequency refers to the number of cyh r cells per ml divided by the total number of cells per ml.
Table 9. Rates and distributions of exchange events in rein J-l~ reml.1 diploids Culture number
1 2 3 4 5 6 7
Interval I
Interval II
Interval III
Events % per cell of division totat x 104
Events % per cell of division total x 104
Events % per cell of division total x 104
1.5 1.3 0.73 1.3 0.39 0.53 1.3
4.8 1.9 2.0 0.94 0.71 0.70 1.2
6 9 23 18 23 10 12
79 17 5.9 2.9 2.0 5.5 7.6
92 84 68 56 64 82 75
9
17 -+ 28
86
2 7 9 26 13 8 12
Arithmetic 1.0 -+ mean 0.44
5
1.8 -+ 1.5
Geometric 0.90 mean
9
1.4
14
7.9
77
15
1.4
18
5.2
67
Median method
1.2
Rates are calculated from frequencies (Table 8) as described in the text.
A Non-Selective Mitotic Map. In order to eliminate the possibility that the recovery of mitotic recombinants was biased due to selection for cyh r recombinants, we also examined a non-selected population which had undergone a crossover between ade5 and the centromere. Such a crossover, when followed by appropriate chromosome segregation to homozygose ade5, results in a white recombinant colony. The frequency of white colonies averaged 2.6 x 10 - 3 . Of 137 white clones examined from 54 cultures of JG44, 70 had crossovers in either intervals I, II, or III. Of these 70, 36% had recombined in interval I, 14% in interval II, and 50% in interval III.
These data are in good agreement with the relative frequencies obtained from selective experiments. A summary of wild type mitotic recombination rates and distributions obtained by all methods is shown in Table 10A.
The Distribution o f Mitotic Exchange Events in r e m l - 1 / reml-1 Diploids. We isolated cyh r colonies from 7 cultures of 2 different strains carrying the r e m l mutation (Table 8). As expected, the frequency of cyh r colonies is increased fiftyfold in the presence of the r e m l mutation. In addition, the distribution of recombination events as determined from the rates (Table 9) is different from the distribution in wildtype. The average of the distribution values calculated by all three methods shows that approximately 10% of the events fall in interval I, 13% in interval II and 77% in interval III. There was clearly a jackpot event in culture 1 in interval III. However, calculation of the relative distribution of the arithmetic means without using culture 1 gives values of 10% for interval I, 13% for interval II, and 76% for interval III. These values are still quite different from the wildtype distribution. A Mitotic Map in rein1-1 Strains f r o m Non-Selected Events. We isolated white (ade5 homozygous) recombinants from 22 cultures of the reml-1/reml-1 diploid, JG43. The frequency of white colonies averaged 2.1%, or about 10 times the wildtype frequency. Of 1,063 white colonies examined, 467 had events in intervals I, II or III; 14% of these were in interval I, 24% were in interval II, and 62% in interval III. This is in reasonably good agreement with the selected distribution, and is also different from the wildtype mitotic distribution. As for the non-selected events observed in wildtype, it must be emphasized that white colonies from a culture are not necessarily independent. A summary of the data found for rein1-1 strains is shown in Table 10B.
247
R. E. Malone et al.: Mitotic Versus Meiotic Exchange Table 10. Summary of mitotic exchange rates and distributions for wild type and reml-1 diploids Experiment
Method of calculation
Interval I
Interval II
Interval III
Events per % of Total cell division x 105
Events per % of Total cell division x 10 s
Events per % of Total cell division x 105
0.73 0.53 0.64
32 28 25
0.32 0.29 0.48
14 15 19
1.2 1.1 1.4
53 57 56
A. Wild type diploids Cultures:
Arithmetic mean Geometric mean Median method
Independent events:
Counting
29
-
10
-
61
Unselected events:
Counting
-
36
-
14
-
50
Cultures:
Arithmetic mean Geometric mean Median method
10.1 9.1 11.9
5 9 15
17.7 14.0 13.5
9 14 18
171 78.7 51.8
86 77 67
Unselected events:
Counting
14
-
24
-
62
B. reml-1 diploicls
Rates and distributions were calculated as described in the text.
a) C
leul
I
trp5
[
28 C leu 1 b) l ~ 5 C c) I
I
16
56
trp5 I
cyh2 I
26
leu 1 [ 10
cyh2
I
trp5 I 13
69 cyh2 I
-77
Fig. 1 a-c. Relative recombination maps from wild type mitotic and meiotic distributions and rein1-1 mitotic distributions for chromosome VII. The relative recombination maps for mitotic values are taken from the averagesof the three methods of calculating rates from cultures. The relative meiotic values are taken from the standard meiotic maps (see Table lb), (a) wildtype mitotic map; (b) wild type meiotic map; (c) reml-1 mitotic map. (C = eentromere)
Discussion
The data presented above demonstrate that the distribution of spontaneous exchange events is different in mitosis and meiosis of wild type yeast strains. In Fig. 1 we have expressed the recombination maps in relative terms to compare the distributions. Regions closer to the centromere have a higher relative rate of recombination in mitosis. Similar results have been reported for Drosophila melanogaster (Stern, 1936; Garcia-Bellido, 1972; Hiraizumi et al., 1973) and Aspergillus nidulans
(Pontecorvo and K/~fer, 1958). In these two species, however, it is not known whether mitotic recombination occurs at the two-strand stage or at the four-strand stage. It has been proposed for Drosophila that the difference in the exchange distribution between meiosis and mitosis is related to the fact that there are large amounts of centromeric heterochromatin (Becker, 1976). These regions are rich in highly repetitive DNA and are throught to be quiescent in meiotic recombination (Baker, 1958). Yeast chromosomes are too small to be easily visualized, and it is not known whether a small amount of highly repetitive DNA might be located in centromeric regions. This question may be answered by current experiments designed to clone yeast centromeres. Such experiments should also determine whether the meiotic or mitotic maps more closely reflect physical distances. Unlike the observations of Campbell and Fogel (1977), we did not find any significant chromosome loss associated with mitotic recombination. Although only 63 different interval I recombinants were dissected (Table 5), in 3 diploids (RM13, RM15, and JG88) chromosome loss would have been signaled by hemizygosis of the A D E 6 marker and ascosporal lethality. We observed no such interval I "recombinants" among thse diploids. We can think of four possible reasons why we did not observe chromosome loss. First, loss of chromosome VII may be lethal, at least most of the time. Second, Fogel and Campbell (1977) were observing gene conversion while this study focused on crossing over. Third, their study was done on chromosome III while ours was on chro-
248 mosome VII; perhaps there are differences in the probabilities for recombination-associated loss among different chromosomes. Finally, their study was done in a disomic (n + 1) strain; ours was done in a normal diploid, tt is possible that recombination in an aneuploid is different or has different consequences from that occurring in euploid strains. Although mitotic recombination in reml-1 hybrids occurs at the two-strand stage as in wild type strains, mitotic exchange events in reml-1 strains exhibit a more meiotic-like distribution (Fig. 1). Since rein1-1 enhances recombination rates and is a semi-dominant mutation, these observations suggest that the reml-1 lesion may turn on some meiotic functions during mitosis. Thus, mitotic exchange in reml-1 cells may be o f a mixed nature. One hypothesis for the reml-1 mutation is that it allows the recognition of putative meiotic recombination sequences analogous to cog in Neurospora (for review, c.f., Catcheside, 1974; Baker et al., 1976) or chi in E. coli (Malone et al., 1978; Stahl et al., 1975). We are isolating mitotic hypo-rec mutations in a reml-1 background; their meiotic phenotypes should be instructive in understanding the reml-1 recombination pathway. Additionally, if reml-1 does turn on meiotic functions, some extragenic suppressors of rein1-1 may be meiotic hypo-rec mutations.
Acknowledgment. We thank Dr. R. Easton Esposito, S. Klapholz, M. Pastorcic and J. Wagstaff for helpful discussions of the experiments described above. This research was supported by NIH grant PHS GM-23277-02, CCRC grant PHS CA 19265-03/Project 508, NIH postdoctoral fellowship PHS GM 05965-03 to R.E.M. and Genetics Training Grant GM 90 to J.E.G.
R.E. Malone et al.: Mitotic Versus Meiotic Exchange
References Baker, W. K.: Am. Nat. 92, 59-60 (1958) Baker, B., Carpenter, A., Esposito, M. S., Esposito, R. E., Sandier, L.: Annu. Rev. Gen. 10, 53-134 (1976) Becker, H. J.: Mitotic recombination. In: The Genetics and Biology of Drosophila M. Ashburner and E. Novitski (eds.), p. 1020. New York: Academic Press 1976 Campbell, D. A., Fogel, S.: Genetics 85,573-585 (1977) Catcheside, D. G.: Fungal genetics. Annu. Rev. Genet. 8, 279 to 300 (1974) Drake, J.: The molecularbasis ofMutation., p. 49, San Francisco: Holden Day 1970 Esposito, M. S.: Proc. Nat. Acad. Sci. USA 75, 4436-4440 (1978) Garcia-Bellido, A.: Mol. Gen. Genet. 115, 54-72 (1972) Golin, J.: Ph.D. Thesis, University of Chicago, Chicago, Iil., 1979 Golin, J., Esposito, M. S.: Mol. Gen. Genet. 150, 127-135 (1977) Hiraizumi, Y., Slatko, B., Langley, C., Nill, A.: Genetics 73, 439-444 (1973) Lea, D. E., Coulson, C. A.: J. Genetics 49, 264-284 (1948) Malone, R. E., Esposito, R. E.: Proc. Nat. Acad. Sci. USA 77, 503-507 (1980) Malone, R. E., Chattoraj, D. K., Faulds, D., Stahl, M. M., Stahl, F. W.: J. Mol. Biol. 131,473-491 (1978) Mortimer, R. K., Hawthorne, D. C.: Genetics 74, 33-54 (1973) Perkins, D. D.: Genetics 34, 607-626 (1949) Pontecorvo, O., K~fer, E.: Genetic analysis by means of mitotic recombination. Adv. Genet. 9, 71-104 (1958) Roman, H. L.: Studies on gene mutation in Saccharomyces. Cold Spring Harbor Symp. Quant. Biol. 21,179-183 (1956) Stahl, F., Crasemann, J. N., Stahl, M. M.: J. Mol. Biol. 94, 203212 (1975) Stern, C.: Genetics 21,625-730 (1936)
Communicated by F. Kaudewitz Received November 29, 1979
~
~
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© by Springer-Verlag 1980
Mitotic Versus Meiotic Recombination in Saccharomyces cerevisiae Robert E. Malone, John E. Golin* and Michael S. Esposito Department of Biology and Committee on Genetics, The University of Chicago, Chicago, Illinois USA
Summary. As part o f a comparative analysis o f spontaneous mitotic and meiotic recombination we have compared the mitotic and meiotic maps o f the wild type and yeast hybrids homozygous for reml-1, a mitosis-specific hyper-rec mutation (Golin and Esposito, 1977; Golin, 1979). In wild type yeast strains recombination in centromere proximal intervals occurs relatively more frequently in mitosis than in meiosis. In reml-1/reml-1 hybrids the distribution of mitotic exchange events is more similar to the distribution observed in meiosis. Key words: Recombination Hyper-rec mutants Yeast
Introduction We have recently begun a comparative analysis of mitotic and meiotic exchange in wild type, hyper-rec (Golin and Esposito, 1977; Golin, 1979), and hypo-rec diploids (Malone and Esposito, 1980). These studies were prompted in part by the observation that spontaneous mitotic recombination in Saccharomyces cerevisiae occurs almost exclusively at the two-strand stage (Esposito, 1978) rather than at the four-strand stage as originally proposed for Drosophila melanogaster by Stem (1936). The purpose o f the experiments described below was to examine the rates and distribution of mitotic exchange events in the wild type and diploids homozygous for the mitosis-specific hyper-rec mutation reml-1 (Golin and Esposito, 1977).Mitotic recombinationinreml-1/reml-1 diploids occurs at the two-strand stage as in the wild * Present address: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA
Offprint requests to." M. S. Esposito
type (Golin, 1979). Meiotic recombination in reml-1 diploids occurs at the wild type level in all genetic intervals thus far tested (Golin, 1979). We have compared the mitotic maps o f chromosome VII generated by wild type and reml-1/reml-1 with the meiotic map. We find: (1) The mitotic and meiotic maps of the wild type differ with respect to the relative distribution o f exchange events. Mitotic recombination occurs relatively more frequently in regions close to the centromere. (2) The rein1-1 mutation not only increases the rate but also alters the distribution of mitotic exchange events resulting in a mitotic map more closely resembling the meiotic map.
Materials and Methods a) Media. The amounts indicated are those required for preparation of one liter of medium. Yeast Peptone Dextrose (YPD): dextrose 20 g, peptone 20 g and yeast extract 10 g. For solid YPD 15 g agar were added. Synthetic Complete: agar 15 g, dextrose 20 g, yeast nitrogen base without amino acids (Difco) 1.7 g, ammonium sulfate 5 g, adenine sulfate 10 rag, uracil 20 rag, Larginine-HCl 50 rag, L-histidine-HC1 20 rag, L-leucine 100 mg, Lqysine-HC1 50 rag, L-methionine 20 rag, L-phenylalanine 50 rag, L-threonine 100 rag, L-tryptophan 50 rag, L-tyrosine 50 rag, adjusted to pH 5.8 with HC1 or KOH. Cycloheximide: synthetic complete medium containing 2 rag/m! of cycloheximide. Sporulation Medium for Replica Plating: agar 15 g, potassium acetate 20 g, dextrose 1 g, yeast extract 2.5 g, 75 mg/1 of each of the following: adenine sulfate, uracil, L-histidine-HC1, Lqeucine, Llysine-HC1, L-methionine, L-phenylalanine, L-tryptophan, Ltyrosine, and adjusted to pH 7 with acetic acid or KOH.
b) Strains Used. The complete genotypes of all strains used in the experiments discussed in this paper are shown in Table la. Note that the cyh2r allele is recessive to wildtype. Strains JG44, JG43, RM13, and RM15 were dissected and at least 40 tetrads analyzed; the meiotic map distances obtained for chromosome VII are shown in Table lb. The standard values for chromosome VII come from the published data of Mortimer and Hawthorne (1973). / O172-8083/80/0001/0241/$ 01.60
242
R. E. Malone et al,: Mitotic Versus Meiotic Exchange
Table la. Genotypes of strains employed Strain
Genotype
REM1/REM1
Strains:
JG44
a D CAN1 -D CAN1
ura3-1 HOM2HIS1 ura3-313 H O M 3 h i s l
lys2-2 t y r l - 2 his7-1 lys2-1 tyrl-1 HIS7
ade5 m e t l 3 - c cyh2 r trp5-c leul-c ADE6 CLY8 ADE5 m e t 1 3 - d CYH2trp5-d l e u l - 1 2 ADE6 CLY8
ade2-1 ade2-1
JG85
a D CAN1 c~ d CAN1
ura3-1 URA3
HOM3 HIS1 HOM3 HIS1
lys2-2 t y r l - 2 his7-1 lys2-! tyrl-1 his7-2
ade5 met13-c cyh2 r trp5-c leul-c ADE6 CLY8 ADE5 met13-d CYH2trp5-d l e u l - 1 2 ade6 cly8
ade2-1 ade2-1
D can1-100 ura3-1 d CAN1 URA3
HOM3 hisl horn3 HIS1
lys2-1 TYR1 his7-7 LYS2 tyrl-1 HIS7
ADE5 MET13 CYH2TRP5 leu1-12 ADE6 CLY8 ADE5 m e t l 3 - c cyh2 r trp5-c leul-c ade6 CLY8
ade2-1 ade2-I
JG88
a
RM7
a d c~ d
can1-100 ura3-1 HOM3 HIS1 CAN1 ura3-313 HOM3 HIS1
lys2-2 t y r l - 2 his7-1 lys2-1 tyrl-1 his7-2
ADE5 met13-c cyh2 r trp5-c leul-c ADE6 CLY8 ade5 m e t l 3 - d CYH2trp5-d l e u l - 1 2 ADE6 CLY8
ade2-1 ade2-1
RM13
a a
d d
c a n l - 1 0 0 ura3-1 h o m 3 HIS1 CAN1 ura3-313 HOM3 h i s l - 1 9
lys2-1 TYR1 HIS7 LYS2 tyrl-1 HIS7
ade5 MET13 CYH2trp5-d leu1-12 ADE6 CLY8 ADE5 m e t l 3 - c cyh2 r trp5-c leul-c ade6 CLY8
ade2-1 ade2-1
RM15
a a
d d
c a n l - 1 0 0 ura3-313 HOM3 HIS1 CAN1 ura3-1 HOM3 HIS1
lys2-1 tyrl-1 his7-2 lys2-2 t y r l - 2 his7-1
ade5 met13-d CYH2trp5-d l e u l - 1 2 ADE6 CLY8 ADE5 m e t l 3 - c cyh2 r trp5-c leul-c ade6 CLY8
ade2-1 ade2-!
f e r a l - I / r e i n 1 - 1 Strains:
JG43 JG83
a
D CAN1 D CAN1
ura3-1 HOM3 HIS1 ura3-313 HOM3 his1
lys2-2 t y r l - 2 his7-1 lys2-1 tyrl-1 HIS7
ade5 m e t l 3 - c cyh2 r trp5-c leul-c ADE6 CLY8 ADE5 m e t l 3 - d CYH2trp5-d l e u l - 1 2 ADE6 CLY8
ade2-1 ade2-1
a D CAN1 c~ D CAN1
ura3-1 HOM3 HIS1 ura3-313 HOM3 HISt
LYS2 t y r l - 2 HIS7 lys2-1 TYR1 HIS7
ade5 m e t l 3 - c cyh2 r trp5-c leul-c ADE6 CLY8 ADE5 m e t l 3 - d CYH2trp5-d l e u l - 1 2 ADE6 CLY8
ade2-1 ade2-!
Gene symbols are as follows: a, ~ - mating type alleles; ade adenine auxotroph; can - canavanine resistance; D - diploidization (homothallism); his - histidine auxotroph; horn - threonine auxotroph; leu - leucine auxotroph; lys - lysine a u x o t r o p h ; m e t methionine a u x o t r o p h ; r e m - hyper-recombination; trp - trptophan auxotroph; and t y r - tyro sine auxotroph.
Table l b , Meiotic map distances on c h r o m o s o m e VII Strain
Standard RM13 RM15 JG44 JG43
No. of tetrads 100 80 200 40
V
IV
III
II
I
0
0'
1/2% TT ura3-leul
> > > >
86.6 50 50 50 50
22.9 24.4 16.2 18.2 26,6
43.2 42.6 33.8 48.7 35,0
16.6 19.7 20.9 21.7 25,0
2.9 -
31.7 -
40.4 43.4 -
9.7 a 10.0 7.5 8.4 12,2
Intervals on c h r o m o s o m e VII are designated as a d e 5 - ( V ) - m e t l 3 ( I V ) - c y h 2 - ( I I I ) - t r p 5 - ( I I ) - l e u l - ( I ) - c e n t r o m e r e - ( O ) - a d e 6 . Region 0' in RM13 and RM15 is the l e u l - a d e 6 distance. Some intervals could not be measured in strains used in these experiments because appro100 x TT + 6NPD priate markers were not present. Map distances were calculated by the formula map distance in centimorgans = of 2(PD + NPD + TT) Perkins (1949) where PD = parental ditype, NPD = non-parental ditype and TT = tetratype asci respectively. One-half of the percent TT asci for the unlinked centromere markers ura3 and l e u l closely approximates the sum of the gene to centromere distances for these markers. The meiotic map distances of R E M 1 / R E M 1 and r e m l - 1 / r e m l - 1 are similar as previously reported (Golin and Esposito, 1977; Golin, 1979). a
S u m of the gene-centromere distance for l e u l and ura3,
c} Isolation a n d A n a l y s i s o f C y c l o h e x i m i d e R e s i s t a n t (cyh r) Colonies f r o m Culture. All cultures were started from single
colonies diluted to a concentration of 1,000 cells/ml in 5 ml of YPD. The cultures were grown at 30 °C, with aeration, to a concentration of approximately 2 x 107. Samples o f the cultures were plated on complete plates (for total cell n u m b e r ) and on cycloheximide plates for c y h r colonies. Approximately
200 c y h r colonies from each culture were picked to YPD masters to be analyzed further. The position of the recombination event which led to the c y h r colony was located by the following procedure. The YPD master was replicated to sporulation m e d i u m and cycloheximide m e d i u m (to verify the p h e n o t y p e again). After 4 days, the sporulation plate was replicated to media lacking leucine or trypto-
243
R. E. Malone et al.: Mitotic Versus Meiotic Exchange Table 2. Frequency, number, and position of exchange events resulting in eyh r colonies in wild type diploids Strain
Culture number
Cells per 5 ml culture x 10 - 8
Total cyh r frequency x 104
Interval I Number
Intervall II Number
Interval III
eyh r
Frequency x 104
cyh r
Frequency x 104
Number
eyh r
Frequency x 104
JG44
1 2 3 4 5 6
1.0 0.5 2.4 3.0 3.4 1.7
3.04 4.85 3.68 4.37 3.07 4.90
55 90 24 31 18 7
1.98 3.40 1.25 0.79 0.61 0.39
3 9 8 85 15 10
0.12 0.34 0.40 2.18 0.52 0.53
26 39 38 53 57 70
0.94 1.50 1.99 1.36 1.93 3.92
JG85
7 8 9
1.0 0.6 1.0
1.84 1.08 1.19
57 36 22
0.66 0.46 0.38
20 9 9
0.24 0.12 0.15
83 38 37
0.96 0.50 0.64
RM7
10 11
0.8 0.6
0.98 1.98
62 58
0.29 0.51
38 35
0.19 0.32
104 128
0.50 1.15
RM13
12 13 14
0.7 2.0 0.6
1.19 0.83 3.40
143 20 28
0.86 0.08 0.67
15 86 41
0.10 0.37 0.99
41 87 72
0.25 0.37 1.74
RM15
15 16
0.7 0.6
1.88 1.59
35 41
0.43 0.37
31 37
0.39 0.33
84 99
1.05 0.89
JG88
17 18
0.2 0.3
3.06 3.21
50 16
1.00 0.29
12 24
0.24 0.44
91 134
1.82 2.45
The number of eyh r ceils is the actual number of cyh r colonies observed. The frequency refers to the number of cyh r cells per ml divided by the total number of cells per ml.
phan or methionine (dropout media). It was assumed that colonies which had undergone gene conversion events of CYH2 s to cyh2 r were those that remained heteroallelic at leul, trp5, and met13. They therefore generated prototrophic papillae on all dropout media after sporulation. Such colonies could also be due to double crossover events; these, however, should be rare even in rein1-1. Colonies which had undergone crossovers in interval I (between the centromere and leul) were detectable because they had become homozygous for all markers on the left arm of chromosome VII. After sporulation, such colonies could not give rise to prototrophs on met, leu, or trp dropout media because they were homoallelic. Crossovers in region II (between leul and trp5) left leul heteroallelic while both trp5 and met13 were homoallelic. Crossovers in region III (between trp5 and cyh2) left leul and trp5 heteroallelic while met13 (which is distal to cyh2) was homoallelic. In at least two cultures of every strain used, the alleles which were homozygosed by the crossover event were tested and shown to be the ones on the cyh2 r marker chromosome. In those strains where the leul, trpS, or met13 loci were heterozygous instead of heteroallelic (e.g., met13 in RM13), the recombination event could be located because the recessive marker was in coupling with the eyh2 r marker chromosome.
d) Isolation andA nalysis of independent cyh r Colonies. A culture of JG88 was grown to a concentration of 107 ceUs/ml and plated on complete medium in order to obtain single colonies (about 50 colonies/plate). (At the same time the frequency of cyh r colonies was measured and found to be normal. Data not shown.) Five hundred colonies were picked and small "patches" of ceils made on YPD masters. Following 2 days of growth, the YPD masters were replicated to cyclohex~mide plates. After 3 to 4 days, a number of cyh r papillae arose (due to mitotic recombination) in
each patch. A single cyh r papillus was picked from each patch for further study; each cyh r colony thus acquired represents an independent event. The cyh r clones were analyzed as described in Section C.
e) Isolation and Analysis of Non-Selected Chromosome VII. Re. combinants. JG44 and JG43 are homozygous for the ade2.1 mutation which causes the production of a red pigment. The production of this pigment can be blocked by a mutation in the ade5 gene (Roman, 1956). JG44 and JG43 are heterozygous for the ade5 mutation, the most distal marker on the left arm of chromosome VII. Recombination events which render the diploid homozygous for the ade5 mutation block the production of pigment and lead to a white colony. In order to study unselected events, white colonies (which appear at an average frequency of 2.6 x 10 - 3 in JG44 and 2.1 x 10 - 2 in JG43) were picked from complete plates to a YPD master and analyzed for the other markers on chromosome VII as discussed above.
Results The Distribution o f Mitotic Exchange Events in Wild T y p e Diploids. In these e x p e r i m e n t s we m e a s u r e d t h e rates/cell division o f m i t o t i c e x c h a n g e events in genetic intervals on c h r o m o s o m e VII e m p l o y i n g six wild t y p e hybrids. Their c o m p l e t e g e n o t y p e s are given in Table la. We isolated r e c o m b i n a n t c y c l o h e x i m i d e resistant (cyh r) colonies f r o m 18 i n d e p e n d e n t cultures. The interval in w h i c h the e x c h a n g e events o c c u r r e d was d e t e r m i n e d as described in Materials and M e t h o d s . The results are
244
R.E. Malone et al.: Mitotic Versus Meiotic Exchange
Table 3. Rates and distributions of exchange events in wild type diploids Culture number
Interval I
Interval II
Interval III
Events % per cell of division total x 105
Events % per cell of division total x 105
Events % per cell of division total x 105
1.7 3.1 1.0 0.62 0.47 0.32 0.57 0.42 0.33 0.26 0.46 0.77 0.066 0.61 0.39 0.39 1.2 0.33
65 70 34 18 20 8 36 43 32 30 26 72 10 21 23 23 33 9
0.10 0.31 0.32 1.7 0.41 0.44 0.21 0.11 0.13 0.17 0.29 0.09 0.30 0.90 0.35 0.35 0.30 0.50
4 7 11 50 17 11 13 11 13 19 16 8 45 31 21 21 8 14
0.82 1.4 1.6 1.1 1.5 3.3 0.83 0.45 0.56 0.44 1.1 0.22 0.30 1.6 0.95 0.95 2.3 2.8
31 23 54 31 63 80 52 46 54 51 58 21 45 48 56 56 59 77
32
0.32 +0.38
14
1.22 -+ 0.84
53
Geometric Mean 0.52
28
0.29
15
1.11
57
Median Method
25
0.48
19
1.4
56
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Arithmetic Mean 0.73 -+ 0.72
0.64
Rates are calculated from frequencies (Table 2) as described in text. Table 4. Sporulation ability of interval I recombinants Strain
Culture number
No. of recombi- Average nant clones % tested sporulation
JG44
1 2 Parent
25 13 -
61 70 70
JG85
7 8 9 Parent
14 14 14 -
91 86 71 81
RM7
10 11 Parent
16 9 -
86 89 83
RM13
12 Parent
11 -
85 83
RM15
15 16 Parent
12 12 -
68 81 83
summarized in Table 2. From the frequencies of Table 2 we calculated the recombination rates shown in Table 3 for each culture from the equation r = 0.4343f/(logN-logNo) where r = r a t e , f = frequency,N = final total cell number, a n d N o = initial cell fiumber (Drake, 1970). Because recombination events occurring early during exponential growth lead to an overestimation of rates, we calculated both the arithmetic and geometric mean rates. The latter reduces the skewing of the means due to cultures with early "jackpot" events. In addition, we applied the method of the median (Lea and Coulson, 1948) to the distribution of frequencies. This gives rates of 6.4 x 10 - 6 for interval I, 4,8 x 10 -6 for II, and 1.4 x 10 - s for III (see Table 3). It is apparent that the relative distribution of rates (i.e., events) is similar no matter which method is used to calculate them. Approximately 28% of the intergenic recombination events occur between the centromere and leul (I), 16% between leul and trp5 (II) and 56% between trp5 and cyh2 (III).
Cyh r Recombinants in Region I are not due to Loss of Chromosome VII. Given the method by which we assign a recombination event to interval I, II, or III, a monosome arising from loss of chromosome VII would appear to have had a crossover event in interval I. In order to determine whether a substantial fraction of our interval I recombinants were really monosomes for chromosome VII, we sporulated and dissected a representative sample for several of the strains used (Tables 4, 5). The spomlation values and spore viability of interval I recombinants were essentially the same as the parental diploid. In every case the spore viability was significantly different than the expected 50% for a monosome; of 63 different interval I recombinants examined, none had lower than 85% spore viability (Table 5). In addition, when the parental diploid was heterozygous for ade6 (on the opposite side of the centromere), the interval I recombinants remained heterozygous for ade6 with one exception. In one culture of JG85, 7/10 interval I recombinants were homozygous for ADE6 and CL YS. All seven had spore viability greater than 90% so they may have been clonally related double recombinants. If chromosome VII was originally lost, then it must have been restored during growth of the clone. We conclude that chromosome loss does not account for a significant fraction of the interval I recombinants observed.
Selective Growth Advantages Cannot Account for the Observed Distribution of Mitotic Exchange Events. It was possible that the distribution of mitotic crossingover we observed was due to growth rate differences between the different classes of recombinants. A reconstruction experiment was done to test this possibility. Approximately equal numbers of interval I, II, and III
245
R. E. Malone et al.: Mitotic Versus Meiotic Exchange Table 5. Spore viability of interval I recombinants Strain
Culture number
No. inter- Average val I no. clones tetrads/ tested clone
Classes of asci observed a 4 :0
3:1
2: 2
i :3
0: 4
Total asci
% viable spores
Range
JG44
1 2
8 9
10 !0
79 64
5 21
1 2
0 0
0 0
85 87
98 93
93-100 85-98
JG85
2 3 4
9 10 8
5 5 5
31 41 28
8 8 10
5 1 2
0 0 0
0 0 0
44 50 40
90 95 91
85-100 85-100 85-100
RM13
1
9
5
38
5
1
0
0
44
96
95-100
RM15
1 2
5 5
5 5
19 20
5 4
1 1
0 0
0 0
25 25
92 93
90-100 85-100
A number of interval I recombinants from several different cultures were sporulated and dissected. a
Number of asci containing 4 viable (4 : 0), 3 viable (3 : 1), 2 viable (2: 2), 1 viable (1 : 3) and no viable ascospores (0:4).
Table 6. Selective growth advantages of various cyh r recombinant classes Recombinant in interval
I II III
Culture 1 Fraction of total B 0.38 0.25 0.37
Culture 2
h
Relative increase A/B
Fraction of total B
0.41 0.34 0.25
1.08 1.36 0.68
0.23 0.39 0.38
Culture 3
h
Relative increase A/B
Fraction of toal B
A
Relative increase A/B
0.21 0.47 0.32
0.91 1.21 0.85
0.36 0.17 0.46
0.5 0 0.18 0.32
1.38 1.06 0.70
Average relative increase
1.12 1.21 0.74
Selective growth advantages of the various cyh r recombinant types were determined by mixing approximately equal amounts of each at a total concentration of 3 x 103 cells/ml in the presence of 105 cells/ml of the parental type. The mixed culture was then grown to a total cell concentration of 2 x 107 ceUs/ml. Cells were plated before and after growth of the cultures to determine the exact ratio of i : II :III. B = before growth; A = after growth.
Table 7. Independent events in a wild type diploid, JG88 Total number cyh r examined Colonies with events in Interval I Colonies with events in Interval II Colonies with events in Interval III Colonies with gene conversions
298 61 (29%) 21 (10%) 132 (61%) 85
Independent cyh2 r colonies were isolated and analyzed as described in the text. The numbers in parenthesis represent percent of total crossover 6vents
r e c o m b i n a n t s were inoculated at a total c o n c e n t r a t i o n o f 3 x 10 3 cells/ml in the presence o f parental cells at a c o n c e n t r a t i o n o f 10 s cells/ml. Such r e c o n s t r u c t e d cultures were t h e n grown to a total c o n c e n t r a t i o n o f app r o x i m a t e l y 2 x 10 7 cells/ml after which the relative ratios o f interval I, II, and III were determined. A comparison o f the ratios o f the various types o f r e c o m b i n a n t s
before and after g r o w t h shows that interval II recombinants have a slight advantage over interval I recombinants which in turn have a m o d e r a t e growth advantage over interval III recombinants (Table 6). These relatively small selective advantages are n o t sufficient to explain the large differences we observe b e t w e e n the m i t o t i c and m e i o t i c maps. A Mitotic Map Obtained f r o m Independent cyh r Reeombinants. Because the r e c o m b i n a t i o n events o b t a i n e d f r o m culture m a y be clonally related, we analyzed 298 i n d e p e n d e n t cyh r colonies f r o m J G 8 8 (see Materials and Methods for details). E a c h cyh r c o l o n y m a y be treated as representative o f an i n d e p e n d e n t r e c o m b i n a t i o n event and the distribution o f such events is shown in Table 7. A p p r o x i m a t e l y 29% o f the crossovers leading to cyh r clones o c c u r r e d in interval I, 10% in interval II, and 61% in interval III. This is similar to the average distribution o b t a i n e d f r o m the 18 cultures e x a m i n e d .
246
R.E. Malone et al.: Mitotic Versus Meiotic Exchange
Table 8. Frequency, number, and position of exchange events resulting in cyh r colonies in rein1-1 diploids Strain
Culture number
Cells per 5 ml culture x 10 -8
Total cyh r frequency x 102
Interval I Number cyh r
Interval II
Interval III
Frequency x 102
Number cyh r
Frequency x 102
Number cyh r
Frequency x 102
JG43
1 2 3 4 5
0.44 1.1 0.55 0.39 1.3
9.41 2.33 0.92 0.54 1.01
29 29 12 130 17
0.16 0.15 0.08 0.14 0.06
32 43 35 94 29
0.53 0.22 0.22 0.10 0.11
505 382 102 295 81
8.42 1.96 0.64 0.31 0.30
JG83
6 7
0.84 0.69
0.77 1.13
17 16
0.06 0.14
24 16
0.08 0.14
187 98
0.62 0.85
The number of cyh r cells is the actual number of cyh r colonies observed. The frequency refers to the number of cyh r cells per ml divided by the total number of cells per ml.
Table 9. Rates and distributions of exchange events in rein J-l~ reml.1 diploids Culture number
1 2 3 4 5 6 7
Interval I
Interval II
Interval III
Events % per cell of division totat x 104
Events % per cell of division total x 104
Events % per cell of division total x 104
1.5 1.3 0.73 1.3 0.39 0.53 1.3
4.8 1.9 2.0 0.94 0.71 0.70 1.2
6 9 23 18 23 10 12
79 17 5.9 2.9 2.0 5.5 7.6
92 84 68 56 64 82 75
9
17 -+ 28
86
2 7 9 26 13 8 12
Arithmetic 1.0 -+ mean 0.44
5
1.8 -+ 1.5
Geometric 0.90 mean
9
1.4
14
7.9
77
15
1.4
18
5.2
67
Median method
1.2
Rates are calculated from frequencies (Table 8) as described in the text.
A Non-Selective Mitotic Map. In order to eliminate the possibility that the recovery of mitotic recombinants was biased due to selection for cyh r recombinants, we also examined a non-selected population which had undergone a crossover between ade5 and the centromere. Such a crossover, when followed by appropriate chromosome segregation to homozygose ade5, results in a white recombinant colony. The frequency of white colonies averaged 2.6 x 10 - 3 . Of 137 white clones examined from 54 cultures of JG44, 70 had crossovers in either intervals I, II, or III. Of these 70, 36% had recombined in interval I, 14% in interval II, and 50% in interval III.
These data are in good agreement with the relative frequencies obtained from selective experiments. A summary of wild type mitotic recombination rates and distributions obtained by all methods is shown in Table 10A.
The Distribution o f Mitotic Exchange Events in r e m l - 1 / reml-1 Diploids. We isolated cyh r colonies from 7 cultures of 2 different strains carrying the r e m l mutation (Table 8). As expected, the frequency of cyh r colonies is increased fiftyfold in the presence of the r e m l mutation. In addition, the distribution of recombination events as determined from the rates (Table 9) is different from the distribution in wildtype. The average of the distribution values calculated by all three methods shows that approximately 10% of the events fall in interval I, 13% in interval II and 77% in interval III. There was clearly a jackpot event in culture 1 in interval III. However, calculation of the relative distribution of the arithmetic means without using culture 1 gives values of 10% for interval I, 13% for interval II, and 76% for interval III. These values are still quite different from the wildtype distribution. A Mitotic Map in rein1-1 Strains f r o m Non-Selected Events. We isolated white (ade5 homozygous) recombinants from 22 cultures of the reml-1/reml-1 diploid, JG43. The frequency of white colonies averaged 2.1%, or about 10 times the wildtype frequency. Of 1,063 white colonies examined, 467 had events in intervals I, II or III; 14% of these were in interval I, 24% were in interval II, and 62% in interval III. This is in reasonably good agreement with the selected distribution, and is also different from the wildtype mitotic distribution. As for the non-selected events observed in wildtype, it must be emphasized that white colonies from a culture are not necessarily independent. A summary of the data found for rein1-1 strains is shown in Table 10B.
247
R. E. Malone et al.: Mitotic Versus Meiotic Exchange Table 10. Summary of mitotic exchange rates and distributions for wild type and reml-1 diploids Experiment
Method of calculation
Interval I
Interval II
Interval III
Events per % of Total cell division x 105
Events per % of Total cell division x 10 s
Events per % of Total cell division x 105
0.73 0.53 0.64
32 28 25
0.32 0.29 0.48
14 15 19
1.2 1.1 1.4
53 57 56
A. Wild type diploids Cultures:
Arithmetic mean Geometric mean Median method
Independent events:
Counting
29
-
10
-
61
Unselected events:
Counting
-
36
-
14
-
50
Cultures:
Arithmetic mean Geometric mean Median method
10.1 9.1 11.9
5 9 15
17.7 14.0 13.5
9 14 18
171 78.7 51.8
86 77 67
Unselected events:
Counting
14
-
24
-
62
B. reml-1 diploicls
Rates and distributions were calculated as described in the text.
a) C
leul
I
trp5
[
28 C leu 1 b) l ~ 5 C c) I
I
16
56
trp5 I
cyh2 I
26
leu 1 [ 10
cyh2
I
trp5 I 13
69 cyh2 I
-77
Fig. 1 a-c. Relative recombination maps from wild type mitotic and meiotic distributions and rein1-1 mitotic distributions for chromosome VII. The relative recombination maps for mitotic values are taken from the averagesof the three methods of calculating rates from cultures. The relative meiotic values are taken from the standard meiotic maps (see Table lb), (a) wildtype mitotic map; (b) wild type meiotic map; (c) reml-1 mitotic map. (C = eentromere)
Discussion
The data presented above demonstrate that the distribution of spontaneous exchange events is different in mitosis and meiosis of wild type yeast strains. In Fig. 1 we have expressed the recombination maps in relative terms to compare the distributions. Regions closer to the centromere have a higher relative rate of recombination in mitosis. Similar results have been reported for Drosophila melanogaster (Stern, 1936; Garcia-Bellido, 1972; Hiraizumi et al., 1973) and Aspergillus nidulans
(Pontecorvo and K/~fer, 1958). In these two species, however, it is not known whether mitotic recombination occurs at the two-strand stage or at the four-strand stage. It has been proposed for Drosophila that the difference in the exchange distribution between meiosis and mitosis is related to the fact that there are large amounts of centromeric heterochromatin (Becker, 1976). These regions are rich in highly repetitive DNA and are throught to be quiescent in meiotic recombination (Baker, 1958). Yeast chromosomes are too small to be easily visualized, and it is not known whether a small amount of highly repetitive DNA might be located in centromeric regions. This question may be answered by current experiments designed to clone yeast centromeres. Such experiments should also determine whether the meiotic or mitotic maps more closely reflect physical distances. Unlike the observations of Campbell and Fogel (1977), we did not find any significant chromosome loss associated with mitotic recombination. Although only 63 different interval I recombinants were dissected (Table 5), in 3 diploids (RM13, RM15, and JG88) chromosome loss would have been signaled by hemizygosis of the A D E 6 marker and ascosporal lethality. We observed no such interval I "recombinants" among thse diploids. We can think of four possible reasons why we did not observe chromosome loss. First, loss of chromosome VII may be lethal, at least most of the time. Second, Fogel and Campbell (1977) were observing gene conversion while this study focused on crossing over. Third, their study was done on chromosome III while ours was on chro-
248 mosome VII; perhaps there are differences in the probabilities for recombination-associated loss among different chromosomes. Finally, their study was done in a disomic (n + 1) strain; ours was done in a normal diploid, tt is possible that recombination in an aneuploid is different or has different consequences from that occurring in euploid strains. Although mitotic recombination in reml-1 hybrids occurs at the two-strand stage as in wild type strains, mitotic exchange events in reml-1 strains exhibit a more meiotic-like distribution (Fig. 1). Since rein1-1 enhances recombination rates and is a semi-dominant mutation, these observations suggest that the reml-1 lesion may turn on some meiotic functions during mitosis. Thus, mitotic exchange in reml-1 cells may be o f a mixed nature. One hypothesis for the reml-1 mutation is that it allows the recognition of putative meiotic recombination sequences analogous to cog in Neurospora (for review, c.f., Catcheside, 1974; Baker et al., 1976) or chi in E. coli (Malone et al., 1978; Stahl et al., 1975). We are isolating mitotic hypo-rec mutations in a reml-1 background; their meiotic phenotypes should be instructive in understanding the reml-1 recombination pathway. Additionally, if reml-1 does turn on meiotic functions, some extragenic suppressors of rein1-1 may be meiotic hypo-rec mutations.
Acknowledgment. We thank Dr. R. Easton Esposito, S. Klapholz, M. Pastorcic and J. Wagstaff for helpful discussions of the experiments described above. This research was supported by NIH grant PHS GM-23277-02, CCRC grant PHS CA 19265-03/Project 508, NIH postdoctoral fellowship PHS GM 05965-03 to R.E.M. and Genetics Training Grant GM 90 to J.E.G.
R.E. Malone et al.: Mitotic Versus Meiotic Exchange
References Baker, W. K.: Am. Nat. 92, 59-60 (1958) Baker, B., Carpenter, A., Esposito, M. S., Esposito, R. E., Sandier, L.: Annu. Rev. Gen. 10, 53-134 (1976) Becker, H. J.: Mitotic recombination. In: The Genetics and Biology of Drosophila M. Ashburner and E. Novitski (eds.), p. 1020. New York: Academic Press 1976 Campbell, D. A., Fogel, S.: Genetics 85,573-585 (1977) Catcheside, D. G.: Fungal genetics. Annu. Rev. Genet. 8, 279 to 300 (1974) Drake, J.: The molecularbasis ofMutation., p. 49, San Francisco: Holden Day 1970 Esposito, M. S.: Proc. Nat. Acad. Sci. USA 75, 4436-4440 (1978) Garcia-Bellido, A.: Mol. Gen. Genet. 115, 54-72 (1972) Golin, J.: Ph.D. Thesis, University of Chicago, Chicago, Iil., 1979 Golin, J., Esposito, M. S.: Mol. Gen. Genet. 150, 127-135 (1977) Hiraizumi, Y., Slatko, B., Langley, C., Nill, A.: Genetics 73, 439-444 (1973) Lea, D. E., Coulson, C. A.: J. Genetics 49, 264-284 (1948) Malone, R. E., Esposito, R. E.: Proc. Nat. Acad. Sci. USA 77, 503-507 (1980) Malone, R. E., Chattoraj, D. K., Faulds, D., Stahl, M. M., Stahl, F. W.: J. Mol. Biol. 131,473-491 (1978) Mortimer, R. K., Hawthorne, D. C.: Genetics 74, 33-54 (1973) Perkins, D. D.: Genetics 34, 607-626 (1949) Pontecorvo, O., K~fer, E.: Genetic analysis by means of mitotic recombination. Adv. Genet. 9, 71-104 (1958) Roman, H. L.: Studies on gene mutation in Saccharomyces. Cold Spring Harbor Symp. Quant. Biol. 21,179-183 (1956) Stahl, F., Crasemann, J. N., Stahl, M. M.: J. Mol. Biol. 94, 203212 (1975) Stern, C.: Genetics 21,625-730 (1936)
Communicated by F. Kaudewitz Received November 29, 1979
